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ADAMTS13: a new link between thrombosis and inflammation

ADAMTS13: a new link between thrombosis and inflammation ARTICLE ADAMTS13: a new link between thrombosis and infl ammation 1,2 1,2 1,2 1 Anil K. Chauhan , Janka Kisucka , Alexander Brill , Meghan T. Walsh , 3 1,2 Friedrich Scheifl inger , and Denisa D. Wagner 1 2 Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115 Baxter Bioscience, 1220 Vienna, Austria von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprote- ase with thrombospondin type I repeats – 13 (ADAMTS13) activity is decreased in both acute and chronic infl ammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and infl ammation. Using intravital microscopy, we show that ADAMTS13 defi ciency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in infl amed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13 mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13 compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13 but not in WT mice. Furthermore, in infl ammatory models, ADAMTS13 defi ciency re- sulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our fi ndings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during infl ammation. Leukocyte rolling, adhesion, and transmigration defi ciency in humans ( 4 ) and defective occlu- CORRESPONDENCE Denisa D. Wagner: at the site of infection or injury are hallmarks of sive thrombus formation in the arteries and [email protected] infl ammation. These steps in the leukocyte ad- veins of Vwf mice ( 5, 6 ). VWF levels are el- hesion cascade are controlled by selectins, integ- evated in both chronic and acute infl ammation Abbreviations used: AD- AMTS13, a disintegrin-like and rins, and other adhesion molecules ( 1 ). It is now ( 7 ). Normally, the glycoprotein GPIb  platelet- metalloprotease with thrombo- evident that several adhesion molecules involved binding domain is not exposed on circulating spondin type I repeats – 13; H & E, in infl ammation are also important in thrombo- VWF, but it becomes exposed under patholog- hematoxylin and eosin; MPO, sis. For example, P-selectin expressed on acti- ical high shear ( 8 ) or upon binding to exposed myeloperoxidase; TTP, throm- botic thrombocytopenia pur- vated platelets and endothelium contributes to subendothelium. Circulating active VWF has also pura; ULVWF, ultralarge VWF; both thrombosis and infl ammation ( 1 – 3 ). P-se- been found in von Willebrand disease type 2B, VWF, von Willebrand factor. lectin and von Willebrand factor (VWF) are malaria, antiphospholipid syndrome, and throm- present in the platelet  -granules and Weibel- botic thrombocytopenia purpura (TTP) ( 9 ). In Palade bodies of endothelial cells. P-selectin me- mice, VWF defi ciency delays the formation diates leukocyte and platelet rolling, and VWF of fatty streaks in two models of atherosclero- mediates initial adhesion of platelets, the fi rst step sis ( 10 ). These studies suggest that VWF could in infl ammation and thrombosis. Thus, Weibel- play an active role in infl ammation in addition Palade bodies constitute a prominent link be- to hemostasis. tween thrombosis and infl ammation. The role of The VWF contained in Weibel-Palade bod- VWF in infl ammation needs to be explored. ies is extremely large ( 11 ) and is designated as The essential role of VWF in hemostasis is illustrated by von Willebrand disease, a bleed- © 2008 Chauhan et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months ing disorder associated with functional VWF after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncom- mercial–Share Alike 3.0 Unported license, as described at http://creativecommons The online version of this article contains supplemental material. .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 205 No. 9 2065-2074 2065 www.jem.org/cgi/doi/10.1084/jem.20080130 The Journal of Experimental Medicine ultralarge VWF (ULVWF;  20,000 kD). Upon cellular acti- or sepsis ( 20 – 22 ). Proinfl ammatory cytokines, such as TNF-  , vation or stimulation with secretagogues (such as histamine, IL-8, and IL-6, released during infl ammation have been shown calcium ionophore, thrombin, and TNF-  ), ULVWF is re- to have distinct eff ects on the endothelial release of ULVWF leased from the storage granules ( 12 ). These ULVWF multi- and its processing ( 23 ). Recently, it was shown in vitro that mers are considered biologically hyperactive because they bind VWF promotes leukocyte adhesion ( 24 ) and that platelets bound better to the extracellular matrix ( 13 ) and form higher strength to ULVWF can support leukocyte tethering and rolling under bonds with platelet GPIb compared with small plasma multi- high shear stress ( 25 ). We hypothesize that ADAMTS13, mers ( 14 ). A disintegrin-like and metalloprotease with throm- by cleaving hyperactive ULVWF, down-regulates not only bospondin type I repeats – 13 (ADAMTS13) inhibits platelet thrombosis, but also infl ammation. To investigate the role of adhesion by quickly cleaving hyperactive ULVWF multimers ADAMTS13 and its substrate VWF in infl ammation, we stud- +/+ +/+ released from the Weibel-Palade bodies under conditions of ied leukocyte rolling and adhesion in Adamts13 /Vwf , /  +/+ +/+  /   / fl uid shear stress ( 15 ). In 1982, Moake et al. found ULVWF Adamts13 /Vwf , Adamts13 /Vwf , and Adamts13 / multimers in the plasma of patients suff ering from chronic Vwf mice using intravital microscopy. We also examined the relapsing TTP ( 16 ), a disorder clinically characterized by micro- role of the ADAMTS13 – VWF axis in neutrophil extravasation angiopathic hemolytic anemia, thrombocytopenia, neurological in two diff erent models of infl ammation (thioglycollate-induced abnormalities, fever, and renal dysfunction. The pathogen- peritonitis and wound healing). esis of TTP has been linked to a defi ciency of ADAMTS13, which can be either familial (a mutation in the ADAMTS13 R ESULTS gene) or acquired (through inhibitory antibodies generated D efi ciency of ADAMTS13 results in increased numbers against ADAMTS13). Studies in mice show that ADAMTS13 of leukocytes rolling per minute in unstimulated veins defi ciency is necessary, but not suffi cient, for the develop- To determine whether ADAMTS13 plays a role in infl am- ment of TTP, suggesting the existence of additional genetic mation, we visualized leukocyte rolling, as a measure of and/or environmental contributing factors ( 17, 18 ). Re- endothelial activation, on unstimulated mesenteric veins. We cently, we and others have found that a complete defi ciency found higher numbers of leukocytes rolling per minute on of ADAMTS13 in mice results in a prothrombotic pheno- the unstimulated endothelium in the Adamts13 mice on a type ( 18, 19 ). mixed background (C57BL/6/129 × 1/SV; mean ± SEM = +/+ Although it is not known whether ADAMTS13 plays a role 115 ± 16) compared with Adamts13 littermates (mean ± in infl ammation, recent studies have found reduced AD- SEM = 43 ± 10; P < 0.001; n = 10 – 12 of each group). To AMTS13 activity in patients with acute systemic infl ammation ensure that the observed phenotype was not caused by the Figure 1. ADAMTS13 defi ciency in mice results in increased leukocyte rolling per minute in unstimulated mesenteric veins . (A) Leukocyte rolling on the vessel wall was recorded in two to three unstimulated veins (200 – 300 μ m diameter) per mouse. There was an  2.5-fold increase in /  +/+ leukocyte rolling per minute on the endothelium in Adamts13 compared with Adamts13 mice, suggesting endothelial activation. The absence of ADAMTS13 activity on a VWF-defi cient background did not increase leukocyte rolling, indicating that the increased leukocyte rolling observed in Adamts13 mice is dependent on the presence of VWF. Data represent the mean ± SEM. (B) P-selectin – dependent rolling in Adamts13 mice. The unstimulated veins of Adamts13 mice were infused with either control Ig or anti – P-selectin Ig ( n = 6 veins from three mice of each group). Endogenous leukocytes and platelets were labeled with Rhodamine 6G. Representative photographs are shown. Lines delineate the blood vessel. The white arrow indicates a labeled leukocyte. Bars, 50 μ m. Video 1 is available at http://www.jem.org/cgi/content/full/jem.20080130/DC1. 2066 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE veins. 1- μ m fl uorescent microspheres coupled to anti – P- mixed background, we next evaluated Adamts13 mice selectin antibody were infused through the retroorbital ve- backcrossed onto a C57BL/6J background for eight genera- nous plexus, and their binding to unstimulated mesenteric tions. We found a similar increase in leukocyte rolling per veins was visualized and quantifi ed. A signifi cantly higher minute in these Adamts13 mice (mean ± SEM = 98 ± 13) +/+ number of microspheres bound to mesenteric veins was ob- compared with Adamts13 mice (mean ± SEM = 35 ± 6; P < /  +/+ served in Adamts 13 mice compared with Adamts13 0.001; n = 10 mice of each group), indicating that the endo- mice ( Fig. 2, C and D ). It is possible that fl uorescent micro- thelium in Adamts13 mice was preactivated ( Fig. 1 A ). spheres coupled to anti – P-selectin antibody were bound We next asked whether platelets on VWF strings were pre- to both endothelial cell and platelet P-selectin. However, sent in the untreated veins and could be responsible for the when we labeled the endogenous platelets and leukocytes observed increase in leukocyte rolling in the Adamts13 with Rhodamine 6G and visualized the unstimulated veins mice. We labeled the endogenous platelets and leukocytes by intravital microscopy, only transient platelet adhesion with Rhodamine 6G and visualized the veins by intravital was observed (Video 1). We did not observe a carpet of microscopy. Fig. 1 B (top) and Video 1 (available at http:// platelets in the unstimulated veins of Adamts13 mice www.jem.org/cgi/content/full/jem.20080130/DC1) show ( Fig. 1 B , top; and Video 1). These observations suggest that the absence of platelet-VWF strings on the endothelium the microspheres coupled to anti – P-selectin antibody were of the unstimulated Adamts13 veins, suggesting that it is most likely bound to endothelial P-selectin. Collectively, most likely an up-regulation of an endothelial selectin that these results suggest that the increased leukocyte rolling ob- is responsible for the increased leukocyte rolling. Indeed, the served in Adamts13 mice is likely caused by the release of observed leukocyte rolling in Adamts13 mice was depen- more Weibel-Palade bodies. dent on P-selectin, because infusion of a blocking antibody to P-selectin completely abolished leukocyte rolling ( Fig. Platelet depletion decreases leukocyte rolling 1 B , bottom; and Video 1). Because the only known substrate in unstimulated veins of Adamts13 mice for ADAMTS13 in thrombosis is VWF ( 26 ), we studied leu- We next examined whether platelets, by binding to ULVWF kocyte rolling in Adamts13 mice on a VWF-defi cient multimers and/or leukocytes, could promote the increase in background to evaluate whether the increase in leukocyte leukocyte rolling. We have previously shown that activated rolling per minute was also VWF dependent. The shear platelets stimulate Weibel-Palade body secretion ( 28 ). We rate and diameter of the evaluated veins were similar for +/+ +/+  /  +/+ +/+ depleted platelets by infusing anti-GPIb Ig, which depletes Adamts13 / Vwf , Adamts13 / Vwf , Adamts13 / /   /   / > 95% of platelets for up to 48 h ( 29 ). Anti-GPIb or control Vwf , and Adamts13 / Vwf mice ( Table I ). We found +/+  / Ig was infused i.v. in the Adamts13 and Adamts13 mice, that the absence of ADAMTS13 activity on a VWF-defi cient and 24 h later leukocyte rolling was visualized. In platelet- background did not result in increased leukocyte rolling +/+ depleted Adamts13 mice ( < 5% normal platelet count), ( Fig. 1 A ). These results suggest that the elevated baseline leukocyte rolling (mean ± SEM = 62 ± 10) was similar to leukocyte rolling observed in the veins of Adamts13 nondepleted mice (mean ± SEM = 57 ± 9; P = 0.75; Fig. 3 ). mice was caused by the combined eff ect of P-selectin and However, in Adamts13 mice, platelet depletion resulted in a VWF, most likely the unprocessed hyperactive ULVWF in twofold decrease in leukocyte rolling (mean ± SEM = 62 ± 8) the circulation. compared with nondepleted mice (mean ± SEM = 143 ± 12; ADAMTS13 defi ciency results in increased Weibel-Palade P < 0.001; Fig. 3 ). These results suggest that platelets con- tribute to the mechanism of increased leukocyte rolling in body release ADAMTS13 defi ciency in mice does not aff ect baseline Adamts13 mice. leukocyte counts in peripheral blood ( 17 ). We measured leukocyte rolling velocity at baseline and did not observe Table I. Hemodynamic parameters of vessels observed any diff erences in the leukocyte velocity in the unstimulated by intravital microscopy /  +/+ veins in the Adamts13 compared with Adamts13 mice Genotype Diameter Newtonian wall (unpublished data), suggesting that P-selectin is likely the ( μ m) shear rate  (s ) up-regulated molecule, as the rolling velocity depends pre- +/+ +/+ dominantly on E-selectin ( 27 ). In addition, increased solu- Adamts13 / Vwf 223.5 ± 21.1 192.4 ± 64.1 ble P-selectin and VWF concentrations were found in the ( n = 18) /  +/+ /  +/+ plasma of Adamts 13 compared with Adamts13 mice Adamts13 / Vwf 234.2 ± 24.2 183.8 ± 59.6 by ELISA, further indicating that the endothelium is acti- ( n = 19) /   / vated in Adamts13 mice ( Fig. 2, A and B ). Because most Adamts13 / Vwf 237.5 ± 36.8 160.6 ± 46 ( n = 18) circulating VWF is derived from the endothelium, these +/+  / 225 ± 25 152.5 ± 39.8 observations also suggest that more Weibel-Palade bodies Adamts13 / Vwf ( n = 21) are released in Adamts13 mice. To confi rm this, we measured endothelial P-selectin expression, as a marker for Hemodynamic parameters were established before recording leukocyte rolling for Fig. 1 . Values are represented as mean ± SD. P = NS. Weibel-Palade body secretion, on unstimulated mesenteric JEM VOL. 205, September 1, 2008 2067 Platelet-VWF strings anchored onto histamine-stimulated tagogue of Weibel-Palade bodies, to release ULVWF multi- endothelium decrease leukocyte rolling velocity mers. We found that leukocyte rolling velocity was slower in /  +/+ Previously, it was shown in vitro that platelets bound to endo- Adamts 13 compared with Adamts13 veins where plate- thelial ULVWF can support leukocyte tethering and rolling let strings do not form ( Fig. 4 ). In Adamts13 veins,  50% ( 25 ). Therefore, we asked whether the presence of VWF- of leukocytes rolled at a velocity of < 30 μ m/s compared with +/+ platelet strings on stimulated endothelium aff ects leukocyte  5% in Adamts13 (P < 0.001; Fig. 4 B ). Moreover, fre- rolling in vivo. Veins were stimulated with histamine, a secre- quent leukocyte interaction with the ULVWF-platelet strings was observed in the mesenteric activated veins ( Fig. 4 A ; and Video 2, available at http://www.jem.org/cgi/content/full/ jem.20080130/DC1), which suggests that the decrease in leukocyte rolling velocity is likely caused by the presence of ULVWF-platelet strings on the endothelium. In contrast to veins, we did not see endothelial VWF-platelet strings in the arteri- oles treated identically with histamine (unpublished data). Next, we assessed whether the ULVWF-platelet strings could support leukocyte rolling in the absence of P-selectin. Infusion of blocking antibody to P-selectin in Adamts13 mice completely abolished leukocyte rolling but had no ef- fect on the presence of ULVWF-platelet strings ( Fig. 4 C ; and Video 3, available at http://www.jem.org/cgi/content/ full/jem.20080130/DC1). These results are in agreement with previous studies where we have observed the formation of platelet-VWF strings in P-selectin mice in the presence of ADAMTS13 inhibitor ( 30 ). Thus, ULVWF multimers only support leukocyte adhesion that was initiated by leuko- cyte binding to P-selectin. ADAMTS13 defi ciency increases leukocyte adhesion in infl amed venules The infl ammatory cytokines TNF-  and IL-8 have been shown to release ULVWF from human umbilical vein endothelial cells in vitro ( 23 ). Similarly, we have observed platelet-VWF strings in Figure 2. Adamts13 mice show an increase in the expression of endothelial P-selectin, and higher soluble P-selectin and plasma VWF. Increased plasma VWF (A) and soluble P-selectin (B) concentrations were /  +/+ found by ELISA in the Adamts13 compared with Adamts13 mice. (C) 1- μ m fl uorescent microspheres coupled to anti – P-selectin or control Ig were infused through the retroorbital venous plexus in the same mouse, and their binding to the unstimulated veins was analyzed. Representative photo- graphs are shown. Lines delineate the blood vessel. Almost no control Ig Figure 3. Platelet depletion decreases leukocyte rolling in beads bound to unstimulated veins, compared with several hundred anti – P- Adamts13 mice. The number of leukocytes rolling per minute was deter- selectin beads. Bars, 100 μ m. (D) Quantifi cation of the number of anti – P- mined by phase-contrast intravital microscopy. Platelet depletion resulted selectin beads binding per square millimeter. A signifi cantly higher number in decreased leukocyte rolling in Adamts13 mice compared with non- /  +/+ of anti – P-selectin beads was observed binding to unstimulated mesenteric depleted in Adamts13 mice but not in Adamts13 mice, indicating /  +/+ veins of Adamts13 than to Adamts13 mice, suggesting increased that platelets are part of the mechanism that results in increased leuko- spontaneous Weibel-Palade body release. Data represent the mean ± SEM. cyte rolling in Adamts13 veins. Data represent the mean ± SEM. 2068 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE (adherent for > 30 s) was increased approximately twofold in the the veins of Adamts13 mice when challenged with TNF- activated microvenules of Adamts 13 mice (mean ± SEM = (unpublished data). We asked whether increased leukocyte +/+ 21 ± 1) when compared with Adamts13 mice (mean ± rolling observed in the unstimulated veins of Adamts13 SEM = 12 ± 1; P < 0.01; Fig. 5 ; and Video 4, available mice would result in increased leukocyte adhesion under in- at http://www.jem.org/cgi/content/full/jem.20080130/DC1). fl ammatory conditions, and if so, whether it is VWF depen- Again, increased leukocyte adhesion was dependent on the dent. To answer these questions, mice were challenged with presence of VWF, because the number of leukocytes adhering TNF-  and mesenteric microvenules were visualized after /   / in microvenules of Adamts13 / Vwf mice was similar to 3.5 h by intravital microscopy. Microvenules were chosen be- +/+  / that in Adamts13 /Vwf mice ( Fig. 5 B ). cause with them we can precisely perform a quantitative anal- ysis of leukocyte adhesion per square micrometer for each Neutrophil infl ux is increased in thioglycollate-induced vessel. The shear rate and diameter of the microvenules +/+ +/+  / peritonitis in Adamts13 mice studied were similar for Adamts13 /Vwf , Adamts13 / +/+ +/+  /   /   / After fi nding that ADAMTS13 defi ciency increases leuko- Vwf , Adamts13 /Vwf , and Adamts13 /Vwf mice cyte rolling and adhesion, we asked whether ADAMTS13 ( Table II ). We found that the number of adherent leukocytes Figure 4. Leukocytes roll more slowly in histamine-stimulated Adamts13 veins. Histamine produced during infl ammation is a secretagogue of Weibel-Palade bodies. Histamine was injected i.p., and the stimulated veins were observed 15 min later by intravital microscopy. Endogenous platelets and leukocytes were labeled with Rhodamine 6G. Representative photographs are shown. Lines delineate the blood vessel. White arrows indicate platelet-VWF strings. Platelet-VWF strings anchoring to endothelium were observed only in Adamts13 mice. (A) Black arrows indicate leukocytes interacting with platelet-VWF strings in the Adamts13 mouse. (B) The cumulative histogram allows direct comparison of rolling velocities of the leukocytes. Leukocyte /  +/+  / rolling velocity was signifi cantly lower in Adamts13 compared with Adamts13 mice veins (P < 0.001). (C) Stimulated veins of Adamts13 mice infused with either control Ig (left) or anti – P-selectin Ig (right). Platelet-VWF strings anchored to endothelium do not support leukocyte rolling if P-selectin is inhibited. The single leukocyte seen in the photograph on the right is fi rmly adherent. Bars, 50 μ m. Videos 2 and 3 are available at http://www .jem.org/cgi/content/full/jem.20080130/DC1. JEM VOL. 205, September 1, 2008 2069 At baseline, peripheral neutrophil counts were not signifi cantly diff erent in Adamts13 mice (mean ± SD = 688 ± 208 × 3 +/+ 10 neutrophils/ml) compared with Adamts13 mice (mean ± SD = 636 ± 232 × 10 neutrophils/ml; P = 0.48; n = 18 – 19 mice of each group). Very few neutrophils were detected /  +/+ in the lavage of Adamts13 and Adamts13 mice that were infused with PBS alone and not challenged with thioglycollate (unpublished data). 4 h after challenge with thioglycollate, Adamts13 mice showed  60% more ex- travastion of neutrophils to infl amed peritoneum compared +/+ with Adamts13 mice (P < 0.03; Fig. 6 A ). The experiment was repeated a second time with the same number of mice in each group, and the results were similar (P < 0.03). The in- creased neutrophil extravasation observed in the peritoneum of Adamts13 mice was dependent on VWF, because neu- trophil counts in the infl amed peritoneum of Adamts13 / /  +/+  / Vwf were similar compared with Adamts13 / Vwf mice (P = 0.23; Fig. 6 B ). These results suggest that ADAMTS13 deficiency results in increased neutrophil extravasation during infl ammation, and this process is also dependent on VWF. Adamts13 mice exhibit increased neutrophil recruitment in excisional skin wounds Because more neutrophils extravasate into infl amed perito- neum in Adamts13 mice, we asked whether more neu- trophils would also be recruited during wound healing. Total counts of neutrophils in the 4-h wounded skin tissue were quantifi ed microscopically in hematoxylin and eosin (H & E) – Figure 5. Increased leukocyte adhesion in the TNF-  – stimulated stained sections. In the Adamts13 mice, signifi cantly more mesenteric venules of Adamts13 mice. Mice were treated with +/+ neutrophils were recruited compared with Adamts13 mice the infl ammatory cytokine TNF-  3.5 h before intravital microscopy. ( Fig. 7, A and B ). These results were con fi rmed in a second A single mesenteric venule (25 – 30 μ m diameter) was studied per mouse. (A) Representative images are shown. Arrows indicate leukocytes adhering to experiment by measuring the myeloperoxidase (MPO) activ- infl amed endothelium. (B) Quantifi cation of the adherent leukocytes. The ity in the excised wounded tissue. Increased MPO activity number of adherent leukocytes was markedly increased in the microve- correlates with an increase in the number of neutrophils ( 32 ). /  +/+ nules of Adamts13 compared with Adamts13 mice. In contrast, the We observed an increase in MPO activity in 4-h wounded tis- /   / number of leukocytes adhering in venules of Adamts13 /Vwf mice /  +/+ sue in Adamts13 compared with Adamts13 mice (P < +/+  / was similar to Adamts13 /Vwf mice, suggesting that VWF plays a 0.01; Fig. 7 C ). Thus, ADAMTS13 defi ciency also results role in increased leukocyte adhesion in Adamts13 vessels. Data repre- in increased extravasation of neutrophils in the early phase of sent the mean ± SEM. Bars, 30 μ m. Video 4 is available at http://www.jem wound healing. We were unable to examine neutrophil re- .org/cgi/content/full/jem.20080130/DC1. +/+  /   /   / cruitment in Adamts13 / Vwf and Adamts13 / Vwf mice because the mice bled excessively. However, on a diff er- defi ciency would also result in increased neutrophil extrava- ent genetic background it was shown that signifi cantly fewer sation under infl ammatory conditions. We used the well- neutrophils were present in tissue sections of 1-h wounds of /  +/+ established model of thioglycollate-induced peritonitis ( 31 ). Vwf compared with Vwf mice ( 33 ). Table II. Hemodynamic parameters of vessels observed by intravital microscopy Genotype Diameter Newtonian wall Interfacial 1  1 ( μ m) shear rate  (s ) shear rate  (1000 s ) w i +/+ +/+ Adamts13 / Vwf ( n = 10) 31.4 ± 4.5 276.1 ± 43.6 1.35 ± 0.45 /  +/+ 30.1 ± 4.3 282.6 ± 83.1 1.38 ± 0.57 Adamts13 / Vwf ( n = 12) /   / 34.7 ± 6 257.9 ± 53 1.26 ± 0.26 Adamts13 / Vwf ( n = 10) +/+  / 31.3 ± 5 265.1 ± 72.3 1.3 ± 0.56 Adamts13 / Vwf ( n = 9) Hemodynamic parameters were established before recording leukocyte adhesion for Fig. 4 . Values are represented as mean ± SD. P = NS. 2070 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 6. Peritoneal neutrophil infl ux in thioglycollate-induced peritonitis is elevated in Adamts13 mice. Total neutrophil count in lavage was counted after 4 h of thioglycollate administration. (A) A signifi cant increase in neutrophil extravasation was observed in Adamts13 compared with +/+  /   /  +/+  / Adamts13 mice. (B) No signifi cant increase in neutrophil infl ux in the peritoneum was observed in Adamts13 /Vwf compared with Adamts13 /Vwf mice, indicating that this effect is dependent on VWF. Data represent the mean ± SEM. DISCUSSION ing and adhesion was dependent on the presence of VWF. In the present study, we document a key role for ADAMTS13 These in vivo fi ndings could be explained in part by recently in down-regulating infl ammation by preventing excessive published in vitro studies by Bernardo et al., who showed leukocyte rolling in unstimulated veins, and leukocyte ad- that platelets bound to endothelial ULVWF could support hesion and extravasation under infl ammatory conditions. leukocyte tethering and rolling ( 25 ), and by Pendu et al., We have previously demonstrated in vivo that ADAMTS13 who showed that VWF acts as a ligand for the leukocyte re- cleaves platelet-VWF strings, regulates platelet interaction ceptors P-selectin glycoprotein ligand 1 and  2 integrin ( 24 ). with the “ activated ” vessel wall in the veins, prevents thrombi This binding involves multiple domains of VWF, including formation in activated microvenules, and modulates throm- D ' -D3 and A1-A2-A3 ( 24 ). botic response in injured arterioles ( 17, 19 ). Thus, these stud- We also found an increase in endothelial P-selectin ex- ies indicate that ADAMTS13 forms a new link between pression, soluble P-selectin, and VWF in the plasma of Ad- thrombosis and infl ammation. The increase in leukocyte roll- amts 13 mice. It is interesting to speculate how ADAMTS13 Figure 7. Neutrophil infl ux in skin excision wounds is increased in Adamts13 mice. The number of neutrophils present in skin tissue sur- rounding the wound 4 h after injury was determined microscopically in H & E-stained sections. (A) Representative images of the wound tissue in /  +/+ Adamts13 and Adamts13 mice are shown. A defi ciency of ADAMTS13 results in an increase in neutrophil extravasation. Bar, 20 μ m. (B) Visual count of the number of neutrophils that had emigrated from the blood vessels is shown. (C) Determination of MPO activity in the wounded tissue shows higher activity in Adamts13 mice samples. Data represent the mean ± SEM. JEM VOL. 205, September 1, 2008 2071 MATERIALS AND METHODS defi ciency results in increased plasma VWF. ADAMTS13 de- Animals. Preliminary experiments to investigate the role of ADAMTS13 on fi ciency could produce slower clearance of ULVWF from leukocyte rolling were done on mixed background mice (C57BL/6J/129 × 1/ the circulation and, thus, elevated VWF levels. Alternatively, SV) using littermates for comparison. Adamts13 mice were then backcrossed ULVWF multimers activate platelets, which in turn may ac- onto the C57BL/6J background for eight generations ( 26 ). The Adamts13 tivate the endothelium. Recently, our laboratory showed that  /   /   / ( 17 ), Vwf ( 5 ), and Adamts13 /Vwf ( 26 ) mice described in this study +/+ activated platelets, by binding to leukocytes, promote the re- are on the C57BL/6J background. The control Adamts13 (WT) mice on a C57BL/6J background were purchased from the Jackson Laboratory. The male lease of Weibel-Palade bodies and stimulate leukocyte rolling /  and female mice used for intravital microscopy were approximately 4 wk old ( 28 ). Interestingly, depletion of platelets in Adamts13 mice and weighed 13 – 15 g. Male and female mice used for experimental thioglycol- resulted in the normalization of leukocyte rolling as com- late-induced peritonitis and excision wounds were 9 – 11 wk old and weighed +/+ pared with Adamts13 mice. This indicates that platelets, 23 – 26 g. Animals were bred at the Immune Disease Institute, and the experi- likely activated by ULVWF either in circulation or directly mental procedures were approved by its Animal Care and Use Committee. on endothelium, stimulate Weibel-Palade bodies secretion. Elevated expression of endothelial P-selectin is also consistent Intravital microscopy. Mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g), and a midline incision was made through the abdominal wall with increased Weibel-Palade body exocytosis. to expose the mesentery and mesenteric veins (200 – 300 μ m diameter). Ve- Leukocytes roll and tether on the endothelium through nules (25 – 30 μ m diameter) were labeled as microvenules and were also exam- P-selectin and P-selectin glycoprotein ligand 1 interaction ined by intravital microscopy. The exposed mesentery vein was kept moist under low shear conditions ( 1 ). We observed that the pres- throughout the experiment by periodic superfusion of warmed (37 ° C) bicar- ence of platelet-ULVWF strings in the veins of Adamts13 bonate-buff ered saline (131.9 mM NaCl, 18 mM NaHCO , 4.7 mM KCl, mice treated with histamine decreased leukocyte velocity in 2 mM CaCl , and 1.2 mM MgCl ) equilibrated with 5% CO in N . The mes- 2 2 2 2 entery vein was transluminated with a 12-V, 100-W, direct current – stabilized the presence of P-selectin. Our in vivo fi ndings are in agree- source. Veins were visualized using an inverted microscope (Axiovert 135; ment with the in vitro study of Bernardo et al. reporting that Carl Zeiss, Inc.) with a 32 × objective connected to an SVHS video recorder the leukocyte rolling on ULVWF-platelet strings was signifi - (model AG-6730; Panasonic) using a charge-coupled device video camera cantly slower than leukocyte rolling on endothelial cells in (model C2400; Hamamatsu Photonics). Leukocyte interaction with the endo- vitro ( 25 ). Remarkably, this study showed that leukocytes thelium vessel wall was recorded in phase contrast for 10 min each in two to three veins per mouse. To study leukocyte rolling velocity, veins were stimu- can tether and roll on platelet-ULVWF strings under high lated with 200 μ l of 1-mM histamine per 15 g of mouse body weight. To shear stress. These results, together with our fi ndings, suggest study leukocyte adhesion under infl ammatory conditions, mice were infused that ULVWF multimers  released together with P-selectin i.p. with 500 ng TNF-  per 15 g of mouse body weight 3.5 h before intravital from Weibel-Palade bodies by many stimuli, including hyp- microscopy. A single mesenteric venule of  30 μ m in diameter was studied oxia ( 34 ), changes in shear stress ( 35 ), or infl ammatory cyto- per mouse. The wall shear rate for mesenteric veins (200 – 300 μ m diameter) kines ( 23 )  could accelerate infl ammatory responses in diseases and microvenules (25 – 30 μ m in diameter) was calculated based on Poiseuille ’ s law for a Newtonian fl uid:  = (8 V /D ). The interfacial shear rate (  ) for such as atherosclerosis by slowing down leukocytes and facili- w m v i microvenules was calculated as follows:  = 4.9(8 V /D ), where V is mean tating their extravasation. i m v m blood velocity, D is the diameter of the venule, and 4.9 is a median empirical Increased plasma VWF levels have been reported in dis- correction factor obtained from velocity profi les measured in microvessels in eases implicating infl ammation, such as rheumatoid arthritis vivo ( 42 ). It is about fi ve times greater than values described in the literature ( 36 ), viral and bacterial infections ( 37, 38 ), coronary artery for wall shear rate because of the slope of the velocity profi le of blood in small disease ( 39 ), and ischemic stroke ( 40 ). In addition, several venules and the presence of the endothelial surface layer ( 42 ). The centerline erythrocyte velocity (V ) was measured using an optical Doppler velocimeter studies suggest that infl ammation is accompanied by a de- rbc (Microcirculation Research Institute). V is estimated from the measured mean crease in ADAMTS13 activity ( 41 ). In the present study, we V by multiplying with an empirical factor of 0.625 ( 43 ). rbc provide evidence that ULVWF that is likely released and present under these circumstances further promotes infl ammation, Quantifi cation of leukocyte rolling, velocity, and adhesion. Re- as ADAMTS13 defi ciency results in increased extravasation corded images for leukocyte rolling were analyzed as follows. First, the num- of neutrophils in both thioglycollate-induced peritonitis and ber of leukocytes passing through a plane perpendicular to the vessel axis wound healing. Our results complement previous studies during a 1-min interval was counted. Leukocyte rolling per minute per vein for each mouse was determined by taking the average of fi ve 1-min counts, that reported decreased extravasation of neutrophils in VWF- as observed on the video screen during the entire 10-min recording. Second, defi cient mice, which was attributed to a lack of P-selectin the rolling velocity was determined by recording the time it took for a leuko- storage ( 33 ), and delayed formation of atherosclerotic lesions in cyte to transverse a certain distance in a vein  250 μ m long and 200 – 300 μ m /   /   / Vwf mice either on an apoE or LDLR background wide, as observed on the video screen. All of the analysis was done by an ( 10 ). The results from our study suggest that ADAMTS13 investigator blinded to genotype. Third, the leukocyte was considered to be down-regulates infl ammation by cleaving hyperactive ULVWF adherent if it remained stationary for > 30 s. The total number of leukocytes adhering per venule per mouse represents an average of adherent leukocytes multimers, and that defi ciency of ADAMTS13 not only can in three diff erent segments per microvenule. induce TTP but also accelerates infl ammatory diseases. Be- cause thrombosis and infl ammation constitute an integral part of P-selectin inhibition. P-selectin was inhibited in the Adamts13 mice the pathogenesis of many diseases, including the major killers by infusing i.v. a blocking rat monoclonal anti – P-selectin antibody (clone atherosclerosis and stroke, the results reported in this paper may RB40.34, containing no azide; BD Biosciences) at a concentration of 2 μ g provide new insights into the possible uses of ADAMTS13 as per gram of body weight. A similar concentration of purifi ed rat Ig (BD Bio- a therapeutic agent. sciences) was used as a control. 2072 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Platelet depletion. Platelets were depleted for 24 h by infusing i.v. anti- paraffi n blocks using standard protocols. 6- μ m tissue sections were stained GPIb antibody at a fi nal concentration of 2 μ g per gram of mouse body with H & E. Extra vascular neutrophils were counted in the entire wound area weight (emfret Analytics). Control rat Ig (emfret Analytics) was used at the using a light microscope (Axioplan; Carl Zeiss, Inc.) at 40 × magnifi cation. same concentration. To ensure that platelets remained depleted for 24 h, whole blood was withdrawn from the eye plexus into tubes containing 5 μ M MPO assay. The wounds were prepared as described in the previous para- EDTA after each surgery. More than 95% of the platelets remained depleted graph. A 6-mm punch of the skin containing the 4-mm wound area was washed after 24 h, as analyzed by FACS. in cold PBS and homogenized in 0.5 ml PBS at 4 ° C using a polytron homoge- nizer (fi ve bursts of 10 s each at maximum speed). 250 μ l of the homogenate Quantifi cation of P-selectin and VWF. Soluble P-selectin in the plasma was added to 250 μ l hexadecyltrimethylammonium bromide, vortexed, and in- was measured by an enzyme immunoassay kit (R & D Systems) according to cubated for 2 min. After centrifugation, the supernatant was collected and as- the manufacturer ’ s guidelines. Plasma VWF levels were measured by an en- sayed for MPO activity by adding 55 μ l TMB substrate to 30 μ l of the zyme immunoassay technique. Microtiter plates were coated overnight at supernatant. The absorbance was read at 630 nm at intervals of 30 s for 2 min. 4 ° C with rabbit anti – human VWF antibody (Dako) at a concentration of 15 μ g/ml diluted in 50 mM of sodium carbonate buff er. Plasma samples (diluted Statistical analysis. Results are reported as the mean ± SEM, unless other- 1:20 in PBS) were incubated for 2 h in the coated wells at room tempera- wise noted. The statistical signifi cance of the diff erence between means was ture. After six washes, the polyclonal anti – human VWF coupled to HRP assessed by using the unpaired Student ’ s t test (for the comparison of two (1:2,000) was added for 2 h. After washing, 3,3  ,5,5  -tetramethylbenzidine groups) or by analysis of variance followed by Boneferroni ’ s multiple com- (TMB) substrate solution (Sigma-Aldrich) was added to the wells, and the parison test. P < 0.05 was considered signifi cant. colorimetric reaction was stopped with H SO after 20 min. Results were 2 4 read in an ELISA microplate reader (model MRXII; Dynex Technologies) Online supplemental material. Video 1 shows P-selectin – dependent leu- at A nm. Normal pooled plasma obtained from 10 WT mice was defi ned kocyte rolling in an Adamts13 mouse. Video 2 shows the interaction and as 1 U VWF antigen per milliliter of plasma. rolling of some leukocytes on ULVWF-platelet strings in a histamine-stimu- lated vein of an Adamts13 mouse. ULVWF-platelet strings are absent in +/+ the Adamts13 mouse. Video 3 shows that ULVWF-platelet strings cannot In vivo detection of endothelial P-selectin. Yellow-green (excitation/ support leukocyte rolling in the absence of P-selectin. Video 4 shows in- emission = 505 nm/515 nm) and red (excitation/emission = 580 nm/605 nm) creased leukocyte adhesion in TNF-  – activated microvenules of Adamts13 carboxylate-modifi ed microspheres (1 μ m diameter; Invitrogen) were +/+ compared with Adamts13 mice. Endogenous leukocytes and platelets were covalently coupled to anti – P-selectin monoclonal antibody RB40.34 or labeled with Rhodamine 6G in Videos 1 – 3. Online supplemental material is control rat IgG (BD Bioscience), according to the manufacturer ’ s instruc- available at http://www.jem.org/cgi/content/full/jem.20080130/DC1. tions (Invitrogen). Mice were infused with 10 microspheres of each color, and mesenteric venules were observed immediately by fl uorescent intra- We thank Lesley Cowan for help in preparing the manuscript. vital microscopy. The order of infusion of yellow and red microspheres was This work was supported by a Sponsored Research Agreement from Baxter reversed between experiments. Bioscience (to A.K. Chauhan and D.D. Wagner), and National Heart, Lung, and Blood Institute grants R37 HL041002 and PO1 HL066105 (to D.D. Wagner). Flow cytometry. For assessing the amount of neutrophils in peripheral F. Scheifl inger is an employee of Baxter Bioscience. The authors have no other blood, 1 ml of RBC lysis buff er was added to 100 μ l of whole blood contain- confl icting fi nancial interests. ing 5 μ M EDTA and incubated for 10 min on ice. The samples were then centrifuged at 400 g , and the pellet was resuspended in 35 μ l of ice-cold PBS. Submitted: 18 January 2008 20 μ l of cell suspension was mixed with an antibody against Gr-1 labeled Accepted: 3 July 2008 with Alexa Fluor 647. After a 5-min incubation at room temperature in the dark, 700 μ l PBS was added to 5 μ l of beads (Spherotech) with a known REFERENCES concentration (12 × 10 beads per μ l). Samples were read by FACS for 1 min 1 . Ley , K. , C. Laudanna , M.I. Cybulsky , and S. Nourshargh . 2007 . Getting at medium pump velocity. The amount of neutrophils (Gr-1 – positive events to the site of infl ammation: the leukocyte adhesion cascade updated. falling in the neutrophil forward and side scatter gate) was calculated based Nat. Rev. Immunol. 7 : 678 – 689 . on the number of beads detected during the same time period. 2 . Wagner , D.D. 2005 . New links between infl ammation and thrombosis. Arterioscler. Thromb. Vasc. Biol. 25 : 1321 – 1324 . Thioglycollate-induced peritonitis. Experimental peritonitis was induced 3 . Denis , C.V. , and D.D. Wagner . 2007 . Platelet adhesion receptors and by injecting 1 ml of 3% thioglycollate (Sigma-Aldrich) i.p. After 4 h, mice their ligands in mouse models of thrombosis. Arterioscler. Thromb. Vasc. were killed by overdosing with isofl urane and 8 ml PBS containing 0.1% BSA, Biol. 27 : 728 – 739 . 4 . Sadler , J.E. 2005 . New concepts in von Willebrand disease. Annu. Rev. and 0.5 mM/liter EDTA was used to lavage the peritoneum. Approximately Med. 56 : 173 – 191 . 6 ml of lavage was collected from each mouse, and 1 ml was used for analysis. 5 . Denis , C. , N. Methia , P.S. Frenette , H. Rayburn , M. Ullman-Cullere , The 1 ml of lavage was spun at 2,600 rpm for 5 min, and the pellet was resus- R.O. Hynes , and D.D. Wagner . 1998 . A mouse model of severe von pended in 40 μ l PBS. For accurate quantifi cation, a 20- μ l sample was taken Willebrand disease: defects in hemostasis and thrombosis. Proc. Natl. from 40 μ l, stained with GR-1 – Alexa Fluor 687 specifi c for neutrophils (BD Acad. Sci. USA . 95 : 9524 – 9529 . Bioscience), and analyzed by FACS as described in the previous paragraph. 6 . Chauhan , A.K. , J. Kisucka , C.B. Lamb , W. Bergmeier , and D.D. Wagner . 2007 . von Willebrand factor and factor VIII are indepen- Wounding and tissue preparation. Wounds on the back of mice were dently required to form stable occlusive thrombi in injured veins. Blood . performed as previously described ( 44 ). In brief, mice were anesthetized with 109 : 2424 – 2429 . 2.5% tribromoethanol (0.15 ml per 10 g of body weight), and hair was re- 7 . Vischer , U.M. 2006 . von Willebrand factor, endothelial dysfunction, moved with an electric razor. The skin was swabbed with 70% ethanol (etha- and cardiovascular disease. J. Thromb. Haemost. 4 : 1186 – 1193 . nol wipes) and two 4-mm full-thickness excision wounds were made by 8 . Ruggeri , Z.M. , J.N. Orje , R. Habermann , A.B. Federici , and A.J. picking up a fold of skin, placing it over dental wax, and punching through Reininger . 2006 . Activation-independent platelet adhesion and aggre- the two layers of skin. Mice were housed in individual cages. After 4 h, the gation under elevated shear stress. Blood . 108 : 1903 – 1910 . mice were killed, and wounds were harvested with 1 – 2 mm of normal skin 9 . Groot , E. , P.G. de Groot , R. Fijnheer , and P.J. Lenting . 2007 . The tissue around them. The wounds were cut in half, fi xed overnight in 4% para- presence of active von Willebrand factor under various pathological formaldehyde, processed through graded ethanol solutions, and embedded in conditions. Curr. Opin. Hematol. 14 : 284 – 289 . JEM VOL. 205, September 1, 2008 2073 10 . Methia , N. , P. Andre , C.V. Denis , M. Economopoulos , and D.D. 27 . Kunkel , E.J. , and K. Ley . 1996 . Distinct phenotype of E-selectin-de- Wagner . 2001 . Localized reduction of atherosclerosis in von Willebrand fi cient mice. E-selectin is required for slow leukocyte rolling in vivo. factor-defi cient mice. Blood . 98 : 1424 – 1428 . Circ. Res. 79 : 1196 – 1204 . 11 . Sporn , L.A. , V.J. Marder , and D.D. Wagner . 1986 . Inducible secre- 28 . Dole , V.S. , W. Bergmeier , H.A. Mitchell , S.C. Eichenberger , and tion of large, biologically potent von Willebrand factor multimers. Cell . D.D. Wagner . 2005 . Activated platelets induce Weibel-Palade-body 46 : 185 – 190 . secretion and leukocyte rolling in vivo: role of P-selectin. Blood . 106 : 12 . Wagner , D.D. 1990 . Cell biology of von Willebrand factor. Annu. Rev. 2334 – 2339 . Cell Biol. 6 : 217 – 246 . 29 . Nieswandt , B. , W. Bergmeier , K. Rackebrandt , J.E. Gessner , and H. 13 . Sporn , L.A. , V.J. Marder , and D.D. Wagner . 1987 . von Willebrand fac- Zirngibl . 2000 . Identifi cation of critical antigen-specifi c mechanisms in tor released from Weibel-Palade bodies binds more avidly to extracel- the development of immune thrombocytopenic purpura in mice. Blood . lular matrix than that secreted constitutively. Blood . 69 : 1531 – 1534 . 96 : 2520 – 2527 . 14 . Arya , M. , B. Anvari , G.M. Romo , M.A. Cruz , J.F. Dong , L.V. 30 . Chauhan , A.K. , T. Goerge , S.W. Schneider , and D.D. Wagner . 2007 . McIntire , J.L. Moake , and J.A. Lopez . 2002 . Ultralarge multimers of Formation of platelet strings and microthrombi in the presence of von Willebrand factor form spontaneous high-strength bonds with the ADAMTS-13 inhibitor does not require P-selectin or beta3 integrin. platelet glycoprotein Ib-IX complex: studies using optical tweezers. J. Thromb. Haemost. 5 : 583 – 589 . Blood . 99 : 3971 – 3977 . 31 . Mayadas , T.N. , R.C. Johnson , H. Rayburn , R.O. Hynes , and D.D. 15 . Dong , J.F. , J.L. Moake , L. Nolasco , A. Bernardo , W. Arceneaux , C.N. Wagner . 1993 . Leukocyte rolling and extravasation are severely com- Shrimpton , A.J. Schade , L.V. McIntire , K. Fujikawa , and J.A. Lopez . promised in P selectin-defi cient mice. Cell . 74 : 541 – 554 . 2002 . ADAMTS-13 rapidly cleaves newly secreted ultralarge von 32 . Bradley , P.P. , D.A. Priebat , R.D. Christensen , and G. Rothstein . 1982 . Willebrand factor multimers on the endothelial surface under fl owing Measurement of cutaneous infl ammation: estimation of neutrophil con- conditions. Blood . 100 : 4033 – 4039 . tent with an enzyme marker. J. Invest. Dermatol. 78 : 206 – 209 . 16 . Moake , J.L. , C.K. Rudy , J.H. Troll , M.J. Weinstein , N.M. Colannino , 33 . Denis , C.V. , P. Andre , S. Saff aripour , and D.D. Wagner . 2001 . Defect J. Azocar , R.H. Seder , S.L. Hong , and D. Deykin . 1982 . Unusually in regulated secretion of P-selectin aff ects leukocyte recruitment in large plasma factor VIII:von Willebrand factor multimers in chronic von Willebrand factor-defi cient mice. Proc. Natl. Acad. Sci. USA . 98 : relapsing thrombotic thrombocytopenic purpura. N. Engl. J. Med. 4072 – 4077 . 307 : 1432 – 1435 . 34 . Pinsky , D.J. , Y. Naka , H. Liao , M.C. Oz , D.D. Wagner , T.N. Mayadas , 17 . Motto , D.G. , A.K. Chauhan , G. Zhu , J. Homeister , C.B. Lamb , R.C. Johnson , R.O. Hynes , M. Heath , C.A. Lawson , and D.M. Stern . K.C. Desch , W. Zhang , H.M. Tsai , D.D. Wagner , and D. Ginsburg . 1996 . Hypoxia-induced exocytosis of endothelial cell Weibel-Palade 2005 . Shigatoxin triggers thrombotic thrombocytopenic purpura in bodies. A mechanism for rapid neutrophil recruitment after cardiac pres- genetically susceptible ADAMTS13-defi cient mice. J. Clin. Invest. ervation. J. Clin. Invest. 97 : 493 – 500 . 115 : 2752 – 2761 . 35 . Galbusera , M. , C. Zoja , R. Donadelli , S. Paris , M. Morigi , A. Benigni , 18 . Banno , F. , K. Kokame , T. Okuda , S. Honda , S. Miyata , H. Kato , Y. M. Figliuzzi , G. Remuzzi , and A. Remuzzi . 1997 . Fluid shear stress Tomiyama , and T. Miyata . 2006 . Complete defi ciency in ADAMTS13 modulates von Willebrand factor release from human vascular endothe- is prothrombotic, but it alone is not suffi cient to cause thrombotic lium. Blood . 90 : 1558 – 1564 . thrombocytopenic purpura. Blood . 107 : 3161 – 3166 . 36 . McEntegart , A. , H.A. Capell , D. Creran , A. Rumley , M. Woodward , 19 . Chauhan , A.K. , D.G. Motto , C.B. Lamb , W. Bergmeier , M. Dockal , and G.D. Lowe . 2001 . Cardiovascular risk factors, including throm- B. Plaimauer , F. Scheifl inger , D. Ginsburg , and D.D. Wagner . botic variables, in a population with rheumatoid arthritis. Rheumatology 2006 . Systemic antithrombotic eff ects of ADAMTS13. J. Exp. Med. (Oxford) . 40 : 640 – 644 . 203 : 767 – 776 . 37 . Tzavara , V. , P.G. Vlachoyiannopoulos , T. Kordossis , D. Galaris , A. Travlou , 20 . Ono , T. , J. Mimuro , S. Madoiwa , K. Soejima , Y. Kashiwakura , A. U. Dafni , and H.M. Moutsopoulos . 1997 . Evidence for non-adaptive im- Ishiwata , K. Takano , T. Ohmori , and Y. Sakata . 2006 . Severe secondary mune response in HIV infection. Eur. J. Clin. Invest. 27 : 846 – 849 . defi ciency of von Willebrand factor-cleaving protease (ADAMTS13) in 38 . Kayal , S. , J.P. Jais , N. Aguini , J. Chaudiere , and J. Labrousse . 1998 . patients with sepsis-induced disseminated intravascular coagulation: its Elevated circulating E-selectin, intercellular adhesion molecule 1, and correlation with development of renal failure. Blood . 107 : 528 – 534 . von Willebrand factor in patients with severe infection. Am. J. Respir. 21 . Nguyen , T.C. , A. Liu , L. Liu , C. Ball , H. Choi , W.S. May , K. Crit. Care Med. 157 : 776 – 784 . Aboulfatova , A.L. Bergeron , and J.F. Dong . 2007 . Acquired ADAMTS- 39 . Jager , A. , V.W. van Hinsbergh , P.J. Kostense , J.J. Emeis , J.S. Yudkin , G. 13 defi ciency in pediatric patients with severe sepsis. Haematologica . Nijpels , J.M. Dekker , R.J. Heine , L.M. Bouter , and C.D. Stehouwer . 92 : 121 – 124 . 1999 . von Willebrand factor, C-reactive protein, and 5-year mortal- 22 . Reiter , R.A. , K. Varadi , P.L. Turecek , B. Jilma , and P. Knobl . 2005 . ity in diabetic and nondiabetic subjects: the Hoorn Study. Arterioscler. Changes in ADAMTS13 (von-Willebrand-factor-cleaving protease) ac- Thromb. Vasc. Biol. 19 : 3071 – 3078 . tivity after induced release of von Willebrand factor during acute sys- 40 . Bongers , T.N. , M.P. de Maat , M.L. van Goor , V. Bhagwanbali , H.H. temic infl ammation. Thromb. Haemost. 93 : 554 – 558 . van Vliet , E.B. Gomez Garcia , D.W. Dippel , and F.W. Leebeek . 2006 . 23 . Bernardo , A. , C. Ball , L. Nolasco , J.F. Moake , and J.F. Dong . 2004 . High von Willebrand factor levels increase the risk of fi rst ischemic Eff ects of infl ammatory cytokines on the release and cleavage of the en- stroke: infl uence of ADAMTS13, infl ammation, and genetic variability. dothelial cell-derived ultralarge von Willebrand factor multimers under Stroke . 37 : 2672 – 2677 . fl ow. Blood . 104 : 100 – 106 . 41 . Mannucci , P.M. , M.T. Canciani , I. Forza , F. Lussana , A. Lattuada , and 24 . Pendu , R. , V. Terraube , O.D. Christophe , C.G. Gahmberg , P.G. de E. Rossi . 2001 . Changes in health and disease of the metalloprotease Groot , P.J. Lenting , and C.V. Denis . 2006 . P-selectin glycoprotein li- that cleaves von Willebrand factor. Blood . 98 : 2730 – 2735 . gand 1 and beta2-integrins cooperate in the adhesion of leukocytes to 42 . Long , D.S. , M.L. Smith , A.R. Pries , K. Ley , and E.R. Damiano . 2004 . von Willebrand factor. Blood . 108 : 3746 – 3752 . Microviscometry reveals reduced blood viscosity and altered shear rate 25 . Bernardo , A. , C. Ball , L. Nolasco , H. Choi , J.L. Moake , and J.F. and shear stress profi les in microvessels after hemodilution. Proc. Natl. Dong . 2005 . Platelets adhered to endothelial cell-bound ultra-large von Acad. Sci. USA . 101 : 10060 – 10065 . Willebrand factor strings support leukocyte tethering and rolling under 43 . Lipowsky , H.H. , and B.W. Zweifach . 1978 . Application of the “ two- high shear stress. J. Thromb. Haemost. 3 : 562 – 570 . slit ” photometric technique to the measurement of microvascular volu- 26 . Chauhan , A.K. , M.T. Walsh , G. Zhu , D. Ginsburg , D.D. Wagner , metric fl ow rates. Microvasc. Res. 15 : 93 – 101 . and D.G. Motto . 2008 . The combined roles of ADAMTS13 and 44 . Subramaniam , M. , S. Saff aripour , L. Van De Water , P.S. Frenette , T.N. VWF in murine models of TTP, endotoxemia, and thrombosis. Blood . Mayadas , R.O. Hynes , and D.D. Wagner . 1997 . Role of endothelial 111 : 3452 – 3457 . selectins in wound repair. Am. J. Pathol. 150 : 1701 – 1709 . 2074 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

ADAMTS13: a new link between thrombosis and inflammation

Chauhan, Anil K.; Kisucka, Janka; Brill, Alexander; Walsh, Meghan T.; Scheiflinger, Friedrich; Wagner, Denisa D.
The Journal of Experimental Medicine , Volume 205 (9) – Sep 1, 2008
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10.1084/jem.20080130
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Abstract

ARTICLE ADAMTS13: a new link between thrombosis and infl ammation 1,2 1,2 1,2 1 Anil K. Chauhan , Janka Kisucka , Alexander Brill , Meghan T. Walsh , 3 1,2 Friedrich Scheifl inger , and Denisa D. Wagner 1 2 Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115 Baxter Bioscience, 1220 Vienna, Austria von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprote- ase with thrombospondin type I repeats – 13 (ADAMTS13) activity is decreased in both acute and chronic infl ammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and infl ammation. Using intravital microscopy, we show that ADAMTS13 defi ciency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in infl amed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13 mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13 compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13 but not in WT mice. Furthermore, in infl ammatory models, ADAMTS13 defi ciency re- sulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our fi ndings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during infl ammation. Leukocyte rolling, adhesion, and transmigration defi ciency in humans ( 4 ) and defective occlu- CORRESPONDENCE Denisa D. Wagner: at the site of infection or injury are hallmarks of sive thrombus formation in the arteries and [email protected] infl ammation. These steps in the leukocyte ad- veins of Vwf mice ( 5, 6 ). VWF levels are el- hesion cascade are controlled by selectins, integ- evated in both chronic and acute infl ammation Abbreviations used: AD- AMTS13, a disintegrin-like and rins, and other adhesion molecules ( 1 ). It is now ( 7 ). Normally, the glycoprotein GPIb  platelet- metalloprotease with thrombo- evident that several adhesion molecules involved binding domain is not exposed on circulating spondin type I repeats – 13; H & E, in infl ammation are also important in thrombo- VWF, but it becomes exposed under patholog- hematoxylin and eosin; MPO, sis. For example, P-selectin expressed on acti- ical high shear ( 8 ) or upon binding to exposed myeloperoxidase; TTP, throm- botic thrombocytopenia pur- vated platelets and endothelium contributes to subendothelium. Circulating active VWF has also pura; ULVWF, ultralarge VWF; both thrombosis and infl ammation ( 1 – 3 ). P-se- been found in von Willebrand disease type 2B, VWF, von Willebrand factor. lectin and von Willebrand factor (VWF) are malaria, antiphospholipid syndrome, and throm- present in the platelet  -granules and Weibel- botic thrombocytopenia purpura (TTP) ( 9 ). In Palade bodies of endothelial cells. P-selectin me- mice, VWF defi ciency delays the formation diates leukocyte and platelet rolling, and VWF of fatty streaks in two models of atherosclero- mediates initial adhesion of platelets, the fi rst step sis ( 10 ). These studies suggest that VWF could in infl ammation and thrombosis. Thus, Weibel- play an active role in infl ammation in addition Palade bodies constitute a prominent link be- to hemostasis. tween thrombosis and infl ammation. The role of The VWF contained in Weibel-Palade bod- VWF in infl ammation needs to be explored. ies is extremely large ( 11 ) and is designated as The essential role of VWF in hemostasis is illustrated by von Willebrand disease, a bleed- © 2008 Chauhan et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months ing disorder associated with functional VWF after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncom- mercial–Share Alike 3.0 Unported license, as described at http://creativecommons The online version of this article contains supplemental material. .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 205 No. 9 2065-2074 2065 www.jem.org/cgi/doi/10.1084/jem.20080130 The Journal of Experimental Medicine ultralarge VWF (ULVWF;  20,000 kD). Upon cellular acti- or sepsis ( 20 – 22 ). Proinfl ammatory cytokines, such as TNF-  , vation or stimulation with secretagogues (such as histamine, IL-8, and IL-6, released during infl ammation have been shown calcium ionophore, thrombin, and TNF-  ), ULVWF is re- to have distinct eff ects on the endothelial release of ULVWF leased from the storage granules ( 12 ). These ULVWF multi- and its processing ( 23 ). Recently, it was shown in vitro that mers are considered biologically hyperactive because they bind VWF promotes leukocyte adhesion ( 24 ) and that platelets bound better to the extracellular matrix ( 13 ) and form higher strength to ULVWF can support leukocyte tethering and rolling under bonds with platelet GPIb compared with small plasma multi- high shear stress ( 25 ). We hypothesize that ADAMTS13, mers ( 14 ). A disintegrin-like and metalloprotease with throm- by cleaving hyperactive ULVWF, down-regulates not only bospondin type I repeats – 13 (ADAMTS13) inhibits platelet thrombosis, but also infl ammation. To investigate the role of adhesion by quickly cleaving hyperactive ULVWF multimers ADAMTS13 and its substrate VWF in infl ammation, we stud- +/+ +/+ released from the Weibel-Palade bodies under conditions of ied leukocyte rolling and adhesion in Adamts13 /Vwf , /  +/+ +/+  /   / fl uid shear stress ( 15 ). In 1982, Moake et al. found ULVWF Adamts13 /Vwf , Adamts13 /Vwf , and Adamts13 / multimers in the plasma of patients suff ering from chronic Vwf mice using intravital microscopy. We also examined the relapsing TTP ( 16 ), a disorder clinically characterized by micro- role of the ADAMTS13 – VWF axis in neutrophil extravasation angiopathic hemolytic anemia, thrombocytopenia, neurological in two diff erent models of infl ammation (thioglycollate-induced abnormalities, fever, and renal dysfunction. The pathogen- peritonitis and wound healing). esis of TTP has been linked to a defi ciency of ADAMTS13, which can be either familial (a mutation in the ADAMTS13 R ESULTS gene) or acquired (through inhibitory antibodies generated D efi ciency of ADAMTS13 results in increased numbers against ADAMTS13). Studies in mice show that ADAMTS13 of leukocytes rolling per minute in unstimulated veins defi ciency is necessary, but not suffi cient, for the develop- To determine whether ADAMTS13 plays a role in infl am- ment of TTP, suggesting the existence of additional genetic mation, we visualized leukocyte rolling, as a measure of and/or environmental contributing factors ( 17, 18 ). Re- endothelial activation, on unstimulated mesenteric veins. We cently, we and others have found that a complete defi ciency found higher numbers of leukocytes rolling per minute on of ADAMTS13 in mice results in a prothrombotic pheno- the unstimulated endothelium in the Adamts13 mice on a type ( 18, 19 ). mixed background (C57BL/6/129 × 1/SV; mean ± SEM = +/+ Although it is not known whether ADAMTS13 plays a role 115 ± 16) compared with Adamts13 littermates (mean ± in infl ammation, recent studies have found reduced AD- SEM = 43 ± 10; P < 0.001; n = 10 – 12 of each group). To AMTS13 activity in patients with acute systemic infl ammation ensure that the observed phenotype was not caused by the Figure 1. ADAMTS13 defi ciency in mice results in increased leukocyte rolling per minute in unstimulated mesenteric veins . (A) Leukocyte rolling on the vessel wall was recorded in two to three unstimulated veins (200 – 300 μ m diameter) per mouse. There was an  2.5-fold increase in /  +/+ leukocyte rolling per minute on the endothelium in Adamts13 compared with Adamts13 mice, suggesting endothelial activation. The absence of ADAMTS13 activity on a VWF-defi cient background did not increase leukocyte rolling, indicating that the increased leukocyte rolling observed in Adamts13 mice is dependent on the presence of VWF. Data represent the mean ± SEM. (B) P-selectin – dependent rolling in Adamts13 mice. The unstimulated veins of Adamts13 mice were infused with either control Ig or anti – P-selectin Ig ( n = 6 veins from three mice of each group). Endogenous leukocytes and platelets were labeled with Rhodamine 6G. Representative photographs are shown. Lines delineate the blood vessel. The white arrow indicates a labeled leukocyte. Bars, 50 μ m. Video 1 is available at http://www.jem.org/cgi/content/full/jem.20080130/DC1. 2066 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE veins. 1- μ m fl uorescent microspheres coupled to anti – P- mixed background, we next evaluated Adamts13 mice selectin antibody were infused through the retroorbital ve- backcrossed onto a C57BL/6J background for eight genera- nous plexus, and their binding to unstimulated mesenteric tions. We found a similar increase in leukocyte rolling per veins was visualized and quantifi ed. A signifi cantly higher minute in these Adamts13 mice (mean ± SEM = 98 ± 13) +/+ number of microspheres bound to mesenteric veins was ob- compared with Adamts13 mice (mean ± SEM = 35 ± 6; P < /  +/+ served in Adamts 13 mice compared with Adamts13 0.001; n = 10 mice of each group), indicating that the endo- mice ( Fig. 2, C and D ). It is possible that fl uorescent micro- thelium in Adamts13 mice was preactivated ( Fig. 1 A ). spheres coupled to anti – P-selectin antibody were bound We next asked whether platelets on VWF strings were pre- to both endothelial cell and platelet P-selectin. However, sent in the untreated veins and could be responsible for the when we labeled the endogenous platelets and leukocytes observed increase in leukocyte rolling in the Adamts13 with Rhodamine 6G and visualized the unstimulated veins mice. We labeled the endogenous platelets and leukocytes by intravital microscopy, only transient platelet adhesion with Rhodamine 6G and visualized the veins by intravital was observed (Video 1). We did not observe a carpet of microscopy. Fig. 1 B (top) and Video 1 (available at http:// platelets in the unstimulated veins of Adamts13 mice www.jem.org/cgi/content/full/jem.20080130/DC1) show ( Fig. 1 B , top; and Video 1). These observations suggest that the absence of platelet-VWF strings on the endothelium the microspheres coupled to anti – P-selectin antibody were of the unstimulated Adamts13 veins, suggesting that it is most likely bound to endothelial P-selectin. Collectively, most likely an up-regulation of an endothelial selectin that these results suggest that the increased leukocyte rolling ob- is responsible for the increased leukocyte rolling. Indeed, the served in Adamts13 mice is likely caused by the release of observed leukocyte rolling in Adamts13 mice was depen- more Weibel-Palade bodies. dent on P-selectin, because infusion of a blocking antibody to P-selectin completely abolished leukocyte rolling ( Fig. Platelet depletion decreases leukocyte rolling 1 B , bottom; and Video 1). Because the only known substrate in unstimulated veins of Adamts13 mice for ADAMTS13 in thrombosis is VWF ( 26 ), we studied leu- We next examined whether platelets, by binding to ULVWF kocyte rolling in Adamts13 mice on a VWF-defi cient multimers and/or leukocytes, could promote the increase in background to evaluate whether the increase in leukocyte leukocyte rolling. We have previously shown that activated rolling per minute was also VWF dependent. The shear platelets stimulate Weibel-Palade body secretion ( 28 ). We rate and diameter of the evaluated veins were similar for +/+ +/+  /  +/+ +/+ depleted platelets by infusing anti-GPIb Ig, which depletes Adamts13 / Vwf , Adamts13 / Vwf , Adamts13 / /   /   / > 95% of platelets for up to 48 h ( 29 ). Anti-GPIb or control Vwf , and Adamts13 / Vwf mice ( Table I ). We found +/+  / Ig was infused i.v. in the Adamts13 and Adamts13 mice, that the absence of ADAMTS13 activity on a VWF-defi cient and 24 h later leukocyte rolling was visualized. In platelet- background did not result in increased leukocyte rolling +/+ depleted Adamts13 mice ( < 5% normal platelet count), ( Fig. 1 A ). These results suggest that the elevated baseline leukocyte rolling (mean ± SEM = 62 ± 10) was similar to leukocyte rolling observed in the veins of Adamts13 nondepleted mice (mean ± SEM = 57 ± 9; P = 0.75; Fig. 3 ). mice was caused by the combined eff ect of P-selectin and However, in Adamts13 mice, platelet depletion resulted in a VWF, most likely the unprocessed hyperactive ULVWF in twofold decrease in leukocyte rolling (mean ± SEM = 62 ± 8) the circulation. compared with nondepleted mice (mean ± SEM = 143 ± 12; ADAMTS13 defi ciency results in increased Weibel-Palade P < 0.001; Fig. 3 ). These results suggest that platelets con- tribute to the mechanism of increased leukocyte rolling in body release ADAMTS13 defi ciency in mice does not aff ect baseline Adamts13 mice. leukocyte counts in peripheral blood ( 17 ). We measured leukocyte rolling velocity at baseline and did not observe Table I. Hemodynamic parameters of vessels observed any diff erences in the leukocyte velocity in the unstimulated by intravital microscopy /  +/+ veins in the Adamts13 compared with Adamts13 mice Genotype Diameter Newtonian wall (unpublished data), suggesting that P-selectin is likely the ( μ m) shear rate  (s ) up-regulated molecule, as the rolling velocity depends pre- +/+ +/+ dominantly on E-selectin ( 27 ). In addition, increased solu- Adamts13 / Vwf 223.5 ± 21.1 192.4 ± 64.1 ble P-selectin and VWF concentrations were found in the ( n = 18) /  +/+ /  +/+ plasma of Adamts 13 compared with Adamts13 mice Adamts13 / Vwf 234.2 ± 24.2 183.8 ± 59.6 by ELISA, further indicating that the endothelium is acti- ( n = 19) /   / vated in Adamts13 mice ( Fig. 2, A and B ). Because most Adamts13 / Vwf 237.5 ± 36.8 160.6 ± 46 ( n = 18) circulating VWF is derived from the endothelium, these +/+  / 225 ± 25 152.5 ± 39.8 observations also suggest that more Weibel-Palade bodies Adamts13 / Vwf ( n = 21) are released in Adamts13 mice. To confi rm this, we measured endothelial P-selectin expression, as a marker for Hemodynamic parameters were established before recording leukocyte rolling for Fig. 1 . Values are represented as mean ± SD. P = NS. Weibel-Palade body secretion, on unstimulated mesenteric JEM VOL. 205, September 1, 2008 2067 Platelet-VWF strings anchored onto histamine-stimulated tagogue of Weibel-Palade bodies, to release ULVWF multi- endothelium decrease leukocyte rolling velocity mers. We found that leukocyte rolling velocity was slower in /  +/+ Previously, it was shown in vitro that platelets bound to endo- Adamts 13 compared with Adamts13 veins where plate- thelial ULVWF can support leukocyte tethering and rolling let strings do not form ( Fig. 4 ). In Adamts13 veins,  50% ( 25 ). Therefore, we asked whether the presence of VWF- of leukocytes rolled at a velocity of < 30 μ m/s compared with +/+ platelet strings on stimulated endothelium aff ects leukocyte  5% in Adamts13 (P < 0.001; Fig. 4 B ). Moreover, fre- rolling in vivo. Veins were stimulated with histamine, a secre- quent leukocyte interaction with the ULVWF-platelet strings was observed in the mesenteric activated veins ( Fig. 4 A ; and Video 2, available at http://www.jem.org/cgi/content/full/ jem.20080130/DC1), which suggests that the decrease in leukocyte rolling velocity is likely caused by the presence of ULVWF-platelet strings on the endothelium. In contrast to veins, we did not see endothelial VWF-platelet strings in the arteri- oles treated identically with histamine (unpublished data). Next, we assessed whether the ULVWF-platelet strings could support leukocyte rolling in the absence of P-selectin. Infusion of blocking antibody to P-selectin in Adamts13 mice completely abolished leukocyte rolling but had no ef- fect on the presence of ULVWF-platelet strings ( Fig. 4 C ; and Video 3, available at http://www.jem.org/cgi/content/ full/jem.20080130/DC1). These results are in agreement with previous studies where we have observed the formation of platelet-VWF strings in P-selectin mice in the presence of ADAMTS13 inhibitor ( 30 ). Thus, ULVWF multimers only support leukocyte adhesion that was initiated by leuko- cyte binding to P-selectin. ADAMTS13 defi ciency increases leukocyte adhesion in infl amed venules The infl ammatory cytokines TNF-  and IL-8 have been shown to release ULVWF from human umbilical vein endothelial cells in vitro ( 23 ). Similarly, we have observed platelet-VWF strings in Figure 2. Adamts13 mice show an increase in the expression of endothelial P-selectin, and higher soluble P-selectin and plasma VWF. Increased plasma VWF (A) and soluble P-selectin (B) concentrations were /  +/+ found by ELISA in the Adamts13 compared with Adamts13 mice. (C) 1- μ m fl uorescent microspheres coupled to anti – P-selectin or control Ig were infused through the retroorbital venous plexus in the same mouse, and their binding to the unstimulated veins was analyzed. Representative photo- graphs are shown. Lines delineate the blood vessel. Almost no control Ig Figure 3. Platelet depletion decreases leukocyte rolling in beads bound to unstimulated veins, compared with several hundred anti – P- Adamts13 mice. The number of leukocytes rolling per minute was deter- selectin beads. Bars, 100 μ m. (D) Quantifi cation of the number of anti – P- mined by phase-contrast intravital microscopy. Platelet depletion resulted selectin beads binding per square millimeter. A signifi cantly higher number in decreased leukocyte rolling in Adamts13 mice compared with non- /  +/+ of anti – P-selectin beads was observed binding to unstimulated mesenteric depleted in Adamts13 mice but not in Adamts13 mice, indicating /  +/+ veins of Adamts13 than to Adamts13 mice, suggesting increased that platelets are part of the mechanism that results in increased leuko- spontaneous Weibel-Palade body release. Data represent the mean ± SEM. cyte rolling in Adamts13 veins. Data represent the mean ± SEM. 2068 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE (adherent for > 30 s) was increased approximately twofold in the the veins of Adamts13 mice when challenged with TNF- activated microvenules of Adamts 13 mice (mean ± SEM = (unpublished data). We asked whether increased leukocyte +/+ 21 ± 1) when compared with Adamts13 mice (mean ± rolling observed in the unstimulated veins of Adamts13 SEM = 12 ± 1; P < 0.01; Fig. 5 ; and Video 4, available mice would result in increased leukocyte adhesion under in- at http://www.jem.org/cgi/content/full/jem.20080130/DC1). fl ammatory conditions, and if so, whether it is VWF depen- Again, increased leukocyte adhesion was dependent on the dent. To answer these questions, mice were challenged with presence of VWF, because the number of leukocytes adhering TNF-  and mesenteric microvenules were visualized after /   / in microvenules of Adamts13 / Vwf mice was similar to 3.5 h by intravital microscopy. Microvenules were chosen be- +/+  / that in Adamts13 /Vwf mice ( Fig. 5 B ). cause with them we can precisely perform a quantitative anal- ysis of leukocyte adhesion per square micrometer for each Neutrophil infl ux is increased in thioglycollate-induced vessel. The shear rate and diameter of the microvenules +/+ +/+  / peritonitis in Adamts13 mice studied were similar for Adamts13 /Vwf , Adamts13 / +/+ +/+  /   /   / After fi nding that ADAMTS13 defi ciency increases leuko- Vwf , Adamts13 /Vwf , and Adamts13 /Vwf mice cyte rolling and adhesion, we asked whether ADAMTS13 ( Table II ). We found that the number of adherent leukocytes Figure 4. Leukocytes roll more slowly in histamine-stimulated Adamts13 veins. Histamine produced during infl ammation is a secretagogue of Weibel-Palade bodies. Histamine was injected i.p., and the stimulated veins were observed 15 min later by intravital microscopy. Endogenous platelets and leukocytes were labeled with Rhodamine 6G. Representative photographs are shown. Lines delineate the blood vessel. White arrows indicate platelet-VWF strings. Platelet-VWF strings anchoring to endothelium were observed only in Adamts13 mice. (A) Black arrows indicate leukocytes interacting with platelet-VWF strings in the Adamts13 mouse. (B) The cumulative histogram allows direct comparison of rolling velocities of the leukocytes. Leukocyte /  +/+  / rolling velocity was signifi cantly lower in Adamts13 compared with Adamts13 mice veins (P < 0.001). (C) Stimulated veins of Adamts13 mice infused with either control Ig (left) or anti – P-selectin Ig (right). Platelet-VWF strings anchored to endothelium do not support leukocyte rolling if P-selectin is inhibited. The single leukocyte seen in the photograph on the right is fi rmly adherent. Bars, 50 μ m. Videos 2 and 3 are available at http://www .jem.org/cgi/content/full/jem.20080130/DC1. JEM VOL. 205, September 1, 2008 2069 At baseline, peripheral neutrophil counts were not signifi cantly diff erent in Adamts13 mice (mean ± SD = 688 ± 208 × 3 +/+ 10 neutrophils/ml) compared with Adamts13 mice (mean ± SD = 636 ± 232 × 10 neutrophils/ml; P = 0.48; n = 18 – 19 mice of each group). Very few neutrophils were detected /  +/+ in the lavage of Adamts13 and Adamts13 mice that were infused with PBS alone and not challenged with thioglycollate (unpublished data). 4 h after challenge with thioglycollate, Adamts13 mice showed  60% more ex- travastion of neutrophils to infl amed peritoneum compared +/+ with Adamts13 mice (P < 0.03; Fig. 6 A ). The experiment was repeated a second time with the same number of mice in each group, and the results were similar (P < 0.03). The in- creased neutrophil extravasation observed in the peritoneum of Adamts13 mice was dependent on VWF, because neu- trophil counts in the infl amed peritoneum of Adamts13 / /  +/+  / Vwf were similar compared with Adamts13 / Vwf mice (P = 0.23; Fig. 6 B ). These results suggest that ADAMTS13 deficiency results in increased neutrophil extravasation during infl ammation, and this process is also dependent on VWF. Adamts13 mice exhibit increased neutrophil recruitment in excisional skin wounds Because more neutrophils extravasate into infl amed perito- neum in Adamts13 mice, we asked whether more neu- trophils would also be recruited during wound healing. Total counts of neutrophils in the 4-h wounded skin tissue were quantifi ed microscopically in hematoxylin and eosin (H & E) – Figure 5. Increased leukocyte adhesion in the TNF-  – stimulated stained sections. In the Adamts13 mice, signifi cantly more mesenteric venules of Adamts13 mice. Mice were treated with +/+ neutrophils were recruited compared with Adamts13 mice the infl ammatory cytokine TNF-  3.5 h before intravital microscopy. ( Fig. 7, A and B ). These results were con fi rmed in a second A single mesenteric venule (25 – 30 μ m diameter) was studied per mouse. (A) Representative images are shown. Arrows indicate leukocytes adhering to experiment by measuring the myeloperoxidase (MPO) activ- infl amed endothelium. (B) Quantifi cation of the adherent leukocytes. The ity in the excised wounded tissue. Increased MPO activity number of adherent leukocytes was markedly increased in the microve- correlates with an increase in the number of neutrophils ( 32 ). /  +/+ nules of Adamts13 compared with Adamts13 mice. In contrast, the We observed an increase in MPO activity in 4-h wounded tis- /   / number of leukocytes adhering in venules of Adamts13 /Vwf mice /  +/+ sue in Adamts13 compared with Adamts13 mice (P < +/+  / was similar to Adamts13 /Vwf mice, suggesting that VWF plays a 0.01; Fig. 7 C ). Thus, ADAMTS13 defi ciency also results role in increased leukocyte adhesion in Adamts13 vessels. Data repre- in increased extravasation of neutrophils in the early phase of sent the mean ± SEM. Bars, 30 μ m. Video 4 is available at http://www.jem wound healing. We were unable to examine neutrophil re- .org/cgi/content/full/jem.20080130/DC1. +/+  /   /   / cruitment in Adamts13 / Vwf and Adamts13 / Vwf mice because the mice bled excessively. However, on a diff er- defi ciency would also result in increased neutrophil extrava- ent genetic background it was shown that signifi cantly fewer sation under infl ammatory conditions. We used the well- neutrophils were present in tissue sections of 1-h wounds of /  +/+ established model of thioglycollate-induced peritonitis ( 31 ). Vwf compared with Vwf mice ( 33 ). Table II. Hemodynamic parameters of vessels observed by intravital microscopy Genotype Diameter Newtonian wall Interfacial 1  1 ( μ m) shear rate  (s ) shear rate  (1000 s ) w i +/+ +/+ Adamts13 / Vwf ( n = 10) 31.4 ± 4.5 276.1 ± 43.6 1.35 ± 0.45 /  +/+ 30.1 ± 4.3 282.6 ± 83.1 1.38 ± 0.57 Adamts13 / Vwf ( n = 12) /   / 34.7 ± 6 257.9 ± 53 1.26 ± 0.26 Adamts13 / Vwf ( n = 10) +/+  / 31.3 ± 5 265.1 ± 72.3 1.3 ± 0.56 Adamts13 / Vwf ( n = 9) Hemodynamic parameters were established before recording leukocyte adhesion for Fig. 4 . Values are represented as mean ± SD. P = NS. 2070 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 6. Peritoneal neutrophil infl ux in thioglycollate-induced peritonitis is elevated in Adamts13 mice. Total neutrophil count in lavage was counted after 4 h of thioglycollate administration. (A) A signifi cant increase in neutrophil extravasation was observed in Adamts13 compared with +/+  /   /  +/+  / Adamts13 mice. (B) No signifi cant increase in neutrophil infl ux in the peritoneum was observed in Adamts13 /Vwf compared with Adamts13 /Vwf mice, indicating that this effect is dependent on VWF. Data represent the mean ± SEM. DISCUSSION ing and adhesion was dependent on the presence of VWF. In the present study, we document a key role for ADAMTS13 These in vivo fi ndings could be explained in part by recently in down-regulating infl ammation by preventing excessive published in vitro studies by Bernardo et al., who showed leukocyte rolling in unstimulated veins, and leukocyte ad- that platelets bound to endothelial ULVWF could support hesion and extravasation under infl ammatory conditions. leukocyte tethering and rolling ( 25 ), and by Pendu et al., We have previously demonstrated in vivo that ADAMTS13 who showed that VWF acts as a ligand for the leukocyte re- cleaves platelet-VWF strings, regulates platelet interaction ceptors P-selectin glycoprotein ligand 1 and  2 integrin ( 24 ). with the “ activated ” vessel wall in the veins, prevents thrombi This binding involves multiple domains of VWF, including formation in activated microvenules, and modulates throm- D ' -D3 and A1-A2-A3 ( 24 ). botic response in injured arterioles ( 17, 19 ). Thus, these stud- We also found an increase in endothelial P-selectin ex- ies indicate that ADAMTS13 forms a new link between pression, soluble P-selectin, and VWF in the plasma of Ad- thrombosis and infl ammation. The increase in leukocyte roll- amts 13 mice. It is interesting to speculate how ADAMTS13 Figure 7. Neutrophil infl ux in skin excision wounds is increased in Adamts13 mice. The number of neutrophils present in skin tissue sur- rounding the wound 4 h after injury was determined microscopically in H & E-stained sections. (A) Representative images of the wound tissue in /  +/+ Adamts13 and Adamts13 mice are shown. A defi ciency of ADAMTS13 results in an increase in neutrophil extravasation. Bar, 20 μ m. (B) Visual count of the number of neutrophils that had emigrated from the blood vessels is shown. (C) Determination of MPO activity in the wounded tissue shows higher activity in Adamts13 mice samples. Data represent the mean ± SEM. JEM VOL. 205, September 1, 2008 2071 MATERIALS AND METHODS defi ciency results in increased plasma VWF. ADAMTS13 de- Animals. Preliminary experiments to investigate the role of ADAMTS13 on fi ciency could produce slower clearance of ULVWF from leukocyte rolling were done on mixed background mice (C57BL/6J/129 × 1/ the circulation and, thus, elevated VWF levels. Alternatively, SV) using littermates for comparison. Adamts13 mice were then backcrossed ULVWF multimers activate platelets, which in turn may ac- onto the C57BL/6J background for eight generations ( 26 ). The Adamts13 tivate the endothelium. Recently, our laboratory showed that  /   /   / ( 17 ), Vwf ( 5 ), and Adamts13 /Vwf ( 26 ) mice described in this study +/+ activated platelets, by binding to leukocytes, promote the re- are on the C57BL/6J background. The control Adamts13 (WT) mice on a C57BL/6J background were purchased from the Jackson Laboratory. The male lease of Weibel-Palade bodies and stimulate leukocyte rolling /  and female mice used for intravital microscopy were approximately 4 wk old ( 28 ). Interestingly, depletion of platelets in Adamts13 mice and weighed 13 – 15 g. Male and female mice used for experimental thioglycol- resulted in the normalization of leukocyte rolling as com- late-induced peritonitis and excision wounds were 9 – 11 wk old and weighed +/+ pared with Adamts13 mice. This indicates that platelets, 23 – 26 g. Animals were bred at the Immune Disease Institute, and the experi- likely activated by ULVWF either in circulation or directly mental procedures were approved by its Animal Care and Use Committee. on endothelium, stimulate Weibel-Palade bodies secretion. Elevated expression of endothelial P-selectin is also consistent Intravital microscopy. Mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g), and a midline incision was made through the abdominal wall with increased Weibel-Palade body exocytosis. to expose the mesentery and mesenteric veins (200 – 300 μ m diameter). Ve- Leukocytes roll and tether on the endothelium through nules (25 – 30 μ m diameter) were labeled as microvenules and were also exam- P-selectin and P-selectin glycoprotein ligand 1 interaction ined by intravital microscopy. The exposed mesentery vein was kept moist under low shear conditions ( 1 ). We observed that the pres- throughout the experiment by periodic superfusion of warmed (37 ° C) bicar- ence of platelet-ULVWF strings in the veins of Adamts13 bonate-buff ered saline (131.9 mM NaCl, 18 mM NaHCO , 4.7 mM KCl, mice treated with histamine decreased leukocyte velocity in 2 mM CaCl , and 1.2 mM MgCl ) equilibrated with 5% CO in N . The mes- 2 2 2 2 entery vein was transluminated with a 12-V, 100-W, direct current – stabilized the presence of P-selectin. Our in vivo fi ndings are in agree- source. Veins were visualized using an inverted microscope (Axiovert 135; ment with the in vitro study of Bernardo et al. reporting that Carl Zeiss, Inc.) with a 32 × objective connected to an SVHS video recorder the leukocyte rolling on ULVWF-platelet strings was signifi - (model AG-6730; Panasonic) using a charge-coupled device video camera cantly slower than leukocyte rolling on endothelial cells in (model C2400; Hamamatsu Photonics). Leukocyte interaction with the endo- vitro ( 25 ). Remarkably, this study showed that leukocytes thelium vessel wall was recorded in phase contrast for 10 min each in two to three veins per mouse. To study leukocyte rolling velocity, veins were stimu- can tether and roll on platelet-ULVWF strings under high lated with 200 μ l of 1-mM histamine per 15 g of mouse body weight. To shear stress. These results, together with our fi ndings, suggest study leukocyte adhesion under infl ammatory conditions, mice were infused that ULVWF multimers  released together with P-selectin i.p. with 500 ng TNF-  per 15 g of mouse body weight 3.5 h before intravital from Weibel-Palade bodies by many stimuli, including hyp- microscopy. A single mesenteric venule of  30 μ m in diameter was studied oxia ( 34 ), changes in shear stress ( 35 ), or infl ammatory cyto- per mouse. The wall shear rate for mesenteric veins (200 – 300 μ m diameter) kines ( 23 )  could accelerate infl ammatory responses in diseases and microvenules (25 – 30 μ m in diameter) was calculated based on Poiseuille ’ s law for a Newtonian fl uid:  = (8 V /D ). The interfacial shear rate (  ) for such as atherosclerosis by slowing down leukocytes and facili- w m v i microvenules was calculated as follows:  = 4.9(8 V /D ), where V is mean tating their extravasation. i m v m blood velocity, D is the diameter of the venule, and 4.9 is a median empirical Increased plasma VWF levels have been reported in dis- correction factor obtained from velocity profi les measured in microvessels in eases implicating infl ammation, such as rheumatoid arthritis vivo ( 42 ). It is about fi ve times greater than values described in the literature ( 36 ), viral and bacterial infections ( 37, 38 ), coronary artery for wall shear rate because of the slope of the velocity profi le of blood in small disease ( 39 ), and ischemic stroke ( 40 ). In addition, several venules and the presence of the endothelial surface layer ( 42 ). The centerline erythrocyte velocity (V ) was measured using an optical Doppler velocimeter studies suggest that infl ammation is accompanied by a de- rbc (Microcirculation Research Institute). V is estimated from the measured mean crease in ADAMTS13 activity ( 41 ). In the present study, we V by multiplying with an empirical factor of 0.625 ( 43 ). rbc provide evidence that ULVWF that is likely released and present under these circumstances further promotes infl ammation, Quantifi cation of leukocyte rolling, velocity, and adhesion. Re- as ADAMTS13 defi ciency results in increased extravasation corded images for leukocyte rolling were analyzed as follows. First, the num- of neutrophils in both thioglycollate-induced peritonitis and ber of leukocytes passing through a plane perpendicular to the vessel axis wound healing. Our results complement previous studies during a 1-min interval was counted. Leukocyte rolling per minute per vein for each mouse was determined by taking the average of fi ve 1-min counts, that reported decreased extravasation of neutrophils in VWF- as observed on the video screen during the entire 10-min recording. Second, defi cient mice, which was attributed to a lack of P-selectin the rolling velocity was determined by recording the time it took for a leuko- storage ( 33 ), and delayed formation of atherosclerotic lesions in cyte to transverse a certain distance in a vein  250 μ m long and 200 – 300 μ m /   /   / Vwf mice either on an apoE or LDLR background wide, as observed on the video screen. All of the analysis was done by an ( 10 ). The results from our study suggest that ADAMTS13 investigator blinded to genotype. Third, the leukocyte was considered to be down-regulates infl ammation by cleaving hyperactive ULVWF adherent if it remained stationary for > 30 s. The total number of leukocytes adhering per venule per mouse represents an average of adherent leukocytes multimers, and that defi ciency of ADAMTS13 not only can in three diff erent segments per microvenule. induce TTP but also accelerates infl ammatory diseases. Be- cause thrombosis and infl ammation constitute an integral part of P-selectin inhibition. P-selectin was inhibited in the Adamts13 mice the pathogenesis of many diseases, including the major killers by infusing i.v. a blocking rat monoclonal anti – P-selectin antibody (clone atherosclerosis and stroke, the results reported in this paper may RB40.34, containing no azide; BD Biosciences) at a concentration of 2 μ g provide new insights into the possible uses of ADAMTS13 as per gram of body weight. A similar concentration of purifi ed rat Ig (BD Bio- a therapeutic agent. sciences) was used as a control. 2072 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Platelet depletion. Platelets were depleted for 24 h by infusing i.v. anti- paraffi n blocks using standard protocols. 6- μ m tissue sections were stained GPIb antibody at a fi nal concentration of 2 μ g per gram of mouse body with H & E. Extra vascular neutrophils were counted in the entire wound area weight (emfret Analytics). Control rat Ig (emfret Analytics) was used at the using a light microscope (Axioplan; Carl Zeiss, Inc.) at 40 × magnifi cation. same concentration. To ensure that platelets remained depleted for 24 h, whole blood was withdrawn from the eye plexus into tubes containing 5 μ M MPO assay. The wounds were prepared as described in the previous para- EDTA after each surgery. More than 95% of the platelets remained depleted graph. A 6-mm punch of the skin containing the 4-mm wound area was washed after 24 h, as analyzed by FACS. in cold PBS and homogenized in 0.5 ml PBS at 4 ° C using a polytron homoge- nizer (fi ve bursts of 10 s each at maximum speed). 250 μ l of the homogenate Quantifi cation of P-selectin and VWF. Soluble P-selectin in the plasma was added to 250 μ l hexadecyltrimethylammonium bromide, vortexed, and in- was measured by an enzyme immunoassay kit (R & D Systems) according to cubated for 2 min. After centrifugation, the supernatant was collected and as- the manufacturer ’ s guidelines. Plasma VWF levels were measured by an en- sayed for MPO activity by adding 55 μ l TMB substrate to 30 μ l of the zyme immunoassay technique. Microtiter plates were coated overnight at supernatant. The absorbance was read at 630 nm at intervals of 30 s for 2 min. 4 ° C with rabbit anti – human VWF antibody (Dako) at a concentration of 15 μ g/ml diluted in 50 mM of sodium carbonate buff er. Plasma samples (diluted Statistical analysis. Results are reported as the mean ± SEM, unless other- 1:20 in PBS) were incubated for 2 h in the coated wells at room tempera- wise noted. The statistical signifi cance of the diff erence between means was ture. After six washes, the polyclonal anti – human VWF coupled to HRP assessed by using the unpaired Student ’ s t test (for the comparison of two (1:2,000) was added for 2 h. After washing, 3,3  ,5,5  -tetramethylbenzidine groups) or by analysis of variance followed by Boneferroni ’ s multiple com- (TMB) substrate solution (Sigma-Aldrich) was added to the wells, and the parison test. P < 0.05 was considered signifi cant. colorimetric reaction was stopped with H SO after 20 min. Results were 2 4 read in an ELISA microplate reader (model MRXII; Dynex Technologies) Online supplemental material. Video 1 shows P-selectin – dependent leu- at A nm. Normal pooled plasma obtained from 10 WT mice was defi ned kocyte rolling in an Adamts13 mouse. Video 2 shows the interaction and as 1 U VWF antigen per milliliter of plasma. rolling of some leukocytes on ULVWF-platelet strings in a histamine-stimu- lated vein of an Adamts13 mouse. ULVWF-platelet strings are absent in +/+ the Adamts13 mouse. Video 3 shows that ULVWF-platelet strings cannot In vivo detection of endothelial P-selectin. Yellow-green (excitation/ support leukocyte rolling in the absence of P-selectin. Video 4 shows in- emission = 505 nm/515 nm) and red (excitation/emission = 580 nm/605 nm) creased leukocyte adhesion in TNF-  – activated microvenules of Adamts13 carboxylate-modifi ed microspheres (1 μ m diameter; Invitrogen) were +/+ compared with Adamts13 mice. Endogenous leukocytes and platelets were covalently coupled to anti – P-selectin monoclonal antibody RB40.34 or labeled with Rhodamine 6G in Videos 1 – 3. Online supplemental material is control rat IgG (BD Bioscience), according to the manufacturer ’ s instruc- available at http://www.jem.org/cgi/content/full/jem.20080130/DC1. tions (Invitrogen). Mice were infused with 10 microspheres of each color, and mesenteric venules were observed immediately by fl uorescent intra- We thank Lesley Cowan for help in preparing the manuscript. vital microscopy. The order of infusion of yellow and red microspheres was This work was supported by a Sponsored Research Agreement from Baxter reversed between experiments. Bioscience (to A.K. Chauhan and D.D. Wagner), and National Heart, Lung, and Blood Institute grants R37 HL041002 and PO1 HL066105 (to D.D. Wagner). Flow cytometry. For assessing the amount of neutrophils in peripheral F. Scheifl inger is an employee of Baxter Bioscience. The authors have no other blood, 1 ml of RBC lysis buff er was added to 100 μ l of whole blood contain- confl icting fi nancial interests. ing 5 μ M EDTA and incubated for 10 min on ice. The samples were then centrifuged at 400 g , and the pellet was resuspended in 35 μ l of ice-cold PBS. Submitted: 18 January 2008 20 μ l of cell suspension was mixed with an antibody against Gr-1 labeled Accepted: 3 July 2008 with Alexa Fluor 647. After a 5-min incubation at room temperature in the dark, 700 μ l PBS was added to 5 μ l of beads (Spherotech) with a known REFERENCES concentration (12 × 10 beads per μ l). Samples were read by FACS for 1 min 1 . Ley , K. , C. Laudanna , M.I. Cybulsky , and S. Nourshargh . 2007 . Getting at medium pump velocity. The amount of neutrophils (Gr-1 – positive events to the site of infl ammation: the leukocyte adhesion cascade updated. falling in the neutrophil forward and side scatter gate) was calculated based Nat. Rev. Immunol. 7 : 678 – 689 . on the number of beads detected during the same time period. 2 . Wagner , D.D. 2005 . New links between infl ammation and thrombosis. Arterioscler. Thromb. Vasc. Biol. 25 : 1321 – 1324 . Thioglycollate-induced peritonitis. Experimental peritonitis was induced 3 . Denis , C.V. , and D.D. Wagner . 2007 . Platelet adhesion receptors and by injecting 1 ml of 3% thioglycollate (Sigma-Aldrich) i.p. After 4 h, mice their ligands in mouse models of thrombosis. Arterioscler. Thromb. Vasc. were killed by overdosing with isofl urane and 8 ml PBS containing 0.1% BSA, Biol. 27 : 728 – 739 . 4 . Sadler , J.E. 2005 . New concepts in von Willebrand disease. Annu. Rev. and 0.5 mM/liter EDTA was used to lavage the peritoneum. Approximately Med. 56 : 173 – 191 . 6 ml of lavage was collected from each mouse, and 1 ml was used for analysis. 5 . Denis , C. , N. Methia , P.S. Frenette , H. Rayburn , M. Ullman-Cullere , The 1 ml of lavage was spun at 2,600 rpm for 5 min, and the pellet was resus- R.O. Hynes , and D.D. Wagner . 1998 . A mouse model of severe von pended in 40 μ l PBS. For accurate quantifi cation, a 20- μ l sample was taken Willebrand disease: defects in hemostasis and thrombosis. Proc. Natl. from 40 μ l, stained with GR-1 – Alexa Fluor 687 specifi c for neutrophils (BD Acad. Sci. USA . 95 : 9524 – 9529 . Bioscience), and analyzed by FACS as described in the previous paragraph. 6 . Chauhan , A.K. , J. Kisucka , C.B. Lamb , W. Bergmeier , and D.D. Wagner . 2007 . von Willebrand factor and factor VIII are indepen- Wounding and tissue preparation. Wounds on the back of mice were dently required to form stable occlusive thrombi in injured veins. Blood . performed as previously described ( 44 ). In brief, mice were anesthetized with 109 : 2424 – 2429 . 2.5% tribromoethanol (0.15 ml per 10 g of body weight), and hair was re- 7 . Vischer , U.M. 2006 . von Willebrand factor, endothelial dysfunction, moved with an electric razor. The skin was swabbed with 70% ethanol (etha- and cardiovascular disease. J. Thromb. Haemost. 4 : 1186 – 1193 . nol wipes) and two 4-mm full-thickness excision wounds were made by 8 . Ruggeri , Z.M. , J.N. Orje , R. Habermann , A.B. Federici , and A.J. picking up a fold of skin, placing it over dental wax, and punching through Reininger . 2006 . Activation-independent platelet adhesion and aggre- the two layers of skin. Mice were housed in individual cages. After 4 h, the gation under elevated shear stress. Blood . 108 : 1903 – 1910 . mice were killed, and wounds were harvested with 1 – 2 mm of normal skin 9 . Groot , E. , P.G. de Groot , R. Fijnheer , and P.J. Lenting . 2007 . The tissue around them. The wounds were cut in half, fi xed overnight in 4% para- presence of active von Willebrand factor under various pathological formaldehyde, processed through graded ethanol solutions, and embedded in conditions. Curr. Opin. Hematol. 14 : 284 – 289 . JEM VOL. 205, September 1, 2008 2073 10 . Methia , N. , P. Andre , C.V. Denis , M. Economopoulos , and D.D. 27 . Kunkel , E.J. , and K. Ley . 1996 . Distinct phenotype of E-selectin-de- Wagner . 2001 . Localized reduction of atherosclerosis in von Willebrand fi cient mice. E-selectin is required for slow leukocyte rolling in vivo. factor-defi cient mice. Blood . 98 : 1424 – 1428 . Circ. Res. 79 : 1196 – 1204 . 11 . Sporn , L.A. , V.J. Marder , and D.D. Wagner . 1986 . Inducible secre- 28 . Dole , V.S. , W. Bergmeier , H.A. Mitchell , S.C. Eichenberger , and tion of large, biologically potent von Willebrand factor multimers. Cell . D.D. Wagner . 2005 . Activated platelets induce Weibel-Palade-body 46 : 185 – 190 . secretion and leukocyte rolling in vivo: role of P-selectin. Blood . 106 : 12 . Wagner , D.D. 1990 . Cell biology of von Willebrand factor. Annu. Rev. 2334 – 2339 . Cell Biol. 6 : 217 – 246 . 29 . Nieswandt , B. , W. Bergmeier , K. Rackebrandt , J.E. Gessner , and H. 13 . Sporn , L.A. , V.J. Marder , and D.D. Wagner . 1987 . von Willebrand fac- Zirngibl . 2000 . Identifi cation of critical antigen-specifi c mechanisms in tor released from Weibel-Palade bodies binds more avidly to extracel- the development of immune thrombocytopenic purpura in mice. Blood . lular matrix than that secreted constitutively. Blood . 69 : 1531 – 1534 . 96 : 2520 – 2527 . 14 . Arya , M. , B. Anvari , G.M. Romo , M.A. Cruz , J.F. Dong , L.V. 30 . Chauhan , A.K. , T. Goerge , S.W. Schneider , and D.D. Wagner . 2007 . McIntire , J.L. Moake , and J.A. Lopez . 2002 . Ultralarge multimers of Formation of platelet strings and microthrombi in the presence of von Willebrand factor form spontaneous high-strength bonds with the ADAMTS-13 inhibitor does not require P-selectin or beta3 integrin. platelet glycoprotein Ib-IX complex: studies using optical tweezers. J. Thromb. Haemost. 5 : 583 – 589 . Blood . 99 : 3971 – 3977 . 31 . Mayadas , T.N. , R.C. Johnson , H. Rayburn , R.O. Hynes , and D.D. 15 . Dong , J.F. , J.L. Moake , L. Nolasco , A. Bernardo , W. Arceneaux , C.N. Wagner . 1993 . Leukocyte rolling and extravasation are severely com- Shrimpton , A.J. Schade , L.V. McIntire , K. Fujikawa , and J.A. Lopez . promised in P selectin-defi cient mice. Cell . 74 : 541 – 554 . 2002 . ADAMTS-13 rapidly cleaves newly secreted ultralarge von 32 . Bradley , P.P. , D.A. Priebat , R.D. Christensen , and G. Rothstein . 1982 . Willebrand factor multimers on the endothelial surface under fl owing Measurement of cutaneous infl ammation: estimation of neutrophil con- conditions. Blood . 100 : 4033 – 4039 . tent with an enzyme marker. J. Invest. Dermatol. 78 : 206 – 209 . 16 . Moake , J.L. , C.K. Rudy , J.H. Troll , M.J. Weinstein , N.M. Colannino , 33 . Denis , C.V. , P. Andre , S. Saff aripour , and D.D. Wagner . 2001 . Defect J. Azocar , R.H. Seder , S.L. Hong , and D. Deykin . 1982 . Unusually in regulated secretion of P-selectin aff ects leukocyte recruitment in large plasma factor VIII:von Willebrand factor multimers in chronic von Willebrand factor-defi cient mice. Proc. Natl. Acad. Sci. USA . 98 : relapsing thrombotic thrombocytopenic purpura. N. Engl. J. Med. 4072 – 4077 . 307 : 1432 – 1435 . 34 . Pinsky , D.J. , Y. Naka , H. Liao , M.C. Oz , D.D. Wagner , T.N. Mayadas , 17 . Motto , D.G. , A.K. Chauhan , G. Zhu , J. Homeister , C.B. Lamb , R.C. Johnson , R.O. Hynes , M. Heath , C.A. Lawson , and D.M. Stern . K.C. Desch , W. Zhang , H.M. Tsai , D.D. Wagner , and D. Ginsburg . 1996 . Hypoxia-induced exocytosis of endothelial cell Weibel-Palade 2005 . Shigatoxin triggers thrombotic thrombocytopenic purpura in bodies. A mechanism for rapid neutrophil recruitment after cardiac pres- genetically susceptible ADAMTS13-defi cient mice. J. Clin. Invest. ervation. J. Clin. Invest. 97 : 493 – 500 . 115 : 2752 – 2761 . 35 . Galbusera , M. , C. Zoja , R. Donadelli , S. Paris , M. Morigi , A. Benigni , 18 . Banno , F. , K. Kokame , T. Okuda , S. Honda , S. Miyata , H. Kato , Y. M. Figliuzzi , G. Remuzzi , and A. Remuzzi . 1997 . Fluid shear stress Tomiyama , and T. Miyata . 2006 . Complete defi ciency in ADAMTS13 modulates von Willebrand factor release from human vascular endothe- is prothrombotic, but it alone is not suffi cient to cause thrombotic lium. Blood . 90 : 1558 – 1564 . thrombocytopenic purpura. Blood . 107 : 3161 – 3166 . 36 . McEntegart , A. , H.A. Capell , D. Creran , A. Rumley , M. Woodward , 19 . Chauhan , A.K. , D.G. Motto , C.B. Lamb , W. Bergmeier , M. Dockal , and G.D. Lowe . 2001 . Cardiovascular risk factors, including throm- B. Plaimauer , F. Scheifl inger , D. Ginsburg , and D.D. Wagner . botic variables, in a population with rheumatoid arthritis. Rheumatology 2006 . Systemic antithrombotic eff ects of ADAMTS13. J. Exp. Med. (Oxford) . 40 : 640 – 644 . 203 : 767 – 776 . 37 . Tzavara , V. , P.G. Vlachoyiannopoulos , T. Kordossis , D. Galaris , A. Travlou , 20 . Ono , T. , J. Mimuro , S. Madoiwa , K. Soejima , Y. Kashiwakura , A. U. Dafni , and H.M. Moutsopoulos . 1997 . Evidence for non-adaptive im- Ishiwata , K. Takano , T. Ohmori , and Y. Sakata . 2006 . Severe secondary mune response in HIV infection. Eur. J. Clin. Invest. 27 : 846 – 849 . defi ciency of von Willebrand factor-cleaving protease (ADAMTS13) in 38 . Kayal , S. , J.P. Jais , N. Aguini , J. Chaudiere , and J. Labrousse . 1998 . patients with sepsis-induced disseminated intravascular coagulation: its Elevated circulating E-selectin, intercellular adhesion molecule 1, and correlation with development of renal failure. Blood . 107 : 528 – 534 . von Willebrand factor in patients with severe infection. Am. J. Respir. 21 . Nguyen , T.C. , A. Liu , L. Liu , C. Ball , H. Choi , W.S. May , K. Crit. Care Med. 157 : 776 – 784 . Aboulfatova , A.L. Bergeron , and J.F. Dong . 2007 . Acquired ADAMTS- 39 . Jager , A. , V.W. van Hinsbergh , P.J. Kostense , J.J. Emeis , J.S. Yudkin , G. 13 defi ciency in pediatric patients with severe sepsis. Haematologica . Nijpels , J.M. Dekker , R.J. Heine , L.M. Bouter , and C.D. Stehouwer . 92 : 121 – 124 . 1999 . von Willebrand factor, C-reactive protein, and 5-year mortal- 22 . Reiter , R.A. , K. Varadi , P.L. Turecek , B. Jilma , and P. Knobl . 2005 . ity in diabetic and nondiabetic subjects: the Hoorn Study. Arterioscler. Changes in ADAMTS13 (von-Willebrand-factor-cleaving protease) ac- Thromb. Vasc. Biol. 19 : 3071 – 3078 . tivity after induced release of von Willebrand factor during acute sys- 40 . Bongers , T.N. , M.P. de Maat , M.L. van Goor , V. Bhagwanbali , H.H. temic infl ammation. Thromb. Haemost. 93 : 554 – 558 . van Vliet , E.B. Gomez Garcia , D.W. Dippel , and F.W. Leebeek . 2006 . 23 . Bernardo , A. , C. Ball , L. Nolasco , J.F. Moake , and J.F. Dong . 2004 . High von Willebrand factor levels increase the risk of fi rst ischemic Eff ects of infl ammatory cytokines on the release and cleavage of the en- stroke: infl uence of ADAMTS13, infl ammation, and genetic variability. dothelial cell-derived ultralarge von Willebrand factor multimers under Stroke . 37 : 2672 – 2677 . fl ow. Blood . 104 : 100 – 106 . 41 . Mannucci , P.M. , M.T. Canciani , I. Forza , F. Lussana , A. Lattuada , and 24 . Pendu , R. , V. Terraube , O.D. Christophe , C.G. Gahmberg , P.G. de E. Rossi . 2001 . Changes in health and disease of the metalloprotease Groot , P.J. Lenting , and C.V. Denis . 2006 . P-selectin glycoprotein li- that cleaves von Willebrand factor. Blood . 98 : 2730 – 2735 . gand 1 and beta2-integrins cooperate in the adhesion of leukocytes to 42 . Long , D.S. , M.L. Smith , A.R. Pries , K. Ley , and E.R. Damiano . 2004 . von Willebrand factor. Blood . 108 : 3746 – 3752 . Microviscometry reveals reduced blood viscosity and altered shear rate 25 . Bernardo , A. , C. Ball , L. Nolasco , H. Choi , J.L. Moake , and J.F. and shear stress profi les in microvessels after hemodilution. Proc. Natl. Dong . 2005 . Platelets adhered to endothelial cell-bound ultra-large von Acad. Sci. USA . 101 : 10060 – 10065 . Willebrand factor strings support leukocyte tethering and rolling under 43 . Lipowsky , H.H. , and B.W. Zweifach . 1978 . Application of the “ two- high shear stress. J. Thromb. Haemost. 3 : 562 – 570 . slit ” photometric technique to the measurement of microvascular volu- 26 . Chauhan , A.K. , M.T. Walsh , G. Zhu , D. Ginsburg , D.D. Wagner , metric fl ow rates. Microvasc. Res. 15 : 93 – 101 . and D.G. Motto . 2008 . The combined roles of ADAMTS13 and 44 . Subramaniam , M. , S. Saff aripour , L. Van De Water , P.S. Frenette , T.N. VWF in murine models of TTP, endotoxemia, and thrombosis. Blood . Mayadas , R.O. Hynes , and D.D. Wagner . 1997 . Role of endothelial 111 : 3452 – 3457 . selectins in wound repair. Am. J. Pathol. 150 : 1701 – 1709 . 2074 ANTIINFLAMMATORY EFFECTS OF ADAMTS13 | Chauhan et al.

Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Sep 1, 2008

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