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Systemic antithrombotic effects of ADAMTS13

Systemic antithrombotic effects of ADAMTS13 ARTICLE <doi>10.1084/jem.20051732</doi><aid>20051732</aid>Systemic antithrombotic eff ects of ADAMTS13 1,2 3 1 Anil K. Chauhan, David G. Motto, Colin B. Lamb, 1,2 5 5 Wolfgang Bergmeier, Michael Dockal, Barbara Plaimauer, 5 4 1,2 Friedrich Scheifl inger, David Ginsburg, and Denisa D. Wagner 1 2 CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115 3 4 Department of Pediatrics, University of Michigan, and Department of Internal Medicine, University of Michigan and Howard Hughes Medical Institute, Ann Arbor, MI 48109 Baxter Bioscience, Vienna, A-1220 Austria The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 defi ciency is linked to a life- threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet- rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in −/− activated microvenules of Adamts13 mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi for- mation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in 𝛂 IIb𝛃 3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our fi ndings reveal that ADAMTS13 has a power- ful natural antithrombotic activity and recombinant ADAMTS13 could be used as an anti- thrombotic agent. Thrombotic thrombocytopenic purpura (TTP) milial TTP (10). Acquired TTP, often caused CORRESPONDENCE Denisa D. Wagner: is a disorder characterized by thrombotic mi- by autoantibodies inhibiting ADAMTS13 ac- [email protected] croangiopathy, thrombocytopenia, and micro- tivity, is a more common disorder that occurs vascular thrombosis that can cause various in adults and older children and can recur at Abbreviations used: ADAMTS, a disintegrin-like and metallo- degrees of tissue ischemia and infarction. Clini- regular intervals in 11–36% of patients (4, 6). protease with thrombospondin cally, TTP patients are diagnosed by signs and Nonneutralizing autoantibodies have been as- type I repeats; r-hu, recombi- symptoms such as thrombocytopenia, micro- sociated with acute acquired TTP (15). In most nant human; TTP, thrombotic angiopathic hemolytic anemia, neurological abnor- patients with familial or acquired TTP, plasma thrombocytopenic purpura; UL-VWF, ultra-large VWF; malities, renal failure, and fever (1, 2). In 1982, ADAMTS13 activity is absent or <5% of normal. VWF, von Willebrand factor. Moake et al. found ultra-large von Willebrand Without treatment, the mortality rate exceeds factor (UL-VWF) multimers in the plasma 90%, but plasma exchange therapy has reduced of patients with chronic relapsing TTP (3). mortality to 20% (2). Most patients suff ering from TTP are defi cient VWF synthesized in megakaryocytes and in a plasma metalloprotease that cleaves UL- endothelial cells is stored in platelet α-granules VWF (4–9). The protease belongs to the AD- and Weibel-Palade bodies, respectively, as UL- AMTS (a disintegrin-like and metalloprotease VWF (16). Once secreted from endothelial with thrombospondin type I repeats) family cells, these UL-VWF multimers are cleaved by and is designated as ADAMTS13, a 190-kD ADAMTS13 in the circulation into a series glycosylated protein produced predominantly of smaller multimers at specifi c cleavage sites by the liver (10–12), specifi cally by hepatic within the VWF molecule (17–19). The prote- stellate cells (13, 14). Mutations in the AD- ase cleaves at the Tyr842–Met843 bond in the AMTS13 gene have been shown to cause fa- central A2 domain of the mature VWF subunit (20) and requires zinc and calcium for activity. The online version of this article contains supplemental material. VWF exists in “ball of yarn” and fi lamentous JEM © The Rockefeller University Press $8.00 767 Vol. 203, No. 3, March 20, 2006 767–776 www.jem.org/cgi/doi/10.1084/jem.20051732 The Journal of Experimental Medicine Table I. Hemodynamic parameters were established before application of A23187 (Fig. 1) on venules and FeCl on arterioles (Fig. 5) −1 Genotype Vessel type Diameter (μm) Centerline velocity (mm/s) Shear rate (s ) +/+ Adamts13 (n = 5) Venule 31.51 ± 1.79 1.43 ± 0.07 213 ± 14.10 −/− Adamts13 (n = 5) Venule 26.36 ± 1.95 1.28 ± 0.13 244 ± 28.29 +/+ Adamts13 (n = 12) Arteriole 103.91 ± 9.29 33.33 ± 1.72 1,688.16 ± 143.32 −/− Adamts13 (n = 12) Arteriole 93.26 ± 10.48 28.05 ± 2.10 1,646.83 ± 157.16 forms as seen by electron microscopy (21). Furthermore, ADAMTS13 could be used to treat TTP and possibly other atomic force microscopy confi rms that VWF exists in a glob- thrombotic conditions. ular conformation under static conditions and may unfold to a fi lamentous state after exposure to shear stress (22). This RESULTS could occur also in vivo when one end of the VWF fi lament Endothelial activation results in thrombi formation −/− is anchored to a surface. UL-VWF multimers have high bio- in microvenules of Adamts13 mice logical activity. They bind better to the extracellular matrix We have previously observed that platelet sticking/trans- than regular multimers (23) and form higher strength bonds location in venules of 200–250 μm in diameter activated with platelet GPIb-IX than plasma VWF (24). It was demon- with calcium ionophore A23187 (a secretagogue of Weibel- −1 strated in vitro that platelets align as beads on the released Palade bodies) at low shear rate (100 s ) was prolonged in −/− +/+ UL-VWF string on the endothelial surface. These strings are Adamts13 mice compared with Adamts13 mice (26). We then cleaved by ADAMTS13 and released from the stimu- investigated whether activation of microvenule endothelium lated endothelial cells (25). We have demonstrated in vivo by A23187 (which does not denude the endothelium; −/− that it is only in Adamts13 mice that strings of platelets re-<CIT>reference 28)</CIT> results in platelet aggregation and subsequent −1 main intact after endothelial activation in veins (26). These thrombus formation. The shear rate (200–250 s ) and diam- strings attach at one end to endothelium and “wave” the eter of all the microvenules (25–30 μm) studied were similar −/− +/+ other end in the blood stream. for Adamts13 and Adamts13 mice (Table I).[ID]TBL1[/ID] In the mi- −/− Thrombi of TTP patients consist of a little fi brin but crovenules of Adamts13 mice, platelet aggregation result- mainly of VWF and platelets, suggesting VWF-mediated ing in thrombus formation was observed from 45 s to 2 min platelet aggregation as a cause of thrombosis (27). We hy- after topical superfusion of A23187 (Fig. 1).[ID]FIG1[/ID] The thrombi pothesized that endothelial activation resulting in elevation of were often unstable and fl ushed away, leading to frequent hyperactive UL-VWF multimers in plasma could be associ- embolization and causing transient downstream occlusion ated with an increased risk of thrombosis in ADAMTS13- usually only lasting 3–4 s. Thus, stimulation of Weibel- defi cient animals. We investigated thrombosis in venules and Palade body secretion can lead to spontaneous thrombus for- −/− −/− arterioles of Adamts13 mice by intravital microscopy. mation in Adamts13 mice in the absence of vascular injury. +/+ Our fi ndings strongly suggest that ADAMTS13 has natural In Adamts13 mice treated identically, platelet strings and antithrombotic activity and that recombinant human (r-hu) very small platelet aggregates could be seen attached to the −/− +/+ Figure 1. Thrombus formation in stimulated microvenules of Adamts13 mice (n = 5). No microthrombi formed in Adamts13 −/− Adamts13 mice. Venules measuring 25–30-μm in diameter were mice treated identically (n = 5). Arrows indicate the microthrombi. See visualized in the mesentery of live mice. 1 min after topical superfusion of Video 1 (available at http://www.jem.org/cgi/content/full/jem.20051732/ −/− calcium ionophore A23187, thrombus formation was observed in DC1) for thrombi in the microvenules of Adamts13 mice. 768 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 2. Antibody to ADAMTS13 increases platelet adhesion and ADAMTS13 Ab, n = 4), more platelets adhered to the vessel wall 4 min +/+ string formation on activated vessel wall. Fluorescently labeled plate- after stimulation compared with Adamts13 control (n = 4). Arrows lets representing 2.5% of total platelets were observed in mesenteric indicate the ≥20-μm strings of platelets attached at one end to the endo- venules (diameter: 200–250 μm) of live mice before (baseline) and after thelium and waving the other end in the blood stream. Inset time points A23187 superfusion. Platelets began to adhere to the endothelium 30–45 s in the lower right corner refer to the time after superfusion of A23187. +/+ after superfusion. In Adamts13 mice (infused with anti–human The bar shown in the middle panel is 50 μm. endothelium for 1–2 s, but thrombi did not form. These ob- before surgical preparation for intravital microscopy. The anti- servations demonstrate that ADAMTS13 is active at low body did not activate the endothelium as normal baseline +/+ shear and, thus, inhibits platelet aggregation and prevents platelet adhesion was found in Adamts13 mice after its thrombus formation in the microvenules. In addition, arteri- infusion (Fig. 2).[ID]FIG2[/ID] After topical superfusion of A23187, many oles (high shear) running parallel to the venules in either platelets stuck/translocated on the endothelium, reaching a −/− +/+ Adamts13 or Adamts13 mice did not show any platelet peak of platelet adhesion from 45 s to 1 min that progres- strings, platelet aggregation, or thrombus formation. sively decreased with time. However, more platelet sticking was observed 4 min after the A23187 application in the +/+ An antibody to ADAMTS13 prolongs adhesion of platelets antibody-infused Adamts13 as compared with control +/+ +/+ to secreted VWF on the vessel wall of Adamts13 mice Adamts13 mice (control IgG [n = 2] or PBS [n = 5]) (Fig. 2). Previous studies have shown that most patients suff ering from The phenomenon observed was similar to that observed −/− the acquired form of TTP have autoimmune inhibitors to in Adamts13 mice (26). Strings of platelets were seen ADAMTS13 in plasma (4, 6). We infused a polyclonal anti– varying from 20 to 40 μm and attached at one end to the en- +/+ human ADAMTS13 antibody in Adamts13 mice 2 h dothelium and waving in the blood stream. These strings +/+ +/+ Figure 3. Thrombus formation in microvenules of Adamts13 Adamts13 mice infused with the anti-ADAMTS13 Ab. The microthrombi −/− mice infused with an anti-ADAMTS13 antibody. Mesenteric venules formation and embolization were similar to that seen in Adamts13 of 25–30-μm in diameter were observed. 1 min after topical superfu- mice (Fig. 1). Arrows indicate a microthrombus. Microthrombi did not +/+ sion with A23187, thrombus formation was observed in four out of six form in Adamts13 control (n = 5). JEM VOL. 203, March 20, 2006 769 strings persisted for up to 5 min. Some strings appeared to coalesce, forming aggregates (Fig. 4 C) that were later re- leased into the blood stream. Infusion of r-hu ADAMTS13 −/− protein in the Adamts13 mice (n = 4; 3 venules per mouse) inhibited platelet string formation in all venules examined (Fig. 4 D), thus demonstrating the activity of ADAMTS13 at low shear. Platelet binding to subendothelium is increased −/− in Adamts13 mice Ferric chloride (FeCl ) injury leads to deendothelization and exposes subendothelium (30). Platelet subendothelial interac- tions after injury at arterial shear are initiated by GPIb–VWF interaction and propagated by other receptors (30). In both +/+ −/− Adamts13 and Adamts13 mice, platelet–vessel wall Figure 4. Recombinant ADAMTS13 inhibits platelet strings in interaction started rapidly after FeCl application to the −/− Adamts13 mice. Rhodamine 6G was used to label endogenous plate- arteriole. The number of animals in which >100 fl uorescent lets and leukocytes. Histamine was administered i.p. 15 min before sur- platelets were deposited 2–3 min after injury was higher in gery and three mesenteric venules of 200–300 μm in diameter were −/− −/− Adamts13 mice. In the Adamts13 , 7 out of 12 mice +/+ visualized per mouse. (A) No platelet strings are seen in Adamts13 showed >100 platelets deposited on the vessel wall com- mice (n = 5). (B) Platelet strings (indicated by arrows) are seen in the +/+ pared with 3 out of 10 in the Adamts13 mice (P < 0.05, −/− Adamts13 mice. Platelet strings anchor up to 1 min on the endothe- Fig. 5 A).[ID]FIG5[/ID] lium (n = 5). (C) The platelet strings could form platelet aggregates in −/− Adamts13 mice as indicated by arrow. (D) Infusion of r-hu ADAMTS13 −/− protein inhibits the platelet strings in Adamts13 mice (n = 4). Thrombus formation is accelerated in injured arterioles −/− of Adamts13 mice After fi nding that ADAMTS13 negatively modulates resting were either not seen or were very short lived (<2 s) in the platelet adhesion to both stimulated endothelium and sub- +/+ Adamts13 mice. endothelium, we asked whether the enzyme aff ects arte- riolar thrombus formation. This process requires platelet ADAMTS13 inhibitor induces thrombi formation activation and employs several ligands aside from VWF (30). +/+ in microvenules of Adamts13 mice The shear rate and diameter of arterioles studied were simi- +/+ −/− +/+ In the Adamts13 mice infused with anti–human ADAMTS13 lar for Adamts13 and Adamts13 mice (Table I). In the −/− antibody 2 h before surgical preparation, microthrombi formed Adamts13 mice, thrombi grew faster as thrombi >30 μm on the vessel wall 45 s to 1 min after topical superfusion of were seen at 6.64 ± 0.93 min compared with 10.78 ± 0.80 +/+ A23187 in four out of six mice (Fig. 3).[ID]FIG3[/ID] The microthrombi min in the Adamts13 mice (P < 0.005, Fig. 5 B). This −/− appearance was similar to those seen in the Adamts13 mice suggests that cleavage of VWF multimers by ADAMTS13 +/+ (Fig. 1). In control Adamts13 mice, short-lived platelet delays thrombus formation. The thrombi grew to occlusive −/− strings could be seen attached to the endothelium, but they did size in 10.56 ± 0.72 min in Adamts13 mice, whereas in +/+ not result in thrombus formation (n = 5). Adamts13 mice all the vessels were still open at this time +/+ (Fig. 5, C and D). In the Adamts13 , the mean vessel oc- Histamine promotes platelet string formation clusion time was 16.69 ± 1.25 min after injury (P < 0.0005). −/− in the venules of Adamts13 mice, a process All the vessels occluded at the site of injury. Of note, in ar- −/− inhibited by recombinant ADAMTS13 terioles of Adamts13 mice, the mean time for formation Histamine produced during infl ammation is a secretagogue of thrombi (>30 μm) as well as the mean occlusion time +/+ of Weibel-Palade bodies and stimulates the endothelium (29). were less than that of any individual Adamts13 mouse We investigated whether activation of venules by injecting (Fig. 5, B and C). −/− histamine i.p. into Adamts13 mice could result in platelet strings. Endogenous platelets were labeled by infusing Rho- ADAMTS13 defi ciency enhances thrombus growth damine 6G i.v. before surgery. Histamine was injected i.p. 15 in an 𝛂 IIb𝛃 3 integrin-dependent manner −/− min before the surgical preparation into Adamts13 (n = 5) To study the importance of integrin αIIbβ3 for thrombus +/+ and Adamts13 (n = 5) mice and venules at a shear rate formation in the absence of ADAMTS13, we performed in −1 +/+ of 100 s were visualized. In the Adamts13 mice, vitro fl ow chamber studies with whole blood in the presence strings of platelets were not seen or were short lived (<5 s; or absence of a blocking antibody (JON/A) against αIIbβ3 −/− Fig. 4 A), whereas, in the Adamts13 platelet strings, vary- (31) (Fig. 6).[ID]FIG6[/ID] To quantify the size of the thrombi, the surface ing from 20 to 100 μm could be seen (Fig. 4 B) anchored to area covered by fl uorescently labeled platelets was deter- −/− the endothelium for 1 min.[ID]FIG4[/ID] In some mice, the platelet mined. As expected, Adamts13 blood formed signifi cantly 770 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 5. Quantitative analysis of platelet adhesion and thrombi +/+ compared with Adamts13 mice (P < 0.0005). (D) Fluorescently labeled +/+ formation in FeCl -injured arterioles of Adamts13 and platelets representing 2.5% of total platelets were observed in mesen- −/− Adamts13 mice. (A) The number of fl uorescent platelets deposited teric arterioles of live mice after FeCl injury. Single adherent platelets are per minute was determined in the interval 2–3 min after injury. Absence +/+ seen in the arteriole at 4 min after injury in the Adamts13 mouse, of ADAMTS13 in the plasma signifi cantly increases early platelet interac- −/− whereas a thrombus (30 μm) can already be seen in the Adamts13 tion with the subendothelium (P < 0.05). (B) Thrombi (>30 μm) appeared mouse at the same time point. The vessel was occluded at 10 min at the −/− +/+ sooner in Adamts13 mice compared with Adamts13 (P < 0.005). −/− +/+ site of injury in the Adamts13 mouse, whereas the Adamts13 (C) The occlusion time (blood fl ow completely stopped for 10 s) was de- mouse arteriole remained opened at that time. Representative fi gures are +/+ −/− termined. Both Adamts13 and Adamts13 mice occluded at the site shown. Blood fl ow was from left to right. −/− of injury; however, in Adamts13 mice, occlusion time was shorter as +/+ −/− larger thrombi than Adamts13 when perfused over colla- Infusion of r-hu ADAMTS13 into Adamts13 or wild-type −1 gen for 2 min at a shear rate of 1,500 s (44.66 ± 3.63% vs. (C57BL/6J) mice inhibits thrombus growth by destabilizing 20.22 ± 3.88%; P < 0.0005), demonstrating again the key the platelet aggregate role of ADAMTS13 in limiting thrombus growth. In the In vitro, r-hu ADAMTS13 cleaves human VWF (18) and presence of the blocking antibody to αIIbβ3, only single mouse plasma VWF into proteolytic fragments with the same platelets adhered to the collagen surface and thrombus for- effi ciency (unpublished data). It has been demonstrated that +/+ mation was completely inhibited in both the Adamts13 r-hu ADAMTS13 corrects the VWF cleavage defect in he- −/− and Adamts13 blood (3.01 ± 0.97% vs. 2.82 ± 0.39%; reditary TTP plasma (33). Because we observed accelerated −/− P > 0.05). growth of thrombi in Adamts13 mice, we hypothesized In addition, we tested whether infusion of ADAMTS13 that ADAMTS13 negatively modulates thrombus growth and, inhibitory antibody into β3 integrin-defi cient mice (32) therefore, infusion of r–hu ADAMTS13 could delay throm- would induce thrombus formation after FeCl injury. We bus formation. We infused r-hu ADAMTS13 into mice and −/− could not detect any thrombi in injured arterioles of β3 determined that the concentration of the circulating human mice (three animals were evaluated) despite the pres- protein was 8.8 U/ml at 17 min after infusion and 1.1 U/ml ence of the anti-ADAMTS13 antibody (unpublished data). at 53 min after infusion. These times correspond approxi- Collectively, these results indicate that, at the arterial shear mately to the onset of FeCl injury and the termination of the rates, UL-VWF enhances thrombus growth in an αIIbβ3- experiment. We examined fi rst whether the prothrombotic −/− dependent manner. phenotype of Adamts13 mice could be reversed. In 5 out JEM VOL. 203, March 20, 2006 771 with half of the arterioles not occluding by 40 min, whereas all arterioles of wild-type mice infused with vehicle occluded by 15 min (Fig. 7 B, P < 0.008). Thus, ADAMTS13 appears to have a signifi cant antithrombotic potential even in wild- type animals. D I S C U S S I O N The studies presented here have defi ned a key role for ADAMTS13 in preventing thrombi formation in activated microvenules and excessive thrombus formation in the injured arterioles of mice. Our in vivo fi ndings of microvascular thrombosis caused by stimulated release of VWF are consistent with the observation that patients suff ering from TTP have thrombi rich in platelet aggregates and VWF (27). It was sug- gested that, in the development of TTP, microvascular endo- thelial activation could be the primary event initiating platelet aggregation in the arterioles and capillaries (2). Various agents, including viruses, bacterial shiga toxins, drugs such as ticlopi- dine and clopidogrel, antibodies, and immune complexes, can trigger vascular activation (34), perhaps inducing Weibel- Palade body release. We did not see thrombi in the arterioles (which have higher shear stress) treated identically with A23187. This is because either Weibel-Palade bodies were not released in these vessels or, more likely, VWF is washed too quickly from the endothelial surface to promote platelet adhe- sion. Venous thrombosis is not generally recognized as a patho- logic characteristic of TTP in human patients and was also not a prominent feature of spontaneous or shigatoxin-induced −/− Figure 6. Inhibition of integrin 𝛂 IIb𝛃 3 blocks thrombus formation TTP in the Adamts13 mouse (26). These observations sug- −/− of ADAMTS13 platelets on collagen under arterial shear rate gest that formation of platelet-rich microthrombi in the ve- +/+ −/− conditions. Adamts13 or Adamts13 whole blood was perfused for nous circulation in the setting of acute TTP is either subclinical −1 2 min over a collagen surface at a shear rate of 1, 500 s . (A) Representa- or transient, or counterbalanced by other regulatory processes tive images are shown. (top) Untreated whole blood; (bottom) whole blood that are not as eff ective in the arteriolar vasculature. pretreated with blocking antibody against αIIbβ3 (JON/A). (B) Quantifi ca- Autoantibodies neutralizing human ADAMTS13 are tion of the surface area covered by platelets after 2 min of perfusion. Four the major cause of acquired TTP. Various epitopes of the frames from different areas of the fl ow chamber were analyzed for each ADAMTS13 protein are recognized by the autoantibodies blood sample. Data represent the mean percentage of surface area cov- ered by fl uorescent platelets ± SEM (n = 3–4). (35, 36). Infusion of anti-ADAMTS13 antibody in the +/+ Adamts13 mice resulted in prolonged adhesion of platelets to secreted VWF and platelet string formation on the stimu- −/− of 13 Adamts13 mice infused with r-hu ADAMTS13, in- lated endothelium (Fig. 2) that was similar to that seen in the −/− jured arterioles did not occlude for up to 40 min when the ex- Adamts13 mice (26). It was shown that P-selectin may periment was terminated (Fig. 7 A).[ID]FIG7[/ID] The eff ect of the infused anchor the newly released UL-VWF multimers in vitro (37); r-hu ADAMTS13 was more than that of endogenous AD- however, this remains to be confi rmed in vivo. Platelet strings +/+ −/− AMTS13 in Adamts13 mice; as in this injury model, all Ad- and aggregates were frequently seen in the Adamts13 mice +/+ amts13 vessels occluded at <24 min (Fig. 5 C). The mean when challenged with Weibel-Palade body secretagogues occlusion time was signifi cantly prolonged in comparison (unpublished data) such as histamine (38), the infl ammatory with the control mice infused with buff er (P < 0.0005). cytokine TNF-α (39), or activated platelets (40). This sug- In all the mice whose arterioles did not occlude, thrombi gests that in patients lacking functional ADAMTS13, TTP formed but were unstable and dissolved (Fig. 7 C). This phe- could be precipitated by infl ammation, by allergic responses, nomenon of thrombi formation and destabilization was pres- or by situations leading to platelet activation. Infusion of anti- +/+ ent during the entire period of observation. ADAMTS13 antibody into Adamts13 mice with activated To examine whether r-hu ADAMTS13 could delay oc- microvenules resulted in platelet aggregation and thrombi clusion in injured arterioles of mice with normal levels of the formation (Fig. 3). However, these thrombi embolized rap- −/− endogenous ADAMTS13 protein, we infused the recom- idly, similar to those in the Adamts13 mice. Thus, the binant protein in C57BL/6J wild-type mice before injury. mouse infused with anti-ADAMTS13 antibody represents a The infused protein caused signifi cant delay in occlusion time new animal model for acquired TTP. 772 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 7. Infusion of r-hu ADAMTS13 inhibits thrombus growth. (B) Occlusion time in injured arterioles of WT (C57BL/6J) mice infused −/− r-hu ADAMTS13 was infused i.v. into the Adamts13 mice 15 min either with r-hu ADAMTS13 (mean occlusion time = 27.99 ± 4.72 min) before the FeCl injury. The occlusion time (blood fl ow completely or buffer (mean occlusion time = 13.12 ± 0.55 min) alone. (C) Repre- −/− −/− stopped for 10 s) was determined. (A) 5 out of 13 Adamts13 mice sentative fl uorescent images of an injured arteriole of an Adamts13 infused with r-hu ADAMTS13 did not occlude in the arteriole at up to mouse treated with r-hu ADAMTS13 are shown. Arrows indicate a 40 min of observation time (mean occlusion time = 23.80 ± 3.71 min), dissolving thrombus. See Video 2 (available at http://www.jem.org/cgi/ −/− whereas all 10 Adamts13 mice infused with recombinant buffer content/full/jem.20051732/DC1) for the effect of r-hu ADAMTS13 on occluded (mean occlusion time = 11.17 ± 0.87 min, P = 0.005). thrombus growth. Our observations that endothelial activation of micro- and wild-type mice before injury. The antithrombotic eff ect −/− venules results in thrombi in the Adamts13 mice led to of the r-hu ADAMTS13, although highly statistically signifi - the hypothesis that ADAMTS13 defi ciency might accelerate cant, varied among the animals (Fig. 7). Some mice did not thrombus formation in injured arterioles. Indeed, the absence respond to r-hu ADAMTS13 treatment. It is possible that in of ADAMTS13 promoted all aspects of thrombus growth. these mice r-hu ADAMTS13 was proteolytically inactivated Unexpectedly, even more platelets were deposited on the de- by thrombin and plasmin (44) produced at the sites of vascular −/− nuded vessel wall after 2–3 min of injury in the Adamts13 injury. IL-6 (39) and high amounts of VWF released after +/+ mice as compared with Adamts13 (Fig. 5 A). Because infl ammation (45) or injury could also reduce ADAMTS13 early platelet deposition in arterioles is VWF dependent (30), activity. Infusion of the r-hu ADAMTS13 protein into the −/− it means that either plasma ADAMTS13 reduces VWF in- histamine-challenged Adamts13 mice inhibited platelet corporation into the basement membrane when it is exposed string and aggregate formation in the activated venules. In to blood or that it digests VWF already present in the extra- vivo, similar to in vitro (46), ADAMTS13 appears to inter- cellular matrix. The rapid thrombus growth and occlusion in act with endothelial UL-VWF. Collectively, our fi ndings −/− Adamts13 mice indicates that ADAMTS13 might cleave suggest that ADAMTS13 could have both antithrombotic VWF multimers incorporated in the thrombus. It has been as well as thrombo- destabilizing activity. In the thrombus, suggested that cleavage of VWF domain A2 by ADAMTS13 ADAMTS13 could be cleaving the UL-VWF multimers is facilitated by the binding of VWF to GPIbα (41). Thus, released from platelets into less adhesive smaller fragments the VWF–GPIb interaction within the thrombus may nega- and/or directly cleaving the VWF molecules bridging the tively regulate thrombus growth. Thrombus formation under platelets. However, we also cannot exclude the possibility venous and arterial fl ow conditions also depends on major that there may be another substrate for ADAMTS13 that is integrin αIIbβ3 (42, 43). Our studies at arteriolar shear rates important in thrombus formation. show that ADAMTS13 modulates the growing thrombus In summary, our results suggest that in vivo ADAMTS13 only when platelets in the thrombus express an active β3 inte- is active at both low venous and high arterial shear stress con- grin. Under our in vitro and in vivo experimental conditions, ditions. It cleaves platelet strings and regulates platelet inter- ADAMTS13 defi ciency did not promote thrombus growth if action with the “activated” vessel wall in the venules, prevents the major platelet integrin was absent or inhibited (Fig. 6). thrombi in activated microvenules, and modulates the throm- −/− To inhibit the fast thrombus growth seen in the Adamts13 botic response in injured arterioles. The antithrombotic eff ect −/− mice, we infused r-hu ADAMTS13 into the Adamts13 of ADAMTS13 suggests that in addition to TTP, recombinant JEM VOL. 203, March 20, 2006 773 ADAMTS13 could also be used to treat patients suff ering was dissolved in PBS. 200 μl of 1 mM histamine (Sigma-Aldrich) was in- jected i.p. to stimulate the endothelium. 100 μl (0.2 mg/ml) of Rhodamine from thrombotic disorders as a result of other hereditary de- 6G (Sigma-Aldrich) was injected i.v. to label the endogenous platelets and fects, infl ammatory disease, or septic conditions. leukocytes before surgery and imaging. MATERIALS AND METHODS Thrombus in arterioles. A previously described model was used with Animals. Mice used in this study were siblings obtained from crosses of slight modifi cations (30). In brief, mice were anesthetized with 2.5% tribro- +/− Adamts13 mice on C57BL/6J/129Sv background (26). The mice of pure moethanol (0.15 ml/10 g) and fl uorescent platelets (1.25 × 10 platelets/kg) C57BL/6J background were purchased from The Jackson Laboratory and were infused through the retro-orbital plexus of the eye. An incision was −/− β3 integrin mice (32) on BALB/c background were a gift from R. Hynes made through the abdominal wall to expose the mesentery, and arterioles of (Massachusetts Institute of Technology, Cambridge, MA). The mice used 100 μm diameter were studied. The shear rate was calculated as described for intravital microscopy were young mice (4 wk old), both male and previously (48). Arterioles were visualized using the aforementioned micro- female, weighing 14–18 g. Infused platelets were isolated from 4–6-mo-old scope, equipped with a 100-W HBO fl uorescent lamp source (Optic Quip). mice of the same genotype. Animals were bred and housed at the CBR Whatman paper saturated with FeCl (10%) solution was applied topically Institute for Biomedical Research and all experimental procedures were ap- for 5 min, which induced denudation of the endothelium, and the vessel was proved by its Animal Care and Use Committee. monitored for 40 min after injury or until occlusion. One arteriole was cho- sen per mouse. Blood sampling and platelet preparation. Blood was harvested from the retro-orbital venous plexus by puncture and collected in 1.5-ml polypropyl- Quantitative analysis of arteriolar thrombus. Analysis of the recorded ene tubes containing 300 μl of heparin (30 U/ml). Platelet-rich plasma was tape was performed blinded to the genotype. We evaluated (1) single plate- obtained by centrifugation at 1,200 revolutions/min for 5 min. The plasma let–vessel wall interaction determined as the number of fl uorescent platelets and buff y coat containing some RBCs were gently transferred to fresh poly- that deposited on the 250 μm vessel wall segment during 1 min (2–3 min af- propylene tubes and recentrifuged at 1,200 revolutions/min for 5 min. The ter injury). Quantitative analysis was performed using the following factors: platelet-rich plasma was transferred to fresh tubes containing 2 μl of PGI platelet counts >100 were counted as 100 for statistics, (2) the time required (2 μg/ml) and incubated at 37°C for 5 min. After centrifugation at 2,800 for formation of a thrombus >30 μm, (3) thrombus stability by determining revolutions/min, pellets were resuspended in 1 ml of modifi ed Tyrode- the number of thrombi of diameter >30 μm embolizing before vessel occlu- Hepes buff er (137 mM NaCl, 0.3 mM Na HPO , 2 mM KCl, 12 mM 2 4 sion, (4) occlusion time of the vessel, that is, time required for blood to stop NaHCO , 5 mM Hepes, 5 mM glucose, 0.35% BSA) containing 2 μl of fl owing for 10 s, and (5) site of vessel occlusion, that is, at the site of injury PGI and incubated at 37°C for 5 min. The suspended pellet was centrifuged or downstream. at 2,800 revolutions/min for 5 min. To remove PGI , the washing step was repeated twice and platelets were fl uorescently labeled with calcein AM 2.5 μg/ml (Invitrogen) for 10 min at room temperature. r-hu ADAMTS13 infusion. r-hu ADAMTS13 protein was dissolved in 150 mmol NaCl/20 mmol histidin/2% sucrose/0.05% Crillet 4HP, Tween 80, Polyclonal anti-ADAMTS13 production and purifi cation. Polyclonal pH 7.4 (Baxter Bioscience). r-hu ADAMTS13 (3,460 U/kg mouse) was injected rabbit anti–human ADAMTS13 IgG was produced by Baxter Bioscience. i.v. Levels of human ADAMTS13 antigen were determined by a slight modifi ca- The antibody was obtained by immunization of New Zealand white rabbits tion of the ELISA method described by Rieger et al. (49) and r-hu ADAMTS13 with purifi ed r-hu ADAMTS13, COOH-terminally tagged with six His activity was determined according to Gerristen et al. (50). 1 U corresponds to the residues. Two rabbits were immunized by injection of 20 μg of r-hu level of ADAMTS13 activity in pooled normal human plasma. ADAMTS13 (6-His) in 200 μl of complete Freund’s adjuvant. The animals were boostered after 2, 4, and 6 wk by injecting 20 μg of r-hu ADAMTS-13 Flow chamber studies. Flow chamber studies were performed as described (6-His) in 200 μl of incomplete Freund’s adjuvant. After 8 wk, the rabbits previously (51). In brief, platelets were isolated from heparinized whole blood, were killed and bled. IgG antibodies were purifi ed by protein G affi nity washed in modifi ed Tyrode-Hepes buff er, and labeled with 2.5 μg/ml calcein. chromatography (HiTrap protein G HP column; GE Healthcare) and for- Platelet-poor whole blood was reconstituted with labeled platelets before mulated in PBS. perfusion in a parallel-plate fl ow chamber system coated with 100 μg/ml col- lagen Horm (NYCOMED) for 1 h at room temperature. Where indicated, Thrombosis in microvenules. Intravital microscopy was as performed as samples were pretreated with 30 μg/ml JON/A (emfret Analytics) (31) for described previously (47). In brief, mice were anesthetized with 2.5% tribro- 10 min before perfusion. Platelet adhesion was visualized with an Axiovert 135 moethanol (0.15 ml/10 g) and an incision was made through the abdominal inverted microscope (Carl Zeiss MicroImaging, Inc.). The percentage of sur- wall to expose the mesentery and a mesenteric venule of 25–30-μm diame- face area covered by fl uorescent platelets was analyzed using National Insti- ter was studied. Exposed mesentery was kept moist by periodic superfusion tutes of Health Image 1.61 software by an individual blinded to genotypes. 2+ 2+ using PBS (without Ca or Mg ) warmed to 37°C. The shear rate was cal- culated using an optical Doppler velocity meter (48). Venules were visual- Statistical analysis. Results are reported as the mean ± SEM. The statisti- ized using an Axiovert 135 inverted microscope (objectives: 10× and 32×; cal signifi cance of the diff erence between means was assessed by the Student’s Carl Zeiss MicroImaging, Inc.) connected to an SVHS video recorder t test. (AG-6730; Panasonic). One venule was chosen per mouse and fi lmed for 3 min for the baseline before the A23187 superfusion (30 μl of a 10 μmol/L Online supplemental material. Video 1 shows stimulated release of −/− solution) and monitored for 10 min. Weibel-Palade bodies in a microvenule of an ADAMTS13 mouse leads to rapid formation of thrombi that embolize downstream. Video 2 depicts −/− Platelet adhesion in large venules. Intravital microscopy was performed arteriolar injury in an ADAMTS13 mouse that results in rapid vessel oc- as described previously (28), except mesenteric venules of 200–300-μm di- clusion and infusion of r-hu ADAMTS13 inhibits thrombus growth. Online ameters were studied. Fluorescent platelets (1.25 × 10 platelets/kg) were in- supplemental material is available at http://www.jem.org/cgi/content/ fused through the tail vein. One venule per animal was fi lmed for 3 min for full/jem.20051732/DC1. the baseline before the A23187 superfusion (30 μl of a 10 μmol/L solution) and fi lming continued until after the platelet sticking and rolling returned We thank L. Cowan for help in preparing the manuscript. to baseline. Purifi ed rabbit polyclonal anti–human ADAMTS13 antibody This work was supported by the National Institutes of Health, National Heart, (5 mg/kg mouse) was dissolved in PBS. Control rabbit IgG (Sigma-Aldrich) Lung, and Blood Institute grant nos. R37 HL41002 (to D.D. Wagner) and R01 774 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE HL39693 and P01 HL057346 (to D. Ginsburg). D. Ginsburg is a Howard Hughes 18. Plaimauer, B., K. Zimmermann, D. Volkel, G. Antoine, R. Kerschbaumer, Medical Institute Investigator. P. Jenab, M. 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Systemic antithrombotic effects of ADAMTS13

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ARTICLE <doi>10.1084/jem.20051732</doi><aid>20051732</aid>Systemic antithrombotic eff ects of ADAMTS13 1,2 3 1 Anil K. Chauhan, David G. Motto, Colin B. Lamb, 1,2 5 5 Wolfgang Bergmeier, Michael Dockal, Barbara Plaimauer, 5 4 1,2 Friedrich Scheifl inger, David Ginsburg, and Denisa D. Wagner 1 2 CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115 3 4 Department of Pediatrics, University of Michigan, and Department of Internal Medicine, University of Michigan and Howard Hughes Medical Institute, Ann Arbor, MI 48109 Baxter Bioscience, Vienna, A-1220 Austria The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 defi ciency is linked to a life- threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet- rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in −/− activated microvenules of Adamts13 mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi for- mation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in 𝛂 IIb𝛃 3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our fi ndings reveal that ADAMTS13 has a power- ful natural antithrombotic activity and recombinant ADAMTS13 could be used as an anti- thrombotic agent. Thrombotic thrombocytopenic purpura (TTP) milial TTP (10). Acquired TTP, often caused CORRESPONDENCE Denisa D. Wagner: is a disorder characterized by thrombotic mi- by autoantibodies inhibiting ADAMTS13 ac- [email protected] croangiopathy, thrombocytopenia, and micro- tivity, is a more common disorder that occurs vascular thrombosis that can cause various in adults and older children and can recur at Abbreviations used: ADAMTS, a disintegrin-like and metallo- degrees of tissue ischemia and infarction. Clini- regular intervals in 11–36% of patients (4, 6). protease with thrombospondin cally, TTP patients are diagnosed by signs and Nonneutralizing autoantibodies have been as- type I repeats; r-hu, recombi- symptoms such as thrombocytopenia, micro- sociated with acute acquired TTP (15). In most nant human; TTP, thrombotic angiopathic hemolytic anemia, neurological abnor- patients with familial or acquired TTP, plasma thrombocytopenic purpura; UL-VWF, ultra-large VWF; malities, renal failure, and fever (1, 2). In 1982, ADAMTS13 activity is absent or <5% of normal. VWF, von Willebrand factor. Moake et al. found ultra-large von Willebrand Without treatment, the mortality rate exceeds factor (UL-VWF) multimers in the plasma 90%, but plasma exchange therapy has reduced of patients with chronic relapsing TTP (3). mortality to 20% (2). Most patients suff ering from TTP are defi cient VWF synthesized in megakaryocytes and in a plasma metalloprotease that cleaves UL- endothelial cells is stored in platelet α-granules VWF (4–9). The protease belongs to the AD- and Weibel-Palade bodies, respectively, as UL- AMTS (a disintegrin-like and metalloprotease VWF (16). Once secreted from endothelial with thrombospondin type I repeats) family cells, these UL-VWF multimers are cleaved by and is designated as ADAMTS13, a 190-kD ADAMTS13 in the circulation into a series glycosylated protein produced predominantly of smaller multimers at specifi c cleavage sites by the liver (10–12), specifi cally by hepatic within the VWF molecule (17–19). The prote- stellate cells (13, 14). Mutations in the AD- ase cleaves at the Tyr842–Met843 bond in the AMTS13 gene have been shown to cause fa- central A2 domain of the mature VWF subunit (20) and requires zinc and calcium for activity. The online version of this article contains supplemental material. VWF exists in “ball of yarn” and fi lamentous JEM © The Rockefeller University Press $8.00 767 Vol. 203, No. 3, March 20, 2006 767–776 www.jem.org/cgi/doi/10.1084/jem.20051732 The Journal of Experimental Medicine Table I. Hemodynamic parameters were established before application of A23187 (Fig. 1) on venules and FeCl on arterioles (Fig. 5) −1 Genotype Vessel type Diameter (μm) Centerline velocity (mm/s) Shear rate (s ) +/+ Adamts13 (n = 5) Venule 31.51 ± 1.79 1.43 ± 0.07 213 ± 14.10 −/− Adamts13 (n = 5) Venule 26.36 ± 1.95 1.28 ± 0.13 244 ± 28.29 +/+ Adamts13 (n = 12) Arteriole 103.91 ± 9.29 33.33 ± 1.72 1,688.16 ± 143.32 −/− Adamts13 (n = 12) Arteriole 93.26 ± 10.48 28.05 ± 2.10 1,646.83 ± 157.16 forms as seen by electron microscopy (21). Furthermore, ADAMTS13 could be used to treat TTP and possibly other atomic force microscopy confi rms that VWF exists in a glob- thrombotic conditions. ular conformation under static conditions and may unfold to a fi lamentous state after exposure to shear stress (22). This RESULTS could occur also in vivo when one end of the VWF fi lament Endothelial activation results in thrombi formation −/− is anchored to a surface. UL-VWF multimers have high bio- in microvenules of Adamts13 mice logical activity. They bind better to the extracellular matrix We have previously observed that platelet sticking/trans- than regular multimers (23) and form higher strength bonds location in venules of 200–250 μm in diameter activated with platelet GPIb-IX than plasma VWF (24). It was demon- with calcium ionophore A23187 (a secretagogue of Weibel- −1 strated in vitro that platelets align as beads on the released Palade bodies) at low shear rate (100 s ) was prolonged in −/− +/+ UL-VWF string on the endothelial surface. These strings are Adamts13 mice compared with Adamts13 mice (26). We then cleaved by ADAMTS13 and released from the stimu- investigated whether activation of microvenule endothelium lated endothelial cells (25). We have demonstrated in vivo by A23187 (which does not denude the endothelium; −/− that it is only in Adamts13 mice that strings of platelets re-<CIT>reference 28)</CIT> results in platelet aggregation and subsequent −1 main intact after endothelial activation in veins (26). These thrombus formation. The shear rate (200–250 s ) and diam- strings attach at one end to endothelium and “wave” the eter of all the microvenules (25–30 μm) studied were similar −/− +/+ other end in the blood stream. for Adamts13 and Adamts13 mice (Table I).[ID]TBL1[/ID] In the mi- −/− Thrombi of TTP patients consist of a little fi brin but crovenules of Adamts13 mice, platelet aggregation result- mainly of VWF and platelets, suggesting VWF-mediated ing in thrombus formation was observed from 45 s to 2 min platelet aggregation as a cause of thrombosis (27). We hy- after topical superfusion of A23187 (Fig. 1).[ID]FIG1[/ID] The thrombi pothesized that endothelial activation resulting in elevation of were often unstable and fl ushed away, leading to frequent hyperactive UL-VWF multimers in plasma could be associ- embolization and causing transient downstream occlusion ated with an increased risk of thrombosis in ADAMTS13- usually only lasting 3–4 s. Thus, stimulation of Weibel- defi cient animals. We investigated thrombosis in venules and Palade body secretion can lead to spontaneous thrombus for- −/− −/− arterioles of Adamts13 mice by intravital microscopy. mation in Adamts13 mice in the absence of vascular injury. +/+ Our fi ndings strongly suggest that ADAMTS13 has natural In Adamts13 mice treated identically, platelet strings and antithrombotic activity and that recombinant human (r-hu) very small platelet aggregates could be seen attached to the −/− +/+ Figure 1. Thrombus formation in stimulated microvenules of Adamts13 mice (n = 5). No microthrombi formed in Adamts13 −/− Adamts13 mice. Venules measuring 25–30-μm in diameter were mice treated identically (n = 5). Arrows indicate the microthrombi. See visualized in the mesentery of live mice. 1 min after topical superfusion of Video 1 (available at http://www.jem.org/cgi/content/full/jem.20051732/ −/− calcium ionophore A23187, thrombus formation was observed in DC1) for thrombi in the microvenules of Adamts13 mice. 768 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 2. Antibody to ADAMTS13 increases platelet adhesion and ADAMTS13 Ab, n = 4), more platelets adhered to the vessel wall 4 min +/+ string formation on activated vessel wall. Fluorescently labeled plate- after stimulation compared with Adamts13 control (n = 4). Arrows lets representing 2.5% of total platelets were observed in mesenteric indicate the ≥20-μm strings of platelets attached at one end to the endo- venules (diameter: 200–250 μm) of live mice before (baseline) and after thelium and waving the other end in the blood stream. Inset time points A23187 superfusion. Platelets began to adhere to the endothelium 30–45 s in the lower right corner refer to the time after superfusion of A23187. +/+ after superfusion. In Adamts13 mice (infused with anti–human The bar shown in the middle panel is 50 μm. endothelium for 1–2 s, but thrombi did not form. These ob- before surgical preparation for intravital microscopy. The anti- servations demonstrate that ADAMTS13 is active at low body did not activate the endothelium as normal baseline +/+ shear and, thus, inhibits platelet aggregation and prevents platelet adhesion was found in Adamts13 mice after its thrombus formation in the microvenules. In addition, arteri- infusion (Fig. 2).[ID]FIG2[/ID] After topical superfusion of A23187, many oles (high shear) running parallel to the venules in either platelets stuck/translocated on the endothelium, reaching a −/− +/+ Adamts13 or Adamts13 mice did not show any platelet peak of platelet adhesion from 45 s to 1 min that progres- strings, platelet aggregation, or thrombus formation. sively decreased with time. However, more platelet sticking was observed 4 min after the A23187 application in the +/+ An antibody to ADAMTS13 prolongs adhesion of platelets antibody-infused Adamts13 as compared with control +/+ +/+ to secreted VWF on the vessel wall of Adamts13 mice Adamts13 mice (control IgG [n = 2] or PBS [n = 5]) (Fig. 2). Previous studies have shown that most patients suff ering from The phenomenon observed was similar to that observed −/− the acquired form of TTP have autoimmune inhibitors to in Adamts13 mice (26). Strings of platelets were seen ADAMTS13 in plasma (4, 6). We infused a polyclonal anti– varying from 20 to 40 μm and attached at one end to the en- +/+ human ADAMTS13 antibody in Adamts13 mice 2 h dothelium and waving in the blood stream. These strings +/+ +/+ Figure 3. Thrombus formation in microvenules of Adamts13 Adamts13 mice infused with the anti-ADAMTS13 Ab. The microthrombi −/− mice infused with an anti-ADAMTS13 antibody. Mesenteric venules formation and embolization were similar to that seen in Adamts13 of 25–30-μm in diameter were observed. 1 min after topical superfu- mice (Fig. 1). Arrows indicate a microthrombus. Microthrombi did not +/+ sion with A23187, thrombus formation was observed in four out of six form in Adamts13 control (n = 5). JEM VOL. 203, March 20, 2006 769 strings persisted for up to 5 min. Some strings appeared to coalesce, forming aggregates (Fig. 4 C) that were later re- leased into the blood stream. Infusion of r-hu ADAMTS13 −/− protein in the Adamts13 mice (n = 4; 3 venules per mouse) inhibited platelet string formation in all venules examined (Fig. 4 D), thus demonstrating the activity of ADAMTS13 at low shear. Platelet binding to subendothelium is increased −/− in Adamts13 mice Ferric chloride (FeCl ) injury leads to deendothelization and exposes subendothelium (30). Platelet subendothelial interac- tions after injury at arterial shear are initiated by GPIb–VWF interaction and propagated by other receptors (30). In both +/+ −/− Adamts13 and Adamts13 mice, platelet–vessel wall Figure 4. Recombinant ADAMTS13 inhibits platelet strings in interaction started rapidly after FeCl application to the −/− Adamts13 mice. Rhodamine 6G was used to label endogenous plate- arteriole. The number of animals in which >100 fl uorescent lets and leukocytes. Histamine was administered i.p. 15 min before sur- platelets were deposited 2–3 min after injury was higher in gery and three mesenteric venules of 200–300 μm in diameter were −/− −/− Adamts13 mice. In the Adamts13 , 7 out of 12 mice +/+ visualized per mouse. (A) No platelet strings are seen in Adamts13 showed >100 platelets deposited on the vessel wall com- mice (n = 5). (B) Platelet strings (indicated by arrows) are seen in the +/+ pared with 3 out of 10 in the Adamts13 mice (P < 0.05, −/− Adamts13 mice. Platelet strings anchor up to 1 min on the endothe- Fig. 5 A).[ID]FIG5[/ID] lium (n = 5). (C) The platelet strings could form platelet aggregates in −/− Adamts13 mice as indicated by arrow. (D) Infusion of r-hu ADAMTS13 −/− protein inhibits the platelet strings in Adamts13 mice (n = 4). Thrombus formation is accelerated in injured arterioles −/− of Adamts13 mice After fi nding that ADAMTS13 negatively modulates resting were either not seen or were very short lived (<2 s) in the platelet adhesion to both stimulated endothelium and sub- +/+ Adamts13 mice. endothelium, we asked whether the enzyme aff ects arte- riolar thrombus formation. This process requires platelet ADAMTS13 inhibitor induces thrombi formation activation and employs several ligands aside from VWF (30). +/+ in microvenules of Adamts13 mice The shear rate and diameter of arterioles studied were simi- +/+ −/− +/+ In the Adamts13 mice infused with anti–human ADAMTS13 lar for Adamts13 and Adamts13 mice (Table I). In the −/− antibody 2 h before surgical preparation, microthrombi formed Adamts13 mice, thrombi grew faster as thrombi >30 μm on the vessel wall 45 s to 1 min after topical superfusion of were seen at 6.64 ± 0.93 min compared with 10.78 ± 0.80 +/+ A23187 in four out of six mice (Fig. 3).[ID]FIG3[/ID] The microthrombi min in the Adamts13 mice (P < 0.005, Fig. 5 B). This −/− appearance was similar to those seen in the Adamts13 mice suggests that cleavage of VWF multimers by ADAMTS13 +/+ (Fig. 1). In control Adamts13 mice, short-lived platelet delays thrombus formation. The thrombi grew to occlusive −/− strings could be seen attached to the endothelium, but they did size in 10.56 ± 0.72 min in Adamts13 mice, whereas in +/+ not result in thrombus formation (n = 5). Adamts13 mice all the vessels were still open at this time +/+ (Fig. 5, C and D). In the Adamts13 , the mean vessel oc- Histamine promotes platelet string formation clusion time was 16.69 ± 1.25 min after injury (P < 0.0005). −/− in the venules of Adamts13 mice, a process All the vessels occluded at the site of injury. Of note, in ar- −/− inhibited by recombinant ADAMTS13 terioles of Adamts13 mice, the mean time for formation Histamine produced during infl ammation is a secretagogue of thrombi (>30 μm) as well as the mean occlusion time +/+ of Weibel-Palade bodies and stimulates the endothelium (29). were less than that of any individual Adamts13 mouse We investigated whether activation of venules by injecting (Fig. 5, B and C). −/− histamine i.p. into Adamts13 mice could result in platelet strings. Endogenous platelets were labeled by infusing Rho- ADAMTS13 defi ciency enhances thrombus growth damine 6G i.v. before surgery. Histamine was injected i.p. 15 in an 𝛂 IIb𝛃 3 integrin-dependent manner −/− min before the surgical preparation into Adamts13 (n = 5) To study the importance of integrin αIIbβ3 for thrombus +/+ and Adamts13 (n = 5) mice and venules at a shear rate formation in the absence of ADAMTS13, we performed in −1 +/+ of 100 s were visualized. In the Adamts13 mice, vitro fl ow chamber studies with whole blood in the presence strings of platelets were not seen or were short lived (<5 s; or absence of a blocking antibody (JON/A) against αIIbβ3 −/− Fig. 4 A), whereas, in the Adamts13 platelet strings, vary- (31) (Fig. 6).[ID]FIG6[/ID] To quantify the size of the thrombi, the surface ing from 20 to 100 μm could be seen (Fig. 4 B) anchored to area covered by fl uorescently labeled platelets was deter- −/− the endothelium for 1 min.[ID]FIG4[/ID] In some mice, the platelet mined. As expected, Adamts13 blood formed signifi cantly 770 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 5. Quantitative analysis of platelet adhesion and thrombi +/+ compared with Adamts13 mice (P < 0.0005). (D) Fluorescently labeled +/+ formation in FeCl -injured arterioles of Adamts13 and platelets representing 2.5% of total platelets were observed in mesen- −/− Adamts13 mice. (A) The number of fl uorescent platelets deposited teric arterioles of live mice after FeCl injury. Single adherent platelets are per minute was determined in the interval 2–3 min after injury. Absence +/+ seen in the arteriole at 4 min after injury in the Adamts13 mouse, of ADAMTS13 in the plasma signifi cantly increases early platelet interac- −/− whereas a thrombus (30 μm) can already be seen in the Adamts13 tion with the subendothelium (P < 0.05). (B) Thrombi (>30 μm) appeared mouse at the same time point. The vessel was occluded at 10 min at the −/− +/+ sooner in Adamts13 mice compared with Adamts13 (P < 0.005). −/− +/+ site of injury in the Adamts13 mouse, whereas the Adamts13 (C) The occlusion time (blood fl ow completely stopped for 10 s) was de- mouse arteriole remained opened at that time. Representative fi gures are +/+ −/− termined. Both Adamts13 and Adamts13 mice occluded at the site shown. Blood fl ow was from left to right. −/− of injury; however, in Adamts13 mice, occlusion time was shorter as +/+ −/− larger thrombi than Adamts13 when perfused over colla- Infusion of r-hu ADAMTS13 into Adamts13 or wild-type −1 gen for 2 min at a shear rate of 1,500 s (44.66 ± 3.63% vs. (C57BL/6J) mice inhibits thrombus growth by destabilizing 20.22 ± 3.88%; P < 0.0005), demonstrating again the key the platelet aggregate role of ADAMTS13 in limiting thrombus growth. In the In vitro, r-hu ADAMTS13 cleaves human VWF (18) and presence of the blocking antibody to αIIbβ3, only single mouse plasma VWF into proteolytic fragments with the same platelets adhered to the collagen surface and thrombus for- effi ciency (unpublished data). It has been demonstrated that +/+ mation was completely inhibited in both the Adamts13 r-hu ADAMTS13 corrects the VWF cleavage defect in he- −/− and Adamts13 blood (3.01 ± 0.97% vs. 2.82 ± 0.39%; reditary TTP plasma (33). Because we observed accelerated −/− P > 0.05). growth of thrombi in Adamts13 mice, we hypothesized In addition, we tested whether infusion of ADAMTS13 that ADAMTS13 negatively modulates thrombus growth and, inhibitory antibody into β3 integrin-defi cient mice (32) therefore, infusion of r–hu ADAMTS13 could delay throm- would induce thrombus formation after FeCl injury. We bus formation. We infused r-hu ADAMTS13 into mice and −/− could not detect any thrombi in injured arterioles of β3 determined that the concentration of the circulating human mice (three animals were evaluated) despite the pres- protein was 8.8 U/ml at 17 min after infusion and 1.1 U/ml ence of the anti-ADAMTS13 antibody (unpublished data). at 53 min after infusion. These times correspond approxi- Collectively, these results indicate that, at the arterial shear mately to the onset of FeCl injury and the termination of the rates, UL-VWF enhances thrombus growth in an αIIbβ3- experiment. We examined fi rst whether the prothrombotic −/− dependent manner. phenotype of Adamts13 mice could be reversed. In 5 out JEM VOL. 203, March 20, 2006 771 with half of the arterioles not occluding by 40 min, whereas all arterioles of wild-type mice infused with vehicle occluded by 15 min (Fig. 7 B, P < 0.008). Thus, ADAMTS13 appears to have a signifi cant antithrombotic potential even in wild- type animals. D I S C U S S I O N The studies presented here have defi ned a key role for ADAMTS13 in preventing thrombi formation in activated microvenules and excessive thrombus formation in the injured arterioles of mice. Our in vivo fi ndings of microvascular thrombosis caused by stimulated release of VWF are consistent with the observation that patients suff ering from TTP have thrombi rich in platelet aggregates and VWF (27). It was sug- gested that, in the development of TTP, microvascular endo- thelial activation could be the primary event initiating platelet aggregation in the arterioles and capillaries (2). Various agents, including viruses, bacterial shiga toxins, drugs such as ticlopi- dine and clopidogrel, antibodies, and immune complexes, can trigger vascular activation (34), perhaps inducing Weibel- Palade body release. We did not see thrombi in the arterioles (which have higher shear stress) treated identically with A23187. This is because either Weibel-Palade bodies were not released in these vessels or, more likely, VWF is washed too quickly from the endothelial surface to promote platelet adhe- sion. Venous thrombosis is not generally recognized as a patho- logic characteristic of TTP in human patients and was also not a prominent feature of spontaneous or shigatoxin-induced −/− Figure 6. Inhibition of integrin 𝛂 IIb𝛃 3 blocks thrombus formation TTP in the Adamts13 mouse (26). These observations sug- −/− of ADAMTS13 platelets on collagen under arterial shear rate gest that formation of platelet-rich microthrombi in the ve- +/+ −/− conditions. Adamts13 or Adamts13 whole blood was perfused for nous circulation in the setting of acute TTP is either subclinical −1 2 min over a collagen surface at a shear rate of 1, 500 s . (A) Representa- or transient, or counterbalanced by other regulatory processes tive images are shown. (top) Untreated whole blood; (bottom) whole blood that are not as eff ective in the arteriolar vasculature. pretreated with blocking antibody against αIIbβ3 (JON/A). (B) Quantifi ca- Autoantibodies neutralizing human ADAMTS13 are tion of the surface area covered by platelets after 2 min of perfusion. Four the major cause of acquired TTP. Various epitopes of the frames from different areas of the fl ow chamber were analyzed for each ADAMTS13 protein are recognized by the autoantibodies blood sample. Data represent the mean percentage of surface area cov- ered by fl uorescent platelets ± SEM (n = 3–4). (35, 36). Infusion of anti-ADAMTS13 antibody in the +/+ Adamts13 mice resulted in prolonged adhesion of platelets to secreted VWF and platelet string formation on the stimu- −/− of 13 Adamts13 mice infused with r-hu ADAMTS13, in- lated endothelium (Fig. 2) that was similar to that seen in the −/− jured arterioles did not occlude for up to 40 min when the ex- Adamts13 mice (26). It was shown that P-selectin may periment was terminated (Fig. 7 A).[ID]FIG7[/ID] The eff ect of the infused anchor the newly released UL-VWF multimers in vitro (37); r-hu ADAMTS13 was more than that of endogenous AD- however, this remains to be confi rmed in vivo. Platelet strings +/+ −/− AMTS13 in Adamts13 mice; as in this injury model, all Ad- and aggregates were frequently seen in the Adamts13 mice +/+ amts13 vessels occluded at <24 min (Fig. 5 C). The mean when challenged with Weibel-Palade body secretagogues occlusion time was signifi cantly prolonged in comparison (unpublished data) such as histamine (38), the infl ammatory with the control mice infused with buff er (P < 0.0005). cytokine TNF-α (39), or activated platelets (40). This sug- In all the mice whose arterioles did not occlude, thrombi gests that in patients lacking functional ADAMTS13, TTP formed but were unstable and dissolved (Fig. 7 C). This phe- could be precipitated by infl ammation, by allergic responses, nomenon of thrombi formation and destabilization was pres- or by situations leading to platelet activation. Infusion of anti- +/+ ent during the entire period of observation. ADAMTS13 antibody into Adamts13 mice with activated To examine whether r-hu ADAMTS13 could delay oc- microvenules resulted in platelet aggregation and thrombi clusion in injured arterioles of mice with normal levels of the formation (Fig. 3). However, these thrombi embolized rap- −/− endogenous ADAMTS13 protein, we infused the recom- idly, similar to those in the Adamts13 mice. Thus, the binant protein in C57BL/6J wild-type mice before injury. mouse infused with anti-ADAMTS13 antibody represents a The infused protein caused signifi cant delay in occlusion time new animal model for acquired TTP. 772 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE Figure 7. Infusion of r-hu ADAMTS13 inhibits thrombus growth. (B) Occlusion time in injured arterioles of WT (C57BL/6J) mice infused −/− r-hu ADAMTS13 was infused i.v. into the Adamts13 mice 15 min either with r-hu ADAMTS13 (mean occlusion time = 27.99 ± 4.72 min) before the FeCl injury. The occlusion time (blood fl ow completely or buffer (mean occlusion time = 13.12 ± 0.55 min) alone. (C) Repre- −/− −/− stopped for 10 s) was determined. (A) 5 out of 13 Adamts13 mice sentative fl uorescent images of an injured arteriole of an Adamts13 infused with r-hu ADAMTS13 did not occlude in the arteriole at up to mouse treated with r-hu ADAMTS13 are shown. Arrows indicate a 40 min of observation time (mean occlusion time = 23.80 ± 3.71 min), dissolving thrombus. See Video 2 (available at http://www.jem.org/cgi/ −/− whereas all 10 Adamts13 mice infused with recombinant buffer content/full/jem.20051732/DC1) for the effect of r-hu ADAMTS13 on occluded (mean occlusion time = 11.17 ± 0.87 min, P = 0.005). thrombus growth. Our observations that endothelial activation of micro- and wild-type mice before injury. The antithrombotic eff ect −/− venules results in thrombi in the Adamts13 mice led to of the r-hu ADAMTS13, although highly statistically signifi - the hypothesis that ADAMTS13 defi ciency might accelerate cant, varied among the animals (Fig. 7). Some mice did not thrombus formation in injured arterioles. Indeed, the absence respond to r-hu ADAMTS13 treatment. It is possible that in of ADAMTS13 promoted all aspects of thrombus growth. these mice r-hu ADAMTS13 was proteolytically inactivated Unexpectedly, even more platelets were deposited on the de- by thrombin and plasmin (44) produced at the sites of vascular −/− nuded vessel wall after 2–3 min of injury in the Adamts13 injury. IL-6 (39) and high amounts of VWF released after +/+ mice as compared with Adamts13 (Fig. 5 A). Because infl ammation (45) or injury could also reduce ADAMTS13 early platelet deposition in arterioles is VWF dependent (30), activity. Infusion of the r-hu ADAMTS13 protein into the −/− it means that either plasma ADAMTS13 reduces VWF in- histamine-challenged Adamts13 mice inhibited platelet corporation into the basement membrane when it is exposed string and aggregate formation in the activated venules. In to blood or that it digests VWF already present in the extra- vivo, similar to in vitro (46), ADAMTS13 appears to inter- cellular matrix. The rapid thrombus growth and occlusion in act with endothelial UL-VWF. Collectively, our fi ndings −/− Adamts13 mice indicates that ADAMTS13 might cleave suggest that ADAMTS13 could have both antithrombotic VWF multimers incorporated in the thrombus. It has been as well as thrombo- destabilizing activity. In the thrombus, suggested that cleavage of VWF domain A2 by ADAMTS13 ADAMTS13 could be cleaving the UL-VWF multimers is facilitated by the binding of VWF to GPIbα (41). Thus, released from platelets into less adhesive smaller fragments the VWF–GPIb interaction within the thrombus may nega- and/or directly cleaving the VWF molecules bridging the tively regulate thrombus growth. Thrombus formation under platelets. However, we also cannot exclude the possibility venous and arterial fl ow conditions also depends on major that there may be another substrate for ADAMTS13 that is integrin αIIbβ3 (42, 43). Our studies at arteriolar shear rates important in thrombus formation. show that ADAMTS13 modulates the growing thrombus In summary, our results suggest that in vivo ADAMTS13 only when platelets in the thrombus express an active β3 inte- is active at both low venous and high arterial shear stress con- grin. Under our in vitro and in vivo experimental conditions, ditions. It cleaves platelet strings and regulates platelet inter- ADAMTS13 defi ciency did not promote thrombus growth if action with the “activated” vessel wall in the venules, prevents the major platelet integrin was absent or inhibited (Fig. 6). thrombi in activated microvenules, and modulates the throm- −/− To inhibit the fast thrombus growth seen in the Adamts13 botic response in injured arterioles. The antithrombotic eff ect −/− mice, we infused r-hu ADAMTS13 into the Adamts13 of ADAMTS13 suggests that in addition to TTP, recombinant JEM VOL. 203, March 20, 2006 773 ADAMTS13 could also be used to treat patients suff ering was dissolved in PBS. 200 μl of 1 mM histamine (Sigma-Aldrich) was in- jected i.p. to stimulate the endothelium. 100 μl (0.2 mg/ml) of Rhodamine from thrombotic disorders as a result of other hereditary de- 6G (Sigma-Aldrich) was injected i.v. to label the endogenous platelets and fects, infl ammatory disease, or septic conditions. leukocytes before surgery and imaging. MATERIALS AND METHODS Thrombus in arterioles. A previously described model was used with Animals. Mice used in this study were siblings obtained from crosses of slight modifi cations (30). In brief, mice were anesthetized with 2.5% tribro- +/− Adamts13 mice on C57BL/6J/129Sv background (26). The mice of pure moethanol (0.15 ml/10 g) and fl uorescent platelets (1.25 × 10 platelets/kg) C57BL/6J background were purchased from The Jackson Laboratory and were infused through the retro-orbital plexus of the eye. An incision was −/− β3 integrin mice (32) on BALB/c background were a gift from R. Hynes made through the abdominal wall to expose the mesentery, and arterioles of (Massachusetts Institute of Technology, Cambridge, MA). The mice used 100 μm diameter were studied. The shear rate was calculated as described for intravital microscopy were young mice (4 wk old), both male and previously (48). Arterioles were visualized using the aforementioned micro- female, weighing 14–18 g. Infused platelets were isolated from 4–6-mo-old scope, equipped with a 100-W HBO fl uorescent lamp source (Optic Quip). mice of the same genotype. Animals were bred and housed at the CBR Whatman paper saturated with FeCl (10%) solution was applied topically Institute for Biomedical Research and all experimental procedures were ap- for 5 min, which induced denudation of the endothelium, and the vessel was proved by its Animal Care and Use Committee. monitored for 40 min after injury or until occlusion. One arteriole was cho- sen per mouse. Blood sampling and platelet preparation. Blood was harvested from the retro-orbital venous plexus by puncture and collected in 1.5-ml polypropyl- Quantitative analysis of arteriolar thrombus. Analysis of the recorded ene tubes containing 300 μl of heparin (30 U/ml). Platelet-rich plasma was tape was performed blinded to the genotype. We evaluated (1) single plate- obtained by centrifugation at 1,200 revolutions/min for 5 min. The plasma let–vessel wall interaction determined as the number of fl uorescent platelets and buff y coat containing some RBCs were gently transferred to fresh poly- that deposited on the 250 μm vessel wall segment during 1 min (2–3 min af- propylene tubes and recentrifuged at 1,200 revolutions/min for 5 min. The ter injury). Quantitative analysis was performed using the following factors: platelet-rich plasma was transferred to fresh tubes containing 2 μl of PGI platelet counts >100 were counted as 100 for statistics, (2) the time required (2 μg/ml) and incubated at 37°C for 5 min. After centrifugation at 2,800 for formation of a thrombus >30 μm, (3) thrombus stability by determining revolutions/min, pellets were resuspended in 1 ml of modifi ed Tyrode- the number of thrombi of diameter >30 μm embolizing before vessel occlu- Hepes buff er (137 mM NaCl, 0.3 mM Na HPO , 2 mM KCl, 12 mM 2 4 sion, (4) occlusion time of the vessel, that is, time required for blood to stop NaHCO , 5 mM Hepes, 5 mM glucose, 0.35% BSA) containing 2 μl of fl owing for 10 s, and (5) site of vessel occlusion, that is, at the site of injury PGI and incubated at 37°C for 5 min. The suspended pellet was centrifuged or downstream. at 2,800 revolutions/min for 5 min. To remove PGI , the washing step was repeated twice and platelets were fl uorescently labeled with calcein AM 2.5 μg/ml (Invitrogen) for 10 min at room temperature. r-hu ADAMTS13 infusion. r-hu ADAMTS13 protein was dissolved in 150 mmol NaCl/20 mmol histidin/2% sucrose/0.05% Crillet 4HP, Tween 80, Polyclonal anti-ADAMTS13 production and purifi cation. Polyclonal pH 7.4 (Baxter Bioscience). r-hu ADAMTS13 (3,460 U/kg mouse) was injected rabbit anti–human ADAMTS13 IgG was produced by Baxter Bioscience. i.v. Levels of human ADAMTS13 antigen were determined by a slight modifi ca- The antibody was obtained by immunization of New Zealand white rabbits tion of the ELISA method described by Rieger et al. (49) and r-hu ADAMTS13 with purifi ed r-hu ADAMTS13, COOH-terminally tagged with six His activity was determined according to Gerristen et al. (50). 1 U corresponds to the residues. Two rabbits were immunized by injection of 20 μg of r-hu level of ADAMTS13 activity in pooled normal human plasma. ADAMTS13 (6-His) in 200 μl of complete Freund’s adjuvant. The animals were boostered after 2, 4, and 6 wk by injecting 20 μg of r-hu ADAMTS-13 Flow chamber studies. Flow chamber studies were performed as described (6-His) in 200 μl of incomplete Freund’s adjuvant. After 8 wk, the rabbits previously (51). In brief, platelets were isolated from heparinized whole blood, were killed and bled. IgG antibodies were purifi ed by protein G affi nity washed in modifi ed Tyrode-Hepes buff er, and labeled with 2.5 μg/ml calcein. chromatography (HiTrap protein G HP column; GE Healthcare) and for- Platelet-poor whole blood was reconstituted with labeled platelets before mulated in PBS. perfusion in a parallel-plate fl ow chamber system coated with 100 μg/ml col- lagen Horm (NYCOMED) for 1 h at room temperature. Where indicated, Thrombosis in microvenules. Intravital microscopy was as performed as samples were pretreated with 30 μg/ml JON/A (emfret Analytics) (31) for described previously (47). In brief, mice were anesthetized with 2.5% tribro- 10 min before perfusion. Platelet adhesion was visualized with an Axiovert 135 moethanol (0.15 ml/10 g) and an incision was made through the abdominal inverted microscope (Carl Zeiss MicroImaging, Inc.). The percentage of sur- wall to expose the mesentery and a mesenteric venule of 25–30-μm diame- face area covered by fl uorescent platelets was analyzed using National Insti- ter was studied. Exposed mesentery was kept moist by periodic superfusion tutes of Health Image 1.61 software by an individual blinded to genotypes. 2+ 2+ using PBS (without Ca or Mg ) warmed to 37°C. The shear rate was cal- culated using an optical Doppler velocity meter (48). Venules were visual- Statistical analysis. Results are reported as the mean ± SEM. The statisti- ized using an Axiovert 135 inverted microscope (objectives: 10× and 32×; cal signifi cance of the diff erence between means was assessed by the Student’s Carl Zeiss MicroImaging, Inc.) connected to an SVHS video recorder t test. (AG-6730; Panasonic). One venule was chosen per mouse and fi lmed for 3 min for the baseline before the A23187 superfusion (30 μl of a 10 μmol/L Online supplemental material. Video 1 shows stimulated release of −/− solution) and monitored for 10 min. Weibel-Palade bodies in a microvenule of an ADAMTS13 mouse leads to rapid formation of thrombi that embolize downstream. Video 2 depicts −/− Platelet adhesion in large venules. Intravital microscopy was performed arteriolar injury in an ADAMTS13 mouse that results in rapid vessel oc- as described previously (28), except mesenteric venules of 200–300-μm di- clusion and infusion of r-hu ADAMTS13 inhibits thrombus growth. Online ameters were studied. Fluorescent platelets (1.25 × 10 platelets/kg) were in- supplemental material is available at http://www.jem.org/cgi/content/ fused through the tail vein. One venule per animal was fi lmed for 3 min for full/jem.20051732/DC1. the baseline before the A23187 superfusion (30 μl of a 10 μmol/L solution) and fi lming continued until after the platelet sticking and rolling returned We thank L. Cowan for help in preparing the manuscript. to baseline. Purifi ed rabbit polyclonal anti–human ADAMTS13 antibody This work was supported by the National Institutes of Health, National Heart, (5 mg/kg mouse) was dissolved in PBS. Control rabbit IgG (Sigma-Aldrich) Lung, and Blood Institute grant nos. R37 HL41002 (to D.D. Wagner) and R01 774 SYSTEMIC ANTITHROMBOTIC EFFECTS OF ADAMTS13 | Chauhan et al. ARTICLE HL39693 and P01 HL057346 (to D. Ginsburg). D. Ginsburg is a Howard Hughes 18. Plaimauer, B., K. Zimmermann, D. Volkel, G. Antoine, R. Kerschbaumer, Medical Institute Investigator. P. Jenab, M. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Mar 20, 2006

References