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 deficiency 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 formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in αIIbβ3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

von Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using genetargeted mice, we show that in ferric chloride-injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF ؊/؊ mice when compared with wild type (WT). We also observed increased embolization in the VWF ؊/؊ mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF ؊/؊ venules did not occlude. Introductionvon Willebrand factor (VWF) is a large adhesive glycoprotein synthesized in megakaryocytes and endothelial cells and stored in platelet ␣-granules and Weibel-Palade bodies, respectively. VWF circulates in plasma as a series of multimers ranging from 500 to 20 000 kDa. The size of the multimers is regulated through proteolysis 1,2 by a specific protease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13). [3][4][5] There are 3 pools of VWF: (1) soluble plasma VWF, (2) subendothelial (ECM) VWF, and (3) cellular VWF in storage granules. 6 VWF is a carrier for factor VIII (FVIII) and protects it from inactivation. 7 The formation of a thrombus on an injured vessel wall is a complex process that involves multiple adhesion molecules (VWF, collagen, fibrinogen, and fibronectin) and their respective receptors on the platelet surface (GPIb␣, GPVI, ␤1 and ␤3 integrins). VWF has 2 main receptors, GPIb␣ in the GPIb-IX-V complex and the integrin ␣IIb␤3. 8 Pathological thrombosis can occur in arteries and veins. Arterial thrombosis is often linked to inappropriate platelet activation and can result in myocardial infarction and ischemic stroke, while venous thrombosis (eg, deep vein thrombosis) is commonly linked to high procoagulant activity, which produces fibrin-rich thrombi. In the clinical setting, elevated levels of FVIII and VWF are associated with an increased risk of venous thrombosis. The Leiden Thrombophilia Study suggested that the effect of elevated VWF on the risk of venous thrombosis was due to increased FVIII levels. 9 In contrast, the Longitudinal Investigation of Thromboembolism Etiology (LITE) Study showed FVIII and VWF were independently associated with venous thromboembolism. 10 Under high shear, the VWF-GPIb␣ interaction is necessary for initial platelet contact with the subendothelium, while irreversible platelet aggregation also requires interaction between the VWF and integrin ␣IIb␤3. 8,[11][12][13] Although an important role for VWF in platelet-platelet cohesion and thrombus formation at arterial shear rates was demonstrated both in vitro 13,14 and in vivo, 15,...

The life threatening disease TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to inherited or acquired deficiency of the ADAMTS13 metalloprotease. Here we show that ADAMTS13-deficient mice generated by gene targeting are viable and exhibit normal survival through 2 years of age. Despite the absence of VWF-cleaving protease activity (<1% of normal), wild-type and ADAMTS13-deficient plasma exhibit identical VWF multimer distributions, and Adamts13−/− mice develop spontaneous TTP at an extremely low rate (2 cases out of 358 mice). However, intravital microscopy demonstrated that VWF-mediated platelet-endothelial interactions are significantly prolonged in Adamts13−/− mice. These observations suggested that additional environmental triggers and/or genetic modifying factors may be required to bring about TTP in the setting of ADAMTS13 deficiency. To address the effect of VWF level on development of TTP, Adamts13−/− mice were crossed to mice of the CASA/Rk strain which exhibit markedly elevated plasma VWF levels. Resulting CASA/Adamts13−/− mice demonstrated plasma VWF ranging from 150% to 600% of C57BL/6 controls, and we found that 21% of these mice were thrombocytopenic at baseline (vs. 0% of controls). Introduction of the CASA/Rk genetic background also resulted in the appearance of UL-VWF in CASA/Adamts13−/− mice, further prolonged VWF-mediated platelet-endothelial cell interactions, increased the rate of spontaneous TTP, and markedly decreased survival. Challenge of CASA/Adamts13−/− mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP, with thrombocytopenia, profound microangiopathic hemolytic anemia, and platelet- and VWF-thrombi seen in multiple organs. Surprisingly, we observed no correlation between plasma VWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from VWF. Our laboratory is pursuing the identification of these genes which may provide insight into the pathogenesis and treatment of TTP in humans. Finally, our data also suggest that microbial-derived toxins, or possibly other sources of endothelial injury, may be among the key factors required to trigger acute TTP in the setting of ADAMTS13 deficiency.

von Willebrand factor (VWF) is a large adhesive glycoprotein synthesized in megakaryocytes and endothelial cells and stored in platelet a-granules and Weibel-Palade bodies respectively. It protects Factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Therefore in order to understand the in vivo role of VWF and FVIII in experimental thrombosis under venous flow conditions, we decided to evaluate thrombosis in VWF−/−, FVIII−/− and transgenic mice lacking the GPIbα extracellular domain which was replaced by human Interleukin-4 receptor (IL4Rα/GPIbα-tg). In ferric chloride-injured veins, platelet adhesion to subendothelium is decreased and thrombus growth is impaired in the VWF−/− mice when compared to wild-type (WT). In the WT mice, thrombi grew to occlusive size with a mean time of 18 min and all injured venules occluded, whereas in VWF−/− mice none of the vessels occluded by 40 min after injury, when observation was terminated. Venules of mice deficient in FVIII treated similarly also did not occlude because of embolization. The infusion of recombinant human-FVIII (r-hu-FVIII) in FVIII−/− mice normalized the occlusion time to WT values. We also observed thrombus instability in the VWF−/− mice, which was due to lower FVIII levels in these mice since r-huFVIII restored thrombus stability i.e. prevented breaking of the thrombi with large platelet aggregates moving downstream. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF−/− thrombi grew at a slower rate than WT and the venules did not occlude. In transgenic mice lacking the GPIbα extracellular domain, all injured venules occluded. Thus, VWF uses other adhesion receptors besides GPIbα in thrombus growth under venous shear conditions. Our studies document crucial independent roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.

The metalloprotease ADAMTS13 cleaves highly adhesive ultra large von Willebrand factor (UL-VWF) multimers after their release from the endothelium. UL-VWF is stored in Weibel-Palade bodies of endothelial cells. ADAMTS13 deficiency is linked to a life threatening disorder, thrombotic thrombocytopenic purpura (TTP), which is characterized by VWF and platelet-rich thrombi in the microvasculature, and may lead to organ ischemia, neurological dysfunction and ultimately death. We previously confirmed in vivo that in Adamts13−/− mice and not in wild-type mice “strings of platelets” formed after endothelial activation. The VWF strings remain attached at one end to the endothelium and “waved” the other end in the blood stream. In addition, we showed prolonged adhesion of platelets to secreted VWF on endothelium of Adamts13−/− mice. The possible role of ADAMTS13 in platelet plug formation has not been addressed previously. We investigated thrombosis in venules and arterioles of Adamts13−/− mice by intravital microscopy. Mesenteric microvenules were stimulated with calcium ionophore A23187, a secretagouge of Weibel-Palade bodies. In the Adamts13−/− mice we observed spontaneous thrombus formation in the activated microvenules. Inhibitory antibody to plasma ADAMTS13 induced thrombi formation in the activated microvenules and prolonged adhesion of platelets to secreted VWF on endothelium of wild-type mice. In the ferric chloride arterial injury model we found that the number of fluorescent platelets deposited within 2–3 min on the sub-endothelium after injury was unexpectedly higher in Adamts13−/− mice as compared to Adamts13+/+ mice (P<0.05). In the Adamts13−/− mice, platelet plug grew faster as thrombi >30 um were seen at 6.6±0.9 min compared to 10.8±0.8 min in the Adamts13+/+ mice (P<0.005). The platelet plug reached occlusive size in 10.6±0.7 min in Adamts13−/− mice, whereas in the Adamts13+/+ mice all the vessels were still open at this time. In the Adamts13+/+, the mean vessel occlusion time was 16.7±1.2 min after injury (P<0.0005). Thus ADAMTS13, by cleaving VWF multimers, down regulates both platelet adhesion to exposed subendothelium and thrombus growth. Furthermore, we observed that recombinant human ADAMTS13 inhibited formation of VWF platelet strings in histamine-activated venules and promoted thrombi dissolution in the Adamts13−/− mice. Our findings reveal that ADAMTS13 has natural anti-thrombotic activity and recombinant ADAMTS13 could be used as an anti-thrombotic agent.