Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib-von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13 ؊/؊ and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13 ؊/؊ mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13 ؊/؊ compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke. (Blood. 2010; 115:1650-1653) Introductionvon Willebrand factor (VWF) is a large multimeric protein that plays a key role in thrombus formation by tethering platelets at sites of vascular injury. 1 Smaller VWF multimers are less active, and the potent thrombogenic activity of ultra-large VWF (ULVWF) secreted from endothelium is regulated in vivo through cleavage by ADAMTS13. 2,3 The importance of this mechanism for normal hemostasis is supported by evidence that patients with deficiency of ADAMTS13 function, diagnosed with thrombotic thrombocytopenic purpura, have ULVWF in circulating blood and VWFdependent microvascular thrombosis. 2 Recently, we demonstrated that ADAMTS13 cleaves VWF on the surface of platelet thrombi in a shear force-dependent manner, which limits thrombus growth in vitro. 4 These data suggest that ADAMTS13 is a key molecule that maintains a physiologic balance between hemostasis and thrombosis through regulation of VWF function in vivo.ADAMTS13 function is crucial for preventing thrombosis in the cerebral microvasculature, as indicated by the occurrence of neurologic deficits in thrombotic thrombocytopenic purpura, but the role of ADMTS13 in the pathogenesis of reperfusion injury after arterial thrombosis has not been established. To address this issue, we investigated the role of ADAMTS13 in a transient middle cerebral arterial occlusion (MCAO) model of ischemia-reperfusion injury in the mouse brain 5 using Adamts Ϫ/Ϫ mice. 6 Because brain ischemia-reperfusion injury is dependent on the platelet glycoprotein Ib-VWF axis 7 and platelet thrombosis adversely affects the postischemic cerebral microcirculation 8-11 leading to secondary brain damage, 10 ADAMTS13 may reduce platelet thrombus growth and thereby ameliorate ischemic brain injury by improving the postischemic no-reflow phenomenon. 12 Here we demonstrate that Adamts13 gene deletion aggravates postischemic cerebral blood reflow, resulting in larger infarct volume. This result suggests that ADAMTS13 may indeed supp...

Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies have revealed that the platelet adhesive process under flow is tightly regulated by multiple ligand-receptor interactions. However, platelet morphological changes during this process, particularly its physiological relevance, remain unknown under blood flow conditions. Using epifluorescence and scanning electron microscopy, we evaluated the real-time changes in platelet morphology during a platelet adhesive process on a von Willebrand factor-coated surface under physiological high shear flow in a perfusion chamber. Here, we show that dynamic platelet shape changes occurring during distinct phases of the adhesive process are precisely regulated by "inside-out" and "outside-in" integrin signals and are also a key regulatory element in successful platelet thrombogenesis opposing rapid blood flow in vivo.

The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a functionblocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWFcleaving activity of ADAMTS13, provided visual evidence for a shear ratedependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis. IntroductionThe adhesive protein von Willebrand factor (VWF) plays a major role in platelet thrombogenesis, a process crucial for hemostasis. However, the excessive function of VWF is thought to increase the risk of fatal arterial thrombosis. 1,2 The thrombogenic activity of VWF is strictly dependent upon its multimeric structure, which is thought to be regulated in vivo by the metalloprotease ADAMTS13 through its cleavage of the A2 domain of the VWF subunit. 3,4 Indeed, patients with congenital deficiency of ADAMTS13 suffer repeated thrombotic complications attributed to excessive function of the ultra-large VWF (ULVWF) multimer, which is not found in normal blood circulation. [3][4][5][6] This concept was recently confirmed by knock-out mouse studies, in which ADAMTS13 Ϫ/Ϫ mice exhibited enhanced thrombogenicity in the ex vivo or in vitro experimental blood flow conditions tested. 7,8 The mechanisms by which ADAMTS13 regulates VWF remain poorly understood. However, recent studies showing that ADAMTS13 under flow conditions can rapidly cleave ULVWF secreted from and anchored to cultured endothelial cell layers 9,10 have raised the possibility that blood flow is critical in activating ADAMTS13. 11 Indeed, the VWF-cleaving activity of ADAMTS13 cannot be reproduced in vitro under static conditions unless the substrate VWF molecule is somewhat modified (eg, denatured by guanidine-HCl or urea). 3,4 Further, the question arises of whether ADAMTS13, in addition to its known action on ULVWF freshly released from endothelial cells, might also act directly at the local sites of thrombus generation to regulate thrombus growth.To address these issues, we analyzed the role and mechanisms of ADAMTS13 action in mural platelet thrombogenesis on a collagen-coated glass surface in an in vitro perfusion chamber system. Our visual evidence demonstrates that ADAMTS13 cleaves VWF and down-regulates mural thrombus growth at the site of ongoing thrombus generation in a shear rate-dependent manner under whole blood flow conditions. Methods Blood collectionThe present work was approved by the i...

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.

ABSTRACTvon Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) lb in the GP Tb-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Tb and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trps50 to Cys550, located in the GP Tb-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trps 5 or a mutant Cys550 residue, bound directly to GP Tb in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal TrpSS( , did not bind to GP Tb directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys5ss) behaved like type UIB vWF-namely, bound to GP Tb even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Tb, may modulate the interaction through conformational changes.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann’s thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s−1). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (≥1,210 s−1) but normal thrombus formation under low shear (≤340 s−1). When shear rate was increased stepwise to 1,500 s−1 during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s−1. However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s−1, whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s−1 during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an “initiator and promoter,” whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against heightened shear forces that could lead to peeling off of platelets from the surface.