International Journal of

Progress in Hematology

HEMATOLOGY

Functional Property of von Willebrand Factor Under Flowing Blood Mitsuhiko Sugimoto,a Shigeki Miyatab a

Department of Pediatrics, Nara Medical University, Kashihara, Nara; bDivision of Transfusion Medicine, National Cardiovascular Center, Suita, Osaka, Japan Received October 19, 2001; accepted October 26, 2001

Abstract

von Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, is stored in the -granule of platelets and in the Weibel-Palade body of endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in both hemostasis and pathological intravascular thrombosis, in which vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple adhesive functions for the platelet membrane receptors, glycoprotein Ib-IX-V complex, integrin IIb3, heparin, various types of collagen, and coagulation factor VIII. Among various functions, the most characteristic feature of vWF is its determinant role on platelet thrombus formation under high–shear-rate conditions. Indeed, at in vivo rheological situations where platelets are flowing with high speed in the bloodstream, the only reaction that can initiate mural thrombogenesis is the interaction of vWF with platelet glycoprotein Ib. The recent x-ray analysis of the crystal structure of various functional domains and functional studies of this protein under experimental flow conditions have rapidly advanced and revised our knowledge of the structure-function relationships of vWF, a key protein for hemostasis and arterial thrombosis. Int J Hematol. 2002;75:19-24. ©2002 The Japanese Society of Hematology Key words: von Willebrand factor (vWF); Platelet adhesion and aggregation; High shear rate; Glycoprotein Ib-IX-V complex; Integrin IIb3

as evidenced by bleeding symptoms seen in patients with von Willebrand disease, a hereditary functional defect of vWF [6]. First, this huge multifunctional glycoprotein plays an essential role in platelet adhesion and aggregation on exposed subendothelial matrices of the vessel walls at sites of injury, thus contributing to the establishment of primary hemostasis. Further, vWF also contributes to secondary hemostasis in an indirect mode by maintaining the procoagulant activity of coagulation factor VIII as its carrier protein in plasma [6,7]. Recent progress in protein and gene technology has accumulated much knowledge of the structure and function of the vWF protein. Indeed, several functional domains of vWF, such as binding sites for platelet glycoprotein (GP) Ib, integrin IIb3, glycosaminoglycans such as heparin, various types of collagen, and factor VIII, have been identified at a molecular level in the last 15 years [6-8]. Functional evaluations of vWF in most of these studies, however, were made based on static experimental systems. Because the in vivo

1. Introduction When vessel walls are ruptured in vivo, hemostatic plugs are formed to repair the damaged sites, ensuring blood flow for vital organs as a function of human defense mechanisms [1-3]. This hemostatic mechanism is also known to trigger fatal intravascular thrombosis such as myocardial infarction or stroke [4,5]. A variety of contributions of von Willebrand factor (vWF) in this mechanism are believed to be essential,

Correspondence and reprint requests: Mitsuhiko Sugimoto, MD, Department of Pediatrics, Nara Medical University, 840 Shijyo-cho, Kashihara, Nara 634-8522, Japan; 81-744-29-8881; fax: 81-744-24-9222 (e-mail: [email protected]); or Shigeki Miyata, MD, Division of Transfusion Medicine, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan; 81-6-6833-5012 ext. 2294; fax: 81-6-6872-8175 (e-mail: [email protected]).

19

20

Sugimoto and Miyata / International Journal of Hematology 75 (2002) 19-24

hemostatic process, including platelet thrombogenesis, in which vWF plays a crucial role, occurs under blood flow conditions, the function of vWF needs to be assessed by an experimental system in which rheological circumstances are taken into consideration. In this regard, more recent flow studies using a perfusion chamber able to reproduce physiologic blood flow conditions in vitro clearly established the new concept for platelet adhesion on a thrombogenic surface under high–shear-rate conditions, which apparently differs from the classic adhesion theory based on previous static experiments, and the functional properties of vWF have now been drastically revised under such flow conditions [9-12]. In this review article, we describe and discuss the recent development in understanding of structure-function relationships of vWF, especially regarding the vWF-GP Ib interaction, under flow conditions with high shear rate, which is thought to be relevant for both in vivo hemostasis and arterial thrombosis.

2. The Function of vWF in Mural Platelet Thrombogenesis Under High-Shear Flow 2.1. Shear Stress Platelets circulate close to the vascular wall in vessel lumens, whereas larger cells, including erythrocytes and leukocytes, flow in the center of the lumen. Theoretically, the speed of blood flow is maximal in the center and exponentially decreases close to zero in the portion close to the vessel wall. Under this condition, platelets are exposed to the shear stress created by the differential speed of laminar blood flow [9,10]. Recent studies have demonstrated that platelets can aggregate under shear-stress conditions without any modulators to form platelet thrombi. In the process of platelet thrombus formation, vWF mediates the initial platelet adhesion on the surface of subendothelial matrices mainly through the functions of A1 and A3 domains and the platelet aggregation through the function of the C-terminal RGD site, depending on the shear stress, as described in detail below.

2.2. vWF in Initial Platelet Adhesion Under HighShear Flow Although many ligand-receptor interactions are known to be involved in the adhesive process, platelet adhesion to a thrombogenic surface under high-shear flow is absolutely dependent on the function of vWF [11-16]. Indeed, the only ligand that can mediate the initial contact of platelets with the exposed subendothelium at sites of vascular damage is vWF immobilized onto subendothelium under high–shearrate conditions, in which blood cells including platelets are flowing with a high speed [11-16]. This observation can be explained by the extremely high association rate of interaction of vWF with the platelet GP Ib-IX-V complex among various ligand-receptor interactions involved in the adhesive process. The dissociation rate of this interaction, however, is also so high that the initial contact of platelets with subendothelium is transient and reversible [15-17]. As a result,

Figure 1. Schematic representation of von Willebrand factor (vWF)dependent platelet thrombogenesis under high shear flow. Platelets roll on subendothelium at sites of vascular injury through the interaction of glycoprotein (GP) Ib with the A1 domain of vWF immobilized onto the subendothelium (top panel). During rolling, platelets gradually become activated by the inside-out signal, stopping on and firmly adhering to the surface by the binding of integrin IIb3 (also refers to GP IIb-IIIa) to the RGD sequence of immobilized vWF and/or by the platelet collagen receptors–collagen interaction (second panel). Then, vWF mediates the 3-dimensional thrombus development in a mode basically similar to the initial platelet adhesion (third, fourth, and bottom panels).

platelets roll on subendothelium, a phenomenon that is analogous to leukocytes rolling on endothelial cell layers (Figure 1). Several lines of evidence indicate that platelet rolling is maintained solely by the interaction of the extracellular domain of GP Ib -chain with vWF A1 domain. In fact, both Chinese hamster ovary (CHO) cells expressing GP Ib by recombinant gene technology and latex beads coated with the soluble amino-terminal fragment of GP Ib have been observed to roll on a vWF-coated glass surface in a manner similar to platelet rolling [18,19]. In addition, the recombinant vWF A1 domain expressed in Escherichia coli, like mul-

vWF Under Flow

timeric whole vWF, was also confirmed to support platelet rolling [17]. During rolling, platelets are gradually activated by the inside-out signal derived from the GP Ib-vWF interaction, eventually stopping on and adhering firmly to the surface by tight binding of integrin IIb3 or 21 to vWF C1 domain containing the RGD amino acid sequence or other adhesive matrices, such as various types of collagen [20] (Figure 1).The precise activation mechanisms organizing the process from rolling to firm adhesion remain to be clarified, but actin-binding protein and 14-3-3 protein, both of which are attached to the cytoplasmic tail of the GP Ib -chain, are suggested to be involved in the signaling pathway from GP Ib [21-24]. Interestingly, recent flow studies indicate that this inside-out signal, in addition to activation of IIb3, also functions to downregulate the GP Ib function itself [25,26]. Regarding the activation mechanisms during the adhesive process on immobilized vWF, cytosolic calcium changes or shape changes in individual platelets have also been studied under high shear [22,27,28]. Although it remains to be determined whether or not the cytosolic calcium elevation in platelets occurs during rolling by the inside-out signal from the vWF-GP Ib interaction, the drastic calcium influx from the extracellular sources is thought to be established after firm adhesion by the outside-in signal from vWF binding to integrin IIb3 [27,28]. During rolling, platelets extrude filopodia in the direction of flow, a mechanism that is also reasonably thought to support subsidiary platelet adhesiveness against rapid blood flow. In this regard, some filopodia are torn off from the cytoplasm during rolling, explaining in part a mechanism for the generation of platelet microparticles under high–shear-rate conditions [29]. After firm adhesion and drastic calcium influx, the platelet adhesive process is finalized by the extensive spreading of platelets on a thrombogenic surface, thus providing a more potent adhesion and a larger thrombogenic base that are essential for the subsequent thrombus development [28]. It should be noted that the whole platelet adhesive process, as described, is precisely up- or down-regulated by the inside-out or outside-in integrin signals triggered by immobilized vWF under high-shear flow (Figure 1).

21

Under flow conditions with high shear rates, the activated IIb3 of platelets irreversibly adhering to a thrombogenic surface as a monolayer is assumed to be insufficient to directly capture adhesive proteins and platelets flowing rapidly in blood. The GP Ib-IX-V complex, which resides near the activated IIb3, functions to enhance the ability of IIb3 to irreversibly capture flowing vWF by slowing vWF transport velocity through its transient interaction with this factor (Figure 1). In light of mechanisms of initial platelet adhesion indicating that platelets moving with high velocity in blood can interact with the surface only through the vWFGP Ib interaction, it also seems reasonable to assume the inverse, that vWF moving with high speed can be captured only through its transient interaction with GP Ib on platelets adhering to and immobilizing on the surface, followed by the irreversible binding of vWF, transiently trapped by GP Ib on platelets, to neighboring activated IIb3. Captured vWF on platelets adhering to the surface may then mediate second-layer platelet adhesion via the vWF-GP Ib interaction and via the vWF-IIb3 interaction, in a mode similar to initial platelet adhesion onto the surface [32,33]. Thus, mural thrombus development under high shear may reflect repeated cycles of these events. Based on this interpretation, the adhesive function of vWF, regardless of whether vWF is in solution or immobilized, is an absolute prerequisite for the overall 3-dimensional mural thrombus development under high shear [11,32-34] (Figure 1). Although the vWF function is apparently essential, fibrinogen binding to IIb3 is also indispensable for proper 3dimensional mural thrombus development under high shear [34-37], as evidenced by the observation that thrombi generated in the absence of fibrinogen were loosely packed and fragile under such high–shear-rate conditions [36,38,39] (Figure 1). It should be stressed again, however, that the adhesive function of fibrinogen in 3-dimensional thrombus development can be fully expressed only in the presence of vWF under high–shear-rate conditions.

3. Structure-Function Relationships of vWF A1 Domain Interacting With Platelet GP Ib Under High-Shear Flow

2.3. vWF in 3-Dimensional Thrombus Development Under High-Shear Flow

3.1. Functional Sites of the A1 Domain

Platelets adhering to a thrombogenic surface aggregate, resulting in mural thrombus development under flow. Platelet aggregation in a soluble phase is basically achieved by platelet-platelet engagement by the function of adhesive proteins that contain a divalent (or multivalent) structure such as fibrinogen and vWF. In a conventional platelet aggregometer, platelet aggregation induced by exogenous agonists is mediated mainly by fibrinogen binding to IIb3 as a molecular glue for platelets. In contrast, vWF, not fibrinogen, plays a pivotal role in platelet aggregation induced by high shear stress observed in a cone-and-plate–type viscometer [30]. Platelet aggregation mediating mural thrombus development under high shear, unlike the soluble-phase platelet aggregation described above, is a solid-phase event, and thus, its mechanisms are different and more complex than are those in a soluble-phase platelet aggregation [31].

The vWF A1 domain can interact with GP Ib-IX-V complex to initiate platelet adhesion and activation. Recently, the crystal structure of vWF A1 domain was solved by 2 different groups using recombinant fragments [40-42]. Overall structure of the A1 domain is cuboid, displaying an / fold with a central hydrophobic parallel -sheet flanked on 2 sides by amphipathic helices (Figure 2). Combined with the results of alanine scanning mutagenesis [43,44], GP Ib binding sites are proposed to locate in shallow groove created by the N-terminal portion of helix 3 with sides formed by helix 4b and N-terminal residues of strand 3. Indeed, docking modeling of the platelet GP Ib peptide containing residues 271-279, a putative functional site of GP Ib, onto the vWF A1 domain suggests that the residues in strand 3 and the preceding loop (residues 559-566), as well as in helix 3 residues (594-603) of the A1 domain, are directly involved in

22

Sugimoto and Miyata / International Journal of Hematology 75 (2002) 19-24

Figure 2. Structure of the von Willebrand factor (vWF) A1 domain (provided by Dr. Taei Matsui, Fujita Health University, Aichi, Japan). Both ribbon modeling (A) and space-filling modeling (B) are displayed in an orientation in which the helix 3 lies at the front face. A, The  helix,  strand, and loop are indicated by blue, yellow, and red, respectively. Amino acid residues crucial for glycoprotein (GP) Ib binding are indicated by green (G561, E596, K599). B, The numbers of some amino acid residues crucial for receptor binding. The 3 strand is indicated by yellow, and the helices 3 and 4b are blue, both of which are candidates for GP Ib binding. As in A, amino acid residues crucial for GP Ib binding are indicated by green.

the interaction with GP Ib. To verify this model, platelet adhesion onto the immobilized mutant A1 domain fragments containing single and multiple side-chain substitutions was investigated under flow conditions [45]. This study showed that each of the residues Tyr565, Glu596, and Lys599 is strictly required for the A1 domain function dependent on Gly651. It is also suggested that a group of positively charged residues, including Arg at positions 629, 632, and 636 and Lys at positions 643 and 645, possibly reacts in concert as an accessory functional site, although there was no evidence that these residues directly participate in GP Ib binding (Figure 2). Another study group [46] investigated GP Ib binding sites with the same approach, by the observation of platelet adhesion onto the mutant A1 domain under flow conditions, showing that Arg524, Gln604, and Ser607 may participate in the GP Ib binding in addition to the residues in strand 3, its preceding loop, and helix 3. Natural mutation sites responsible for von Willebrand disease (vWD) type 2B, showing enhanced soluble vWF binding to GP Ib (“gain of function”), locate on the lower surface of the domain involved in salt bridges or hydrophobic packing [40-42]. Thus, it appears that these residues are not directly involved in the binding sites for GP Ib but may be part of a region that regulates function indirectly by interfacing with other parts of the intact vWF molecule, modulating the conformation of the GP Ib binding groove [45,47]. For another A1 domain function, Mazzucato et al [48] reported that A1 may play an important role in vWF binding to tetrameric collagen type VI, one of the components in the subendothelial matrix. The A1 domain interacts with the globular domain of collagen type VI, inducing platelet arrest

on the collagen surface through a concerted action of the A1 and A3 domains.

3.2. Adhesive Property of the A1 Domain Under Flow The vWF Al domain induces platelet adhesion and aggregation at an injured vascular wall under high–shear-stress conditions and/or after immobilization on a denuded subendothelium through the interaction with platelet GP Ib, whereas the vWF Al domain in normal blood circulation cannot initiate platelet activation. Thus, it has been generally assumed that a conformational change of the Al domain with the exposure of GP Ib binding sites normally cryptic inside the molecule is required to promote platelet adhesion and activation [49], supported by evidence that single-point mutation within the Al domain associated with vWD type 2B confers high affinity on the Al domain to GP Ib in solution [50,51]. However, crystal modeling of the A1 domain shows that the residues responsible for vWD type 2B are not directly involved in the GP Ib binding as described above. Using a parallel-plate flow-chamber system, the functional effect of shearing on vWF molecules was investigated by exposing immobilized vWF to high shear immediately before assay [18]. The results showed that preshearing vWF did not significantly change the function of immobilized vWF molecules to initiate platelet rolling to the surface. The studies using recombinant A1 domain with conformational alterations by chemical modification showed that conformational changes resulting in the high affinity for GP Ib in solution did not lead to effective platelet rolling under high shear [17,52]. In

vWF Under Flow

contrast, the altered molecule, the conformation of which was most similar to the native molecule and did not bind to GP Ib in solution, appears to initiate platelet rolling most effectively under high shear, with high association and dissociation rates of the bond formation between the vWF A1 domain and GP Ib [17,52]. The local density and the multimeric structure of vWF molecules at a site of thrombogenic surface, rather than the conformational changes, were crucial for platelet rolling against high shear stress, which also has the strength to detach platelets from the surface [17]. According to the above results, we hypothesize the mechanisms to recruit platelets freely flowing with a high speed onto a site of vascular injury as follows: vWF in normal circulation may be able to interact with GP Ib but cannot activate platelets because of one of its properties, a high dissociation rate of the bond formation with GP Ib. At the site of vascular injury, vWF in the circulating blood immediately binds through the A3 domain, a collagen binding site, to exposed subendothelial matrices. Together with internal vWF existing in subendothelium, the density of vWF gradually increases and becomes sufficient to capture the freeflowing platelets in blood against the shear stress by another property of the vWF A1 domain, a high association rate. Finally, platelets start to roll on the surface via the binding of the GP Ib-IX-V complex with the vWF A1 domain, inducing intraplatelet signaling [53]. The initial tethering is rapidly followed by the synergistic binding of integrins to vWF or other substrates, making stable platelet adhesion [20,35].

4. Concluding Remarks To date, the adhesive property of vWF cannot be discussed without consideration of blood rheological circumstances. Indeed, the physiologic relevance of this unique protein under flow became paramount in molecular mechanisms of mural thrombogenesis. Because fatal thrombosis such as myocardial infarction or stroke is assumed to be established under flow conditions with high shear rates, the clarification of structure-function relationships of vWF under high shear would give significant insight into an antithrombotic strategy against such diseases. It could be promising to develop a new molecular design of antithrombotic drugs aiming at the regulation of vWF function under high-shear flow.

Acknowledgments This work was supported in part by a grant from the Ministry of Education, Science, and Culture of Japan to M.S. (No. 11670780 and No. 13671074) and by the Research Grant for Cardiovascular Diseases (12C-9) from the Ministry of Health, Labor, and Welfare to S.M. We thank Dr. Taei Matsui (Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan) for allowing us to use a figure for structure of the vWF A1 domain.

References 1. Sixma JJ, Waster J. The hemostatic plug. Semin Hematol. 1977;14: 265-299.

23

2. Weiss HJ. Platelet physiology and abnormalities of platelet function, I. N Engl J Med. 1975;293:531-541. 3. Weiss HJ. Platelet physiology and abnormalities of platelet function, II. N Engl J Med. 1975;293:580-588. 4. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and acute coronary syndromes, I. N Engl J Med. 1992;326:242-250. 5. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and acute coronary syndromes, II. N Engl J Med. 1992;326:310-318. 6. Ruggeri ZM, Zimmerman TS. von Willebrand factor and von Willebrand disease. Blood. 1987;70:895-904. 7. Sadler JE. von Willebrand factor. J Biol Chem. 1991;266: 22777-22780. 8. Ginsberg D, Bowie EJW. Molecular genetics of von Willebrand disease. Blood. 1992;79:2507-2519. 9. Usami S, Chen HH, Zhao Y, Chein S, Skalak R. Design and construction of a linear shear stress flow chamber. Ann Biomed Eng. 1993;21:77-83. 10. Kroll MH, Hellums JD, McIntire LV, et al. Platelets and shear stress. Blood. 1996;88:1525-1541. 11. Ruggeri ZM. von Willebrand factor. J Clin Invest. 1997;99:559-564. 12. Ruggeri ZM. Old concepts and new developments in the study of platelet aggregation. J Clin Invest. 2000;105:699-701. 13. Aleviadou BR, Moake JL, Tunner NA, et al. Real-time analysis of shear-dependent thrombus formation and its blockage by inhibitors of von Willebrand factor binding to platelets. Blood. 1993;81:1263-1276. 14. Goto S, Salomon DR, Ikeda Y, Ruggeri ZM. Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Willebrand factor to platelets. J Biol Chem. 1995;270: 23352-23361. 15. Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by attachment onto fibrinogen or translocation on von Willebrand factor. Cell. 1996;84:289-297. 16. Moroi M, Jung SM, Nomura S, et al. Analysis of the involvement of the von Willebrand factor-glycoprotein Ib interaction in platelet adhesion to a collagen-coated surface under flow conditions. Blood. 1997;90:4413-4424. 17. Miyata S, Ruggeri ZM. Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ib under flow. J Biol Chem. 1999;274:6586-6593. 18. Fredrickson BJ, Dong JF, Mclntire LV, et al. Shear-dependent rolling on von Willebrand factor or mammalian cells expressing the platelet glycoprotein Ib-IX-V complex. Blood. 1998;92:3684-3693. 19. Marchese P, Saldivar E, Ware J, Ruggeri ZM. Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ib in a flow field. Proc Natl Acad Sci U S A. 1999;96:7837-7842. 20. Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interaction in platelet thrombus formation under flow. Cell. 1998;94:657-666. 21. Cranmer SL, Ulsemer P, Cooke BM, et al. Glycoprotein (GP) IbIX-transfected cells roll on a von Willebrand factor matrix under flow: importance of the GPIb/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear. J Biol Chem. 1999;274:6097-6106. 22. Yuan Y, Kulkarni S, Ulsemer P, et al. The von Willebrand factorglycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells. J Biol Chem. 1999;274:36241-36251. 23. Afshar-Kharghan V, Gineys G, Schade AJ, et al. Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib for the proper conformation and function of the ligand-binding region. Biochemistry. 2000;39:3384-3391. 24. Shen Y, Romo GM, Dong JF, et al. Requirement of leucine-rich repeats of glycoprotein (GP) Ib a for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP IbIX-V complex. Blood. 2000;95:903-910. 25. Mistry N, Cranmer SL, Yuan Y, et al. Cytoskeletal regulation of the

24

26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

36.

37. 38.

39.

40.

Sugimoto and Miyata / International Journal of Hematology 75 (2002) 19-24 platelet glycoprotein Ib-V-IX-von Willebrand factor interaction. Blood. 2000;96:3480-3489. England GE, Bodnar RD, Li Z, Ruggeri ZM, Du X. Regulation of vWf binding function of the platelet glycoprotein Ib-IX by the membrane skeleton-dependent inside-out signal. J Biol Chem. 2001;276:16952-16959. Kuwahara M, Sugimoto M, Miyata S, et al. Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions. Blood. 1999;94:1149-1155. Kuwahara M, Sugimoto M, Tsuji S, et al. Platelet shape changes and adhesion under high shear flow. Arterioscler Thromb Vasc Biol. In press. Reininger KB, Ruggeri ZM. Mechanism of platelet adhesion to von Willebrand factor through glycoprotein Ib dependent tethering, serving of tethers and microparticle formation [abstract]. Blood. 2000;96:621a. Ikeda Y, Handa M, Kawano K, et al. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest. 1991;87:1234-1240. Tsuji S, Sugimoto M, Kuwahara M, et al. Role and initiation mechanism of the interaction of glycoprotein Ib with surfaceimmobilized von Willebrand factor in a solid-phase platelet cohesion process. Blood. 1996;88:3854-3861. Goto S, Ikeda Y, Saldivar E, Ruggeri ZM. Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest. 1998;101:479-486. Sugimoto M, Tsuji S, Kuwahara M, et al. Shear-dependent functions of the interaction between soluble von Willebrand factor and platelet glycoprotein Ib in mural thrombus formation on a collagen surface. Int J Hematol. 1999;69:48-53. Ruggeri ZM. Role of von Willebrand factor in platelet thrombus formation. Ann Med. 2000;32:2-9. Ruggeri ZM, Dent JA, Saldivar E. Contribution of distinct adhesive interactions to platelet aggregation in flowing blood. Blood. 1999;94:1172-1178. Tsuji S, Sugimoto M, Miyata S, et al. Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow. Blood. 1999;94:968-975. Kulkarni S, Dopheide SM, Yap CL, et al. A revised model of platelet aggregation. J Clin Invest. 2000;105:783-791. Remijn JA, Wu YP, Ijsseldijk MJW, Zwaginga JJ, Sixma JJ, de Groot PG. Absence of fibrinogen in afibrinogenemia results in large but loosely packed thrombi under flow conditions. Thromb Haemost. 2001;85:736-742. Matsui H, Sugimoto M, Tsuji S, et al. Distinct and concerted functions of von Willebrand factor and fibrinogen for three-dimensional thrombus development under high shear flow [abstract]. Blood. 2000;96:620a. Celikel R, Varughese KI, Madhusdan A, Yoshioka A, Ware J, Ruggeri ZM. Crystal structure of von Willebrand factor A1 domain in

complex with the functional blocking NMC-4 Fab. Nat Struct Biol. 1998;5:189-194. 41. Celikel R, Ruggeri ZM, Varughese KI. Modulation of von Willebrand factor conformation and adhesive function by internalized water molecule. Nat Struct Biol. 2000;7:881-884. 42. Emsley J, Cruz M, Handin R, et al. Crystal structure of the von Willebrand factor A1 domain and implications for the binding of platelet glycoprotein Ib. J Biol Chem. 1998;273:10396-10401. 43. Matsushita T, Sadler JE. Identification of amino acid residues essential for von Willebrand factor binding to platelet glycoprotein Ib: charged-to-alanine scanning mutagenesis of the A1 domain of human von Willebrand factor. J Biol Chem. 1995;270:13406-13414. 44. Matsushita T, Meyer D, Sadler JE. Localization of von Willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis. J Biol Chem. 2000;275: 11044-11049. 45. Vasudevan S, Roberts J, Ruggeri ZM, et al. Modeling and functional analysis of the interaction between von Willebrand factor A1 domain and glycoprotein Ib. J Biol Chem. 2000;275:12763-12768. 46. Cruz MA, Diacovo TG, Emsley J, et al. Mapping the glycoprotein Ib-binding site in the von Willebrand factor A1 domain. J Biol Chem. 2000;275:19098-19105. 47. Miura S, Li CQ, Sadler JE, et al. Interaction of von Willebrand factor domain A1 with platelet glycoprotein Ib (1-289): slow intrinsic binding kinetics mediate rapid platelet adhesion. J Biol Chem. 2000;275:7539-7546. 48. Mazzucato M, Spessotto P, Masotti A, et al. Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow. J Biol Chem. 1999:274:3033-3041. 49. Siedlecki CA, Lestini BJ, Kottke-Marchant KK, et al. Sheardependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939-2950. 50. Inbal A, Kornbrot N, Harrison P, Randi AM, Sadler JE. Effect of type IIB von Willebrand disease mutation Arg(545)Cys on platelet glycoprotein Ib binding-studies with recombinant von Willebrand factor. Thromb Haemost. 1993;70:1058-1062. 51. Christphe O, Ribba AS, Baruch D, et al. Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor. Blood. 1994;83:3553-3561. 52. Miyata S, Goto S, Federici AB, Ware J, Ruggeri ZM. Conformational changes in the A1 domain of von Willebrand factor modulating the interaction with platelet glycoprotein Ib. J Biol Chem. 1996;271:9046-9053. 53. Yap CL, Hughan SC, Cramer SL, et al. Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin IIb3. J Biol Chem. 2000;275: 41377-41388.