Thrombin bound to platelets contributes to stop bleeding and, in pathological conditions, may cause vascular thrombosis. We have determined the structure of platelet glycoprotein Ibalpha (GpIbalpha) bound to thrombin at 2.3 angstrom resolution and defined two sites in GpIbalpha that bind to exosite II and exosite I of two distinct alpha-thrombin molecules, respectively. GpIbalpha occupancy may be sequential, as the site binding to alpha-thrombin exosite I appears to be cryptic in the unoccupied receptor but exposed when a first thrombin molecule is bound through exosite II. These interactions may modulate alpha-thrombin function by mediating GpIbalpha clustering and cleavage of protease-activated receptors, which promote platelet activation, while limiting fibrinogen clotting through blockade of exosite I.

The presence of one or more copies of von Willebrand factor type A domains identifies a superfamily of proteins usually involved in biological processes controlled by specific molecular interactions, often adhesive in nature. We have solved the crystal structure of the prototypic von Willebrand factor A1 domain, essential for the antihemorrhagic activity of platelets, in complex with the function blocking antibody, NMC-4, at 2.2 A resolution. This has led to the recognition of a putative binding groove for the platelet receptor, glycoprotein Ib alpha, formed by two adjacent alpha-helices and a beta-strand. The structure also shows a contact interface between A1 domain pairs, suggesting a hypothetical mechanism for the regulation of protein assembly and heterologous ligand binding mediated by homophilic interactions of type A domains.

The binding of von Willebrand factor (vWF) to the platelet receptor, glycoprotein (GP) Ib-IX-V complex, has a key role in the initiation of thrombus formation and is regulated by interactions with extracellular matrix components under the influence of hemodynamic forces. To a certain extent, these effects can be mimicked in vitro by two nonphysiologic modulators, ristocetin and botrocetin. The latter, isolated from the venom of the snake Bothrops jararaca, is a 31-kDa heterodimeric protein that forms a soluble complex with vWF. As an initial step toward understanding the mechanisms that regulate vWF function, we have solved the crystal structure of botrocetin at 1.8 A resolution. Botrocetin exhibits homology with other snake proteins, but contains only one metal binding site as compared to two in Factor IX binding protein and Factor IX/X binding protein and none in flavocetin. A distinctive feature of botrocetin is the presence of a negatively charged surface that may play a role in the association with the vWF A1 domain.

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr 276 , which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr 278 or Tyr 279 , which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands-aptamer HD1, hirugen, and lepirudin-did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp 148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys 279 . These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.tyrosine sulfation | tyrosylprotein sulfotransferase-2 | protease-activated receptor | platelet activation | platelet aggregation

Binding of the von Willebrand factor (vWF) A1 domain to the glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to reactive substrates under high shear stress conditions, a key event in hemostasis and thrombosis. We have now used the known three-dimensional structure of the A1 domain to model the interaction with the GP Ib␣ sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand ␤3 and preceding loop (residues 559 -566) as well as in helix ␣3 (residues 594 -603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ib␣. To verify this model, 14 mutant A1 domain fragments containing single or multiple side chain substitutions were tested for their ability to mediate platelet adhesion under flow. Each of the vWF residues Tyr 565 , Glu 596 , and Lys 599 proved to be strictly required for A1 domain function, which, in agreement with previous findings, was also dependent on Gly 561 . Moreover, an accessory functional role was apparent for a group of positively charged residues, including Arg at positions 629, 632, 636 and Lys at positions 643 and 645, possibly acting in concert. There was, however, no evidence from the model that these residues directly participate in forming the complex with GP Ib␣. These results provide a partial model of the vWF-GP Ib␣ interaction linked to the manifestation of functional activity in platelet adhesion. Binding of von Willebrand factor (vWF)1 to the platelet membrane glycoprotein (GP) Ib␣, a component of the GP Ib-IX-V complex (1), supports the arrest of bleeding after tissue trauma (2, 3) and may also contribute to the acute thrombotic occlusion of diseased arteries (4, 5). The interaction between GP Ib␣ and immobilized vWF can tether platelets to surfaces in rapidly flowing blood but is inherently short-lived, mediating transient periods of arrest that alternate with slow translocation (6). During this rolling motion, however, additional synergistic bonds can form through specific integrin receptors and establish firm platelet adhesion to extravascular matrix constituents (6, 7). The rapid association of GP Ib␣ with vWF not only initiates platelet deposition at sites of vessel injury but is also essential for maintaining thrombus growth in blood flowing with high shear rates (8). A detailed explanation of the molecular mechanisms underlying these processes may, therefore, elucidate key regulatory aspects of normal and pathological platelet function as well as indicate new approaches for antithrombotic therapy.The notion that the GP Ib␣ binding site is located between vWF residues 449 and 728 (9, 10), comprising the entire A1 domain and the preceding carboxyl-terminal portion of domain D3 (11, 12), was established over a decade ago. Since then, studies attempting to identify A1 domain residues involved in interacting with the receptor have yielded inconclusive and sometimes conflicting results (13-16). One reason for this outcome is that the assays employed to e...