Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex.

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.

Binding of alpha-thrombin to surface-anchored platelet glycoprotein Ib(alpha) sulfotyrosines through a two-site mechanism involving exosite I.

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.

Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib.

The main question concerning the mechanism of a-thrombin binding to platelet membrane glycoprotein (GP)Ib is whether it involves both thrombin exosite I and exosite II. The solution of two independent crystal structures suggests alternative explanations that may actually reflect different modes of binding with distinct pathophysiological significance. With respect to function, it is still unclear whether thrombin binding to GPIb promotes procoagulant and prothrombotic pathways of response to vascular injury or limits such responses by sequestering, at least temporarily, the active enzyme. We review here published information on these topics and touch upon ongoing studies aimed at finding definitive answers to outstanding questions relevant for a better understanding of thrombosis and haemostasis.

Adhesion mechanisms in platelet function.

Platelet adhesion is an essential function in response to vascular injury and is generally viewed as the first step during which single platelets bind through specific membrane receptors to cellular and extracellular matrix constituents of the vessel wall and tissues. This response initiates thrombus formation that arrests hemorrhage and permits wound healing. Pathological conditions that cause vascular alterations and blood flow disturbances may turn this beneficial process into a disease mechanism that results in arterial occlusion, most frequently in atherosclerotic vessels of the heart and brain. Besides their relevant role in hemostasis and thrombosis, platelet adhesive properties are central to a variety of pathophysiological processes that extend from inflammation to immune-mediated host defense and pathogenic mechanisms as well as cancer metastasis. All of these activities depend on the ability of platelets to circulate in blood as sentinels of vascular integrity, adhere where alterations are detected, and signal the abnormality to other platelets and blood cells. In this respect, therefore, platelet adhesion to vascular wall structures, to one another (aggregation), or to other blood cells, represent different aspects of the same fundamental biological process. Detailed studies by many investigators over the past several years have been aimed to dissect the complexity of these functions, and the results obtained now permit an attempt to integrate all the available information into a picture that highlights the balanced diversity and synergy of distinct platelet adhesive interactions.

Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibalpha.

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.