Platelet aggregation, which contributes to bleeding arrest and also to thrombovascular disorders, is thought to initiate after signaling-induced activation. We found that this paradigm does not apply under blood flow conditions comparable to those existing in stenotic coronary arteries. Platelets interacting with immobilized von Willebrand factor (VWF) aggregate independently of activation when soluble VWF is present and the shear rate exceeds 10 000 s ؊1 (shear stress ‫؍‬ 400 dyn/ cm 2 ). Above this threshold, active A1 domains become exposed in soluble VWF multimers and can bind to glycoprotein Ib␣, promoting additional platelet recruitment. Aggregates thus formed are unstable until the shear rate approaches 20 000 s ؊1 (shear stress ‫؍‬ 800 dyn/cm. 2 ). Above this threshold, adherent platelets at the interface of surface-immobilized and membrane-bound VWF are stretched into elongated structures and become the core of aggregates that can persist on the surface for minutes. When isolated dimeric A1 domain is present instead of native VWF multimers, activation-independent platelet aggregation occurs without requiring shear stress above a threshold level, but aggregates never become firmly attached to the surface and progressively disaggregate as shear rate exceeds 6000 s ؊1 . Platelet and VWF modulation by hydrodynamic force is a mechanism for activation-independent aggregation that may support thrombotic arterial occlusion. IntroductionPlatelets aggregate at sites of vascular injury, forming thrombi that contribute to arrest bleeding but also occlude atherosclerotic arteries causing cardiac and cerebrovascular diseases. 1,2 Platelet thrombus formation is thought to occur in successive stages. First, individual platelets adhere to altered vascular surfaces and are activated, after which the integrin ␣IIb␤3 can bind plasma proteins, notably fibrinogen, von Willebrand factor (VWF), and fibronectin; these adhesive substrates immobilized on the membrane surface then recruit additional platelets, resulting in aggregation and thrombus growth. 2 Such events take place in flowing blood that generates shear forces. At shear rates exceeding 1000 s Ϫ1 in the human circulation, initial platelet arrest depends on glycoprotein (GP) Ib␣ binding to immobilized VWF even when extracellular matrices 3 or vascular structures 4 present multiple reactive components. Continued platelet recruitment also becomes dependent on VWF-GP Ib␣ as growing thrombi narrow the lumen where blood flows, locally increasing the shear rate. 5 Current knowledge, therefore, is that rapidly forming but short-lived VWF-GP Ib␣ bonds can keep platelets in contact with a surface or with one another for a limited time, until additional bonds, established mostly through integrin receptors, stabilize adhesion and aggregation. 3,5,6 A feature distinguishing hemostasis from arterial thrombosis is their occurrence in different hemodynamic environments. A 90% lumen reduction in a coronary artery may cause shear rates of 20 000-40 000 s Ϫ1 at or just upstream of the ...

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collageninduced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VI null platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VI null or FcR-␥ null animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VI null platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils. IntroductionPlatelet membrane receptors interact with surface-bound adhesive ligands and, as such, become essential for hemostasis and thrombosis. 1 There are numerous unique receptors interacting with different adhesive ligands suggesting that a large opportunity exists for functional redundancy in platelet adhesion. However, an emerging theme of platelet biology is the relevance of different membrane receptors in different areas of the vasculature. 2,3 A specific example is the exclusive role for the platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor in areas of the vascular system where flow rates and high shear occur, such as in small arteries and arterioles. 4 Thus, defining the physiologic relevance of an individual receptor and its ligand is an important aspect for understanding participation of the platelet in hemostasis and thrombosis.Among adhesive ligands of the extravascular matrix, collagen is a significant component with a number of known collagen receptors on the platelet surface. 5,6 One of the more recently characterized collagen receptors is GP VI. 7 The molecular cloning of GP VI revealed it to be a member of the immunoglobulin superfamily of type I transmembrane proteins. [8][9][10] The surface expression of GP VI requires the concomitant expression of the ␥-subunit of the FcR receptor (FcR-␥) and their association is functionally relevant as collagen binding to GP VI results in platelet signaling via the immunoreceptor tyrosine-based activation motif (ITAM) located in the FcR-␥ subunit. 8,[11][12][13][14] As with many of the platelet receptors, the in vivo relevance of GP VI was established prior to its description and recognition as a p...

We describe here the mechanism of platelet adhesion to immobilized von Willebrand factor (VWF) and subsequent formation of platelet-derived microparticles mediated by glycoprotein Ib␣ (GPIb␣) under high shear stress. As visualized in whole blood perfused in a flow chamber, platelet attachment to VWF involved one or few membrane areas of 0.05 to 0.1 m 2 that formed discrete adhesion points (DAPs) capable of resisting force in excess of 160 pN. Under the influence of hydrodynamic drag, membrane tethers developed between the moving platelet body and DAPs firmly adherent to immobilized VWF. Continued stretching eventually caused the separation of many such tethers, leaving on the surface tubeshaped or spherical microparticles with a diameter as low as 50 to 100 nm. Adhesion receptors (GPIb␣, ␣IIb␤3) and phosphatidylserine were expressed on the surface of these microparticles, which were procoagulant. Shearing platelet-rich plasma at the rate of 10 000 s ؊1 in a cone-and-plate viscosimeter increased microparticle counts up to 55-fold above baseline. Blocking the GPIb-VWF interaction abolished microparticle generation in both experimental conditions. Thus, a biomechanical process mediated by GPIb␣-VWF bonds in rapidly flowing blood may not only initiate platelet arrest onto reactive vascular surfaces but also generate procoagulant microparticles that further enhance thrombus formation. IntroductionThe integrity of the vessel wall is key for the normal circulation of blood and is constantly surveyed by platelets. 1 In arterial flow, platelets are positioned at high density near the endothelial-cell layer, while erythrocytes are lifted away from it through a hemodynamic process called axial migration. 2,3 When damage to the vascular surface occurs, von Willebrand factor (VWF) binds rapidly to exposed subendothelial structures 4,5 and enables platelet arrest from fast-flowing blood through the interaction of its A1 domain (VWFA1) with the platelet glycoprotein Ib␣ (GPIb␣) receptor. 6 The VWFA1-GPIb␣ bond has a short half-life and by itself cannot provide irreversible adhesion. Consequently, the torque imposed by flowing blood causes platelets to translocate over immobilized VWF until receptors such as glycoprotein VI or integrin ␣IIb␤3 engage their respective ligands and mediate permanent adhesion, spreading, and aggregation. 7 Under the effect of shear stress, platelet-derived microparticles (PMPs) can be generated in blood through a process that was reported to be dependent on the VWF-GPIb␣ interaction by some investigators 8 but not others. 9 Owing to their ability to bind coagulation factors, and the exposure on their surface of phosphatidylserine 9 as well as adhesion receptors 10 and possibly tissue factor, 11,12 PMPs have been suggested to play a role in blood clotting and thrombus formation. 13 Lacking so far, however, is a direct visualization and explanation of how shear stress can induce the generation of microparticles from platelets and how this may be linked to the subsequent development of thrombi. Becaus...

Lipid-rich atherosclerotic plaques are vulnerable, and their rupture can cause the formation of a platelet- and fibrin-rich thrombus leading to myocardial infarction and ischemic stroke. Although the role of plaque-based tissue factor as stimulator of blood coagulation has been recognized, it is not known whether plaques can cause thrombus formation through direct activation of platelets. We isolated lipid-rich atheromatous plaques from 60 patients with carotid stenosis and identified morphologically diverse collagen type I- and type III-positive structures in the plaques that directly stimulated adhesion, dense granule secretion, and aggregation of platelets in buffer, plasma, and blood. This material also elicited platelet-monocyte aggregation and platelet-dependent blood coagulation. Plaques exposed to flowing blood at arterial wall shear rate induced platelets to adhere to and spread on the collagenous structures, triggering subsequent thrombus formation. Plaque-induced platelet thrombus formation was observed in fully anticoagulated blood (i.e., in the absence of tissue factor-mediated coagulation). Mice platelets lacking glycoprotein VI (GPVI) were unable to adhere to atheromatous plaque or form thrombi. Human platelet thrombus formation onto plaques in flowing blood was completely blocked by GPVI inhibition with the antibody 10B12 but not affected by integrin alpha2beta1 inhibition with 6F1 mAb. Moreover, the initial platelet response, shape change, induced by plaque was blocked by GPVI inhibition but not with alpha2beta1 antagonists (6F1 mAb or GFOGER-GPP peptide). Pretreatment of plaques with collagenase or anti-collagen type I and anti-collagen type III antibodies abolished plaque-induced platelet activation. Our results indicate that morphologically diverse collagen type I- and collagen type III-containing structures in lipid-rich atherosclerotic plaques stimulate thrombus formation by activating platelet GPVI. This platelet collagen receptor, essential for plaque-induced thrombus formation, presents a promising new anti-thrombotic target for the prevention of ischemic cardiovascular diseases.

Summary. The physiological protection against bleeding is secured by platelet adhesion to the site of injury and sealing of the defect. The first step involves the arrest of platelets that have adhered to subendothelial structures, primarily collagen, at the site of injury. Under conditions of low shear rates, platelet adhesion to the damaged vessel wall is mediated by several proteins, including von Willebrand factor (VWF). However, under conditions of high shear, aggregation occurs only in the presence of soluble VWF. In solution, VWF becomes immobilized via its A3 domain on the fibrillar collagen of the vessel wall and acts as an intermediary between collagen and the platelet receptor glycoprotein Iba (GPIba), which is the only platelet receptor that does not require prior activation for bond formation. After GPIba binds to the A1 domain of its main ligand VWF, further activation of the platelet via intracellular signalling occurs, allowing other receptors to engage VWF and collagen and thereby reinforcing permanent adhesion. On this first layer of adherent platelets, soluble VWF binds and uncoils, thereby attracting more platelets. Platelet interaction with immobilized and soluble VWF may also generate platelet-derived microparticles that exhibit procoagulant activity. Full growth of a multilayered platelet aggregate comprises binding of the platelet receptor integrin aIIbb3 to VWF and fibrinogen. In addition, the surface of the activated platelets accelerates the coagulation cascade, which, by its end product fibrin, stabilizes the growing platelet thrombus. This article summarizes the characteristics and role of VWF in the coagulation cascade.