Objective-Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results-High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated ␣IIb␤3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active ␣IIb␤3. Conclusions-These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. Key Words: aggregation Ⅲ coagulation Ⅲ microdomains Ⅲ platelets Ⅲ integrin activation A ctivated platelets have a dual role in hemostasis and thrombosis. They form the building blocks of a thrombus and provide the membrane surface for coagulation factor activation, which results in thrombin and fibrin formation. 1 Once formed, thrombin greatly enhances platelet activation and aggregation. Given the strong interdependency of thrombin generation and platelet activation, it is intuitively assumed that those platelets that participate in aggregate formation are also involved in the coagulation process, but this has not been investigated.The mechanism(s) by which platelets contribute to thrombin generation and coagulation have been investigated for decades. Kinetic evidence shows that collagen/thrombin-activated platelets expose phosphatidylserine (PS) at their outer surface and then bind Gla domain-containing coagulation factors, mediating factor Xa and thrombin generation. 2 However, even after activation with strong agonists, only a subpopulation of the platelets tends to expose PS. 3 Conversely, it has been argued that PS exposure alone is insufficient to explain the procoagulant contribution of platelets. 4,5 There is also evidence that subfractions of activated platelets have different roles in the coagulation process. Several reports indicate an imperfect relation of PS exposure and the binding of coagulation factors Va, VIIIa, IXa, and Xa to the platelet surface. 6 -8 Another report characterizes a subfraction of activated platelets according to their so-called SCIP morphology (for susta...

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l ؊/؊ ) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l ؊/؊ platelets into Apoe ؊/؊ mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wildtype platelets. Moreover, Cd40l ؊/؊ platelets failed to form proinflammatory plateletleukocyte aggregates. Expression of CD40L on platelets was required for plateletinduced atherosclerosis as injection of Cd40l ؊/؊ platelets in contrast to Cd40l ؉/؉ platelets did not promote lesion formation. Remarkably, injection of Cd40l ؉/؉ , but not Cd40l ؊/؊ , platelets transiently decreased the amount of regulatory T cells

The platelet glycoproteins (GPs) Ib, integrin ␣ 2 ␤ 1 , and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s ؊1 ), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb␣ (12G1 Fab 2 ) and ␣ 2 ␤ 1 (6F1 mAb or GFOGER-GPP peptide).GPVI was found to be crucial for aggregate formation, Ca 2؉ signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting ␣ 2 ␤ 1 revealed its involvement in regulating Ca 2؉ signaling, PS exposure, and aggregate size. Both GPIb␣ and ␣ 2 ␤ 1 contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb␣ or ␣ 2 ␤ 1 . In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with ␣ 2 ␤ 1 mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation. IntroductionThe platelet response to exposed subendothelial matrix is fundamental to thrombosis and hemostasis. Uniquely, collagen, the most abundant vessel wall protein, mediates platelet adhesion and activation, localizing and regulating the hemostatic response at sites of injury. Discovering the molecular mechanisms that control platelet-collagen interaction is crucial for understanding the pathogenesis of arteriothrombotic diseases such as stroke and myocardial infarction. Under high shear rate conditions, the glycoprotein (GP) Ib/V/IX complex allows initial platelet rolling over von Willebrand factor (VWF) bound to subendothelial collagen fibers, and subsequently collagen receptors come into contact with their specific binding sequences in the collagen. For the next step, platelet arrest and activation, firm evidence exists of a role for only 2 receptors, integrin ␣ 2 ␤ 1 and immunoglobulin superfamily member GPVI, despite the apparent redundancy in collagen receptors (for a review, see Siljander PR-M and Farndale RW 1 ).According to the 2-site, 2-step model, high-affinity interaction through ␣ 2 ␤ 1 stops the platelet, allowing low-affinity binding of GPVI, which generates signaling required for the subsequent thrombus formation. Platelet deposition under flow was found to be dependent on GPIb/V/IX and ␣ 2 ␤ 1 , 2-4 whereas no platelet deposition occurred on the GPVI-specific substrate collagen-related peptide (CRP), even under low shear rates. 5 The limited number of studies with human platelets deficient in either GPVI or ␣ 2 ␤ 1 support the 2-site, ...