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...

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, ...

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

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dosedependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase C␥2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factorVIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLC␥2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi. (Blood. 2009; 114:881-890) IntroductionVascular injury leads to exposure of hemostatically active components, such as tissue factor and collagen, to the bloodstream. For a long time, it has been considered that exposed tissue factor is the main vascular activator of the coagulation cascade. In the extrinsic pathway of coagulation, de-encrypted tissue factor complexes with circulating factor (F)VII(a), which activates a multistep cascade of serine proteases to form thrombin and fibrin. 1 The exposed collagen is thought to function as a substrate for the adhesion and activation of platelets via the glycoprotein VI (GPVI) receptor, which evokes an important signal transduction pathway in platelets. 2 Independently of tissue factor, the second intrinsic coagulation pathway is initiated by activation of plasma FXII (Hageman factor). Proteolytically active FXIIa mediates the sequential activation of the serine proteases, FXI and FIX, which also lead to thrombin generation. Until recently, the physiologic relevance of this pathway has remained obscure, as the initial trigger in vivo was not known. On the other hand, in vitro the intrinsic system is easily triggered in plasma using negatively charged materials, such as kaolin, glass, and ellagic acid. Common clotting tests, such as the activated partial thromboplastin time (aPTT), rely on activation of the intrinsic coagulation pathway. In the 1980s, it was proposed that polyanionic vascular components, such as cerebroside sulfates and glycosaminoglycans, function to stimulate FXII activation. 3,4 A similar role for collagen was suggested, 5,6 but this was refuted by other authors. [7][8][9] Recent studies with mice have greatly increased the inte...

Signaling from collagen and G proteincoupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin ␣ IIb ␤ 3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting ␣ IIb ␤ 3 ; (3) by blocking P2Y 12 receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) ␤. In murine blood, absence of PI3K␥ led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3K␤ delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y 12 or PI3K isoforms, resulting in single platelets that had inactivated ␣ IIb ␤ 3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y 12 and both PI3K␤ and PI3K␥ isoforms is required for perpetuated ␣ IIb IntroductionPlatelet plug formation at sites of vascular injury is considered to consist of 3 phases: initiation, propagation, and perpetuation. 1,2 The initiation phase involves platelet adhesion to von Willebrand factor (VWF) and to collagen exposed in the vessel wall. In the propagation phase, activated platelets secrete mediators such as ADP, thromboxane A 2 (TxA 2 ), and Gas6, which activate other platelets to form aggregates. In the perpetuation phase, fibrin formation and less well-understood postaggregation events are assumed to accomplish stabilization of the thrombus.At arterial shear rates, the thrombotic process is initiated by the tethering of platelets via glycoprotein (GP) Ib-IX-V to VWF, bound to collagen. 3 In human and murine platelets, 2 interacting collagen receptors, GPVI and integrin ␣ 2 ␤ 1 , mediate stable adhesion to collagen. [4][5][6][7] The signaling receptor GPVI triggers series of activation events, including integrin ␣ IIb ␤ 3 activation (providing binding sites, for example, for fibrinogen) and Ca 2ϩ mobilization and secretion, which all function to recruit other platelets. 8,9 During the propagation phase of thrombus formation, released ADP and TxA 2 in a paracrine way pursue the activation process, mediated by P2Y 1 , P2Y 12 , and TP␣ receptors. 8,10,11 The Gq-coupled P2Y 1 and TP␣ receptors evoke Ca 2ϩ mobilization and protein kinase C activity. The P2Y 12 signaling pathway involves Gimediated inhibition of adenylyl cyclase, which lowers cyclic AMP and indirectly enhances Ca 2ϩ signal generation. 12-14 P2Y 12 signaling via Gi also leads to activation of the phosphoinositide 3-kinase (PI3K) and protein kinase B signaling pathways. However, it is still unc...

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Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase C␥2, which bears similarity to the signaling pathway evoked by T-and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3K␣ or -␤ strongly and selectively suppressed GPVI-induced Ca 2؉ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase C␥2 by PI3K␣/␤. That PI3K␣ and -␤ have a non-redundant function in GPVIinduced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca 2؉ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3K␣/␤ was blocked or p85␣ was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3K␦ or -␥. Furthermore, PI3K␣/␤, but not PI3K␥, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3K␣ and -␤ isoforms are required for full GPVI-dependent platelet Ca 2؉ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3K␣ an interesting new target for anti-platelet therapy.Exposed collagen in a damaged vessel wall activates platelets via their immunoglobulin family receptor, glycoprotein VI (GPVI), 3 by using a complex signal transduction pathway, which is reminiscent to the pathway employed by immune receptors in T and B cells (1, 2). In platelets, tyrosine phosphorylation of the Fc receptor ␥-chain, linked to GPVI via Src family kinases, leads to a cascade of protein phosphorylation events, cumulating in the activation of phospholipase C␥2 (PLC␥2). This key effector enzyme triggers many downstream events, including production of inositol 1,4,5-trisphosphate (InsP 3 ), mobilization of cytosolic Ca 2ϩ , activation of integrin ␣ IIb ␤ 3 , secretion of platelet granules loaded with autocrine-stimulating agents (ADP and ATP), and exposure of negatively charged phosphatidylserine (PS) at the platelet surface to ensure coagulation (1, 3, 4). All these responses are potently triggered by GPVI ligands, which, besides collagen, include collagen-related peptides and the snake venom convulxin (5-7).One of the GPVI-induced signaling events contributing to PLC␥2 activation is activation of the protein/lipid kinase, phosphoinositide 3-kinase (PI3K) in both human and mouse platelets (8 -11). Evidence for this role ca...