Summary. Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell-based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca 2+ and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated a IIb b 3 regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet-based coagulation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann’s thrombasthenia, Hermansky–Pudlak syndrome, May–Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

, or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. Conclusions-Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Background: Inactivation of integrin ␣ IIb ␤ 3 reverses platelet aggregate formation upon coagulation. Results and conclusion: Platelets from patient (Scott) and mouse (Capn1 Ϫ/Ϫ and Ppif Ϫ/Ϫ ) blood reveal a dual mechanism of ␣ IIb ␤ 3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. Significance: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets.

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...

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCα and PKCβ and novel isoforms PKCδ and PKCθ. Here, we used a genetic approach to definitively demonstrate the role played by PKCα in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca -/-mice, and PKCα was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca -/-platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCα expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca -/-mice. In summary, the effects of PKCα ablation on thrombus formation and granule secretion may implicate PKCα as a drug target for antithrombotic therapy.

Custom-made and commercial parallel-plate flow chambers are widely used for studies of platelet activation and thrombus formation in whole blood at defined shear rates. When used in a reproducible way, such flow chamber devices give valuable information on the thrombogenic potential of human, mouse, or rat blood. This article aims to provide a practical guide for the use of parallel-plate flow chambers in combination with routine microscopic imaging techniques. The following methodological aspects are addressed: preparation of surface coatings, calculation of blood flow and shear rate, control of pre-analytical variables, protocols for routine performing of flow chamber tests with non-coagulating or coagulating blood, and procedures for real-time and end-point analysis of thrombus formation. Frequently encountered experimental problems and artifacts are discussed, as well as possibilities for using flow chamber devices as a diagnostic tool to test antithrombotic medication.