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SwedenTo cite this article: van der Meijden PEJ, van Schilfgaarde M, van Oerle R, Renné T, ten Cate H, Spronk HMH. Platelet-and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa. J Thromb Haemost 2012; 10: 1355-62.See also Shapiro S, Laffan M. Making contact with microparticles. This issue, pp 1352-4.Summary. Background: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. Objectives: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. Methods: Microparticles were isolated from calcium ionophore-stimulated platelets, erythrocytes and monocytic THP-1 cells. MPs were quantified, characterized for cellspecific antigens and analyzed for TF, PS exposure and their thrombin-generating potential. Results: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet-and erythrocyte-derived MPs (< 1 fM nM )1 PS), whereas monocyte-derived MPs exposed TF (32 fM nM )1 PS). Platelet-, erythrocyte-and monocyte-derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet-and erythrocyte-derived MPs, but interfered with monocyte MP-triggered coagulation. Platelet-and erythrocyte-derived MPs completely failed to induce thrombin generation in FXII-deficient plasma. In contrast, monocyte-derived MPs induced similar thrombin generation in normal vs. FXII-deficient plasma. Conclusion: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner. In contrast, monocyte-derived MPs trigger coagulation predominantly via TF.

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...

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca 2؉ entry (SOCE) with Orai1 as principal Ca 2؉ entry channel. Both proteins contribute to collagendependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca 2؉ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1 ] i rises are required for the procoagulant response (1, 2). The latter is achieved by a Ca 2ϩ -activated scramblase mechanism disturbing the normal phospholipid asymmetry in the plasma membrane, with, as a result, the exposure of phosphatidylserine (PS) 5 at the outer membrane surface (3, 4). Exposed PS provides high affinity binding sites for key coagulation factors and, thereby, facilitates the assembly of tenase and prothrombinase complexes, which are responsible for the formation of factor Xa and thrombin, respectively (3). Because thrombin is one of the most potent platelet agonists, the procoagulant platelet response triggers a potent positive feedback loop of platelet and coagulation activation. Recent in vivo studies have indicated that PS exposure and ensuing thrombin generation are key regulatory events in murine arterial thrombus formation (5, 6).Whereas stored platelets may expose procoagulant PS in a Ca 2ϩ -independent way, PS exposure in activated platelets relies on a high and prolonged rise in cytosolic [Ca 2ϩ ] i (7). Platelet stimulation with single G protein-coupled agonists, like thrombin and ADP, results in limited PS exposure (8, 9), but stimulation of the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI), with ligands such as collagen-related peptide (CRP) or convulxin, results in appreciable procoagulant activity (10, 11). Combined stimulation of the collagen and thrombin receptors though results in high PS exposure, likely because these agonists use different signaling pathways for mobilizing cytosolic Ca 2ϩ (1). Although thrombin transiently activates G q ␣ and phospholipase C␤2/␤3 isoforms, activation of GPVI causes a more persistent activation of the phospholipase C␥2 isoform (2, 12). For PS exposure, entry of extracellular Ca 2ϩ is required, complementing the Ca 2ϩ -mobilizing effect of phospholipase C stimulation, to reach sufficiently high [Ca 2ϩ ] i (10,13,14).

Key Points• Under physiological flow rates, plasminogen primarily accumulates on fibrin(ogen), emanating from platelets and initiates fibrinolysis.• Plasminogen is localized to defined "caps" on the surface of PS-exposing platelets in a fibrin(ogen)-dependent manner.The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and GlyPro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PSexposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding "cap." These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. (Blood. 2015;125(16):2568-2578 IntroductionPlatelet accumulation is central to the hemostatic response. Platelets are activated in vivo by numerous agonists of varying potency, including thrombin, collagen, adenosine 59diphosphate, and thromboxane A2. Platelets exhibit a nonuniform response to activation, with distinct populations forming with different surface characteristics.1 Aggregating platelets are characterized by a spherical shape, binding of fibrinogen, and expression of the active integrin a IIb b 3, and predominantly function in clot retraction. Highly activated platelets are observed on collagen fibers 1 and in the core region of a thrombus nearest the vascular injury.2 These platelets are characterized by membrane exposure of phosphatidylserine (PS), a rounded balloon-like structure, sustained increase in cytosolic Ca 21, and binding of coagulation factors. 3,4 PS-exposing platelets, also termed procoagulant platelets, substantially enhance the activity of the prothrombinase complex, 5,6 and subsequent thrombin and fibrin formation. 7 An additional subpopulation of platelets, termed "coated" platelets, are generated in response to strong dual agonist stimulatio...

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...

A therothrombosis, characterized by atherosclerotic lesion disruption with superimposed thrombus formation, is the major cause of acute coronary syndromes and cardiovascular death. Predominantly, platelet-rich (white) thrombi are formed at the ruptured area, because platelet recruitment preferentially occurs at regions of high shear rate and disturbed flow, whereas maximal fibrin generation occurs in regions of low flow.1 Activated platelets support the tissue factor (TF) pathway of blood coagulation by binding various (anti)coagulation factors.2 There is emerging evidence that platelets also support the intrinsic coagulation pathway mediated by factors XII (FXII) and XI (FXI), for example, by releasing polyphosphates, although the exact mechanism is still unclear.3-5 Better understanding of these multifaceted roles of platelets in coagulation stimulation can lead to new approaches to selectively inhibit the pathways most relevant in atherothrombosis. See accompanying editorial on page 1607Collagen type I is not only the most potent platelet activating component in atherosclerotic plaques by binding and activating the glycoprotein VI receptor, 6-8 but has also has been shown to bind to and activate FXII. 9 Several mouse studies using nonatherosclerotic vessels point to a consolidating role of the FXII pathway in arterial thrombus formation and thrombus stabilization, as concluded from experiments with genetic ablation or pharmacological inhibition of FXII.4,10-13 Interestingly, deficiency in FXII in man or mouse is not accompanied by abnormal bleeding.3,14 On the contrary, individuals with partly reduced FXII levels have an increased risk of cardiovascular disease (reviewed in Renné et al 15 and Woodruff et al 16 ), which indicates that the clinical consequences of (partial) FXII deficiency are more complex than the reported antithrombotic effects of FXII ablation in mice.To date, no experimental data exist on a role of the intrinsic coagulation pathway in pathological thrombus formation after atherosclerotic plaque rupture. A recent report studying plaque-induced thrombin generation in vitro suggested that the intrinsic FXII pathway may not play a key role in this process, such in contrast to a predominant role for plaque-derived TF triggering the extrinsic pathway. 8 In the present article, we therefore compared the roles of both the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques in vivo and ex vivo. Objective-Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo. Approach and Results-Plaque rupture after ultrasound treatment evoked immediate formation of subocclu...

Platelets interact with the coagulation system in a multitude of ways, not only during the phases of thrombus formation, but also in specific areas within a formed thrombus. This review discusses current concepts of platelet control of thrombin generation, fibrin formation and structure, and anticoagulation. Indicated are how combined signalling via the platelet receptors for collagen (glycoprotein VI) and thrombin induces the secretion of (anti)coagulation factors, as well as surface exposure of phosphatidylserine, thereby catalysing thrombin generation. This procoagulant platelet response is also facilitated by the adhesive complexes glycoprotein Ib‐V‐IX and integrin αIIbβ3. In the buildup of a platelet‐fibrin thrombus, the extrinsic, tissue factor–driven coagulation pathway is predominant in early stages, while the intrinsic, factor XII pathway seems to promote at later time points. Already early generation of thrombin enforces platelet responses and stimulates intra‐thrombus heterogeneity with patches of loosely aggregated, contracted, and phosphatidylserine‐exposing platelets. Fibrin actively formed on the surface of activated platelets supports thrombus growth, but also captures thrombin. The fibrin distribution in a thrombus appears to rely on the local procoagulant trigger and the blood flow rate. Clinical studies support the importance of the platelet‐coagulation interplay, by showing beneficial effects of combination therapy in the secondary prevention of cardiovascular disease.