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See also Brill A. A ride with ferric chloride. This issue, pp 776–8. Summary.  Background: The FeCl3‐induced vascular injury model is widely used to study thrombogenesis in vivo, but the processes leading to vascular injury and thrombosis are poorly defined. Objectives: The aim of our study was to better characterize the mechanisms of FeCl3‐induced vascular injury and thrombus formation, in order to evaluate the pathophysiological relevance of this model. Methods: FeCl3 was applied at different concentrations (from 7.5% to 20%) and for different time periods (up to 5 min) to mouse carotid or mesenteric arteries. Results: Under all the conditions tested, ultrastructural analysis revealed that FeCl3 diffused through the vessel wall, resulting in endothelial cell denudation without exposure of the inner layers. Hence, only the basement membrane components were exposed to circulating blood cells and might have contributed to thrombus formation. Shortly after FeCl3 application, numerous ferric ion‐filled spherical bodies appeared on the endothelial cells. Interestingly, platelets could adhere to these spheres and form aggregates. Immunogold labeling revealed important amounts of tissue factor at their surface, suggesting that these spheres may play a role in thrombin generation. Invitro experiments indicated that FeCl3 altered the ability of adhesive proteins, including collagen, fibrinogen and von Willebrand factor, to support platelet adhesion. Finally, real‐time intravital microscopy showed no protection against thrombosis in GPVI‐immunodepleted and β1−/− mice, suggesting that GPVI and β1 integrins, known to be involved in initial platelet adhesion and activation, do not play a critical role in FeCl3‐induced thrombus formation. Conclusion: This model should be used cautiously, in particular to study the earliest stage of thrombus formation.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9⌬ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin ␤3 phosphorylation and PtdIns(3,4)P 2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9⌬ platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl 3 -induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outsidein signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9⌬ mice. IntroductionImportant morphologic changes occur in platelets during their activation at sites of vascular injury. The cells lose their resting discoid shape to become spheroid and contracted, emitting membrane blebs and longer extensions. [1][2][3][4] Once in contact with a surface, the spheroid platelets extend long filopodia and finally spread over it by emitting thin, sheet-like lamellipodia. 1,2 Myosin activation plays a central role in the cytoskeletal rearrangements underlying these changes in morphology. Myosin becomes activated after phosphorylation of the myosin regulatory light chain (RLC), which results from both calcium-regulated myosin lightchain kinase activity and Rho kinase-regulated myosin phosphatase activity. [5][6][7][8] Activated myosin assembles into short filaments through the myosin heavy chain and interacts mainly with central actin filaments. Myosin has been proposed to participate in several platelet contractile functions such as platelet spheration, contraction and stress-fiber formation, and fibrin clot retraction. Platelet spheration and contraction, as observed in the aggregometer, closely correlate with phosphorylation of the RLC 9,10 and are prevented when RLC phosphorylation is inhibited. 6,7,9,10 Myosin has also been shown to be associated with stress fiber-like structures in spreading adherent platelets. 11 In addition, myosin could play a role in platelet secretion, as it is decreased by inhibition of myosin RLC phosphorylation. 5,[12][13][14][15] Finally, a role of myosin in clot retraction has long been suspected in view of the necessity for a contractile force and was ...

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Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of ␣ granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling. (Blood. 2010;115(16): 3364-3373) IntroductionAt sites of tissue trauma, platelets become activated and rapidly aggregate to form a plug that seals the wound and limits blood loss. On the other hand, platelet activation in pathologic situations can lead to thrombosis, causing myocardial infarction or stroke. Platelet activation by multiple signaling pathways leads to shape change, release of intracellularly stored granules, and spreading on immobilized ligands. Small GTPases of the Rho family, namely RhoA, Cdc42, and Rac1, are thought to play important roles in the cytoskeletal rearrangements occurring during platelet activation by facilitating the formation of stress fibers, filopodia and lamellipodia, respectively. 1 In platelets, signaling from G protein-coupled receptors, such as the thromboxane or thrombin receptors, as well as immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors (GPVI, CLEC-2) was shown to induce activation of Rho GTPases. 2,3 Cdc42 is a small (ϳ 23 kDa) protein that cycles between a GDP-bound inactive and a GTP-bound active state. 4 Cdc42 is an important mediator of filopodia formation in various cell types. According to the "convergent elongation model," active Cdc42 induces activation of Wiskott-Aldrich Syndrome protein (WASP). WASP subsequently activates the ARP2/3 complex, thereby increasing actin turnover and initiating the formation of parallel actin bundles. [5][6][7] Furthermore, Cdc42 can also bind to and activate IRSp53, which recruits the Ena/vasodilator-stimulated phosphoprotein (VASP) family protein Mena, thus promoting filopodi...

SummaryPlatelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y1 receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADP β S. Platelets incubated for 1 h at 37° C with 1 mM ADP β S and resuspended in Tyrode’s buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADP β S treated platelets loaded with fura2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT2A receptor while adrenaline had no such effect. These results suggested that the refractory state of ADP β S treated platelets was entirely due to desensitization of the P2Y1 receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y1 and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y1 and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y1 sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y1 receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y1 receptor, where ADP β S treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y1 and P2cyc receptors are differently regulated during platelet activation.

PI3KC2a is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2a in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2a-deficient platelets. These findings demonstrate an important role for PI3KC2a in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca 2؉ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP 3 ) and that treating platelets with the IP 3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca 2؉ flux and platelet shape change. Normal shape change was observed in G␣ q ؊/؊ mouse platelets, excluding a role for PLC␤ isoforms in this process. However, decreased shape change and Ca 2؉ mobilization were observed in mice lacking PLC␥2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca 2؉ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca 2؉ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc␥RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR␥ chain. Taken together, these studies define an important role for PLC␥2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.