The effects of arterial flow on platelet activation, thrombus growth, and stabilization

Injury of an arterial vessel wall acutely triggers a multifaceted process of thrombus formation, which is dictated by the high-shear flow conditions in the artery. In this overview, we describe how the classical concept of arterial thrombus formation and vascular occlusion, driven by platelet activation and fibrin formation, can be extended and fine-tuned. This has become possible because of recent insight into the mechanisms of: (i) platelet-vessel wall and platelet-platelet communication, (ii) autocrine platelet activation, and (iii) platelet-coagulation interactions, in relation to blood flow dynamics. We list over 40 studies with genetically modified mice showing a role of platelet and plasma proteins in the control of thrombus stability after vascular injury. These include multiple platelet adhesive receptors and other junctional molecules, components of the ADP receptor signalling cascade to integrin activation, proteins controlling platelet shape, and autocrine activation processes, as well as multiple plasma proteins binding to platelets and proteins of the intrinsic coagulation cascade. Regulatory roles herein of the endothelium and other blood cells are recapitulated as well. Patient studies support the contribution of platelet- and coagulation activation in the regulation of thrombus stability. Analysis of the factors determining flow-dependent thrombus stabilization and embolus formation in mice will help to understand the regulation of this process in human arterial diseas

Combined quantification of the global proteome, phosphoproteome, and proteolytic cleavage to characterize altered platelet functions in the human Scott syndrome

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca(2+)-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca(2+)-dependent changes that are normally associated with phosphatidylserine exposure

Coordinated membrane ballooning and procoagulant spreading in human platelets

Background—Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. Methods and Results—Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na+, Cl–, and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na+, Cl–, 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