Key Points: Myeloid cell TF-dependent venous thrombosis is under control of PDI and the complement cascade. C5 deficiency reduces fibrin formation and leukocyte PS exposure with normal platelet deposition in flow-restricted vessels.

The symbiotic gut microbiota plays pivotal roles in host physiology and the development of cardiovascular diseases, but microbiota-triggered pattern recognition signaling mechanisms impacting thrombosis are poorly defined. Here, we show that germ-free and Toll-like receptor (Tlr) 2-deficient mice have reduced thrombus growth following carotid artery injury relative to conventionally raised controls. Germ-free Tlr2^-/- and wild-type mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between genotypes. We identify reduced plasma levels of von Willebrand Factor (VWF) and reduced VWF synthesis specifically in hepatic endothelial cells as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2^-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in germ-free wild-type, Tlr2^-/-, and heterozygous Vwf^+/- mice that are all characterized by a modest reduction in plasma VWF level. Defective platelet matrix interaction can be restored by exposure to wild-type plasma or to purified VWF dependent on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2^-/- mice in vivo. These experiments delineate an unexpected pathway, in which microbiota-triggered TLR2 signaling alters the synthesis of pro-adhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth. Key points VWF synthesis in liver sinusoidal endothelial cells is regulated by gut microbiota through TLR2 signaling. Reduced plasma VWF in germ-free and Tlr2^-/- mice cause reduced thrombus formation at the ligation injured carotid artery.

Red blood cells (RBCs) influence rheology, and release ADP, ATP, and nitric oxide, suggesting a role for RBCs in hemostasis and thrombosis. Here, we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL/FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation, and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts between platelets and RBCs via FasL/FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL^–/– mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL/FasR pathway.

Aims: In a randomized, parallel, blinded study, we investigate the impact of clopidogrel, prasugrel, or ticagrelor on peripheral endothelial function in patients undergoing stenting for an acute coronary syndrome. Methods and results: The primary endpoint of the study was the change in endothelium-dependent flow-mediated dilation (FMD) following stenting. A total of 90 patients (age 62 ± 9 years, 81 males, 22 diabetics, 49 non-ST elevation myocardial infarctions) were enrolled. There were no significant differences among groups in any clinical parameter. Acutely before stenting, all three drugs improved FMD without differences between groups (P = 0.73). Stenting blunted FMD in the clopidogrel and ticagrelor group (both P < 0.01), but not in the prasugrel group. During follow-up, prasugrel was superior to clopidogrel [mean difference 2.13, 95% confidence interval (CI) 0.68–3.58; P = 0.0047] and ticagrelor (mean difference 1.57, 95% CI 0.31–2.83; P = 0.0155), but this difference was limited to patients who received the study therapy 2 h before stenting. Ticagrelor was not significantly superior to clopidogrel (mean difference 0.55, 95% CI −0.73 to 1.82; P = 0.39). No significant differences were seen among groups for low-flow-mediated dilation. Plasma interleukin (IL)-6 (P = 0.02 and P = 0.01, respectively) and platelet aggregation reactivity in response to adenosine diphosphate (P = 0.002 and P = 0.035) were lower in the prasugrel compared to clopidogrel and ticagrelor group. Conclusion: As compared to ticagrelor and clopidogrel, therapy with prasugrel in patients undergoing stenting for an acute coronary syndrome is associated with improved endothelial function, stronger platelet inhibition, and reduced IL-6 levels, all of which may have prognostic implications. This effect was lost in patients who received the study medication immediately after stenting. EUDRACT-No: 2011-005305-73

Human platelets are key players in a multitude of physiological and pathological processes. Upon activation they release cargo from different types of granules as well as microparticles in an apparently well-regulated and orchestrated manner. The resulting specific platelet releasates create microenvironments of biologically active compounds and proteins during platelet aggregation and thrombus formation, allowing efficient delivery of growth factors and immune modulators to their sites of effect and enhancing the coagulative response in a positive feedback loop. Thus, platelet releasates play a central role in the regulation of platelet homeostasis and heterotypic cell interaction. Additionally, it recently emerged that both the qualitative and quantitative composition of the releasate as well as release dynamics may be stimulus dependent and therefore more complex than expected. Mass spectrometry-based proteomics is an important asset for studying platelet releasates in vitro, as it allows not only (i) identifying released proteins, but moreover (ii) determining their quantities and the dynamics of release as well as (iii) differentially comparing releasates across a variety of conditions. Though owing to the high sensitivity and comprehensiveness of modern proteomic techniques, a thorough experimental design and a standardized and robust sample preparation are essential to obtain highly confident and reliable insights into platelet biology and pathology. Here, we review releasate proteome studies and crucial sample preparation strategies to summarize possible achievements of state-of-the-art technologies and furthermore discuss potential pitfalls and limitations. We provide a future perspective of platelet releasate proteomics including targeted analyses, post-translational modifications and multi-omics approaches that should be adopted by platelet releasate researchers due to their tremendous depth and comprehensiveness.