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.

, 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.

Key Points• Temporal profiles of .4000 phosphopeptides after stimulating human platelets (a) with ADP and (b) consecutively with ADP and Iloprost.• Reciprocal phosphorylation profiles of ADP and Iloprost point to central players of platelet homeostasis.Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin aIIbb3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPaseactivating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3A Ser312 , CALDAG-GEFI Ser587 , ENSA Ser109 ), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. (Blood. 2017;129(2):e1-e12)

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...

Key Points• TMEM16F is not essential for apoptosis-induced phosphatidylserine exposure in platelets.• Collagen plus thrombininduced phosphatidylserine exposure in platelets results from 2 distinct pathways, one being TMEM16F dependent.Scott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis-or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets. High, but not moderate, PS exposure in platelets was suppressed by chelation of intracellular Ca 21, whereas caspase inhibition completely abolished ABT737-induced PS exposure in both Scott and control platelets. On the other hand, high PS exposure induced by the Ca 21 -mobilizing agonists convulxin/thrombin fully relied on mitochondrial depolarization and was virtually absent in Scott platelets. Finally, PS exposure induced by collagen/thrombin was partly affected in Scott platelets, and the residual PS positive fraction was insensitive to inhibition of caspases or mitochondrial depolarization. In conclusion, TMEM16F is not required for, but enhances, caspase-dependent PS exposure; convulxin-/thrombininduced PS exposure is entirely dependent on TMEM16F, whereas collagen/thrombin-induced PS exposure results from 2 distinct pathways, one of which involves mitochondrial depolarization and is mediated by

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 disease.

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl−) and cation currents and is required for Ca2+-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl− currents and PS are related, whether both processes are Ca2+ dependent, and whether Ca2+-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca2+ increase by ionomycin activated Ano6 Cl− currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca2+, but activated Cl− currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl− channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl− currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl− ions. Remarkably, Ca2+-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl− currents are activated by increase in cytosolic Ca2+, or Ca2+ independent by stimulation of Fas receptors; (ii) Ca2+-dependent PS induced by Ano6 does not require Cl− currents; (iii) Ca2+-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca2+-dependent PS, but not by increasing intracellular Ca2+.