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Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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 αIIbβ3 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/GTPase-activating 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., (© 2017 by The American Society of Hematology.)

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., Competing Interests: We declare no conflicts of interest., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)β3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)β3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)β3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)β3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)β3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)β3., (Copyright© Ferrata Storti Foundation.)

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome., (© FASEB.)

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., (© 2015 American Heart Association, Inc.)

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet-platelet interactions and thrombus formation under conditions relevant for atherothrombosis., Approach and Results: Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg(-/-)Apoe(-/-)) showed diminished αIIbβ3 activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40(-/-)Apoe(-/-)) were not decreased. Using blood from Cd40lg(-/-)Apoe(-/-) and Cd40(-/-)Apoe(-/-) mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe(-/-) blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI-induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb(R/R) platelets or by inhibiting phosphatidylinositol 3-kinase β (PI3Kβ). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kβ. Finally, platelets from Chuk1(A/A)Apoe(-/-) mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation., Conclusions: Under atherogenic conditions, CD40L enhances collagen-induced platelet-platelet interactions by supporting integrin αIIbβ3 activation, secretion and thrombus growth via PI3Kβ, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation., (© 2015 American Heart Association, Inc.)

Rationale: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbβ3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity., Objective: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development., Methods and Results: JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbβ3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbβ3 signaling in vitro., Conclusions: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease., (© 2014 American Heart Association, Inc.)

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 subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas., Conclusions: The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability., (© 2014 American Heart Association, Inc.)

Background: Cold-stored platelets are increasingly being used to treat bleeding. Differences in manufacturing processes and storage solutions can affect platelet quality and may influence the shelf life of cold-stored platelets. PAS-E and PAS-F are approved platelet additive solutions (PAS) in Europe and Australia, or the United States respectively. Comparative data are required to facilitate international transferability of laboratory and clinical data., Study Design and Methods: Single apheresis platelets from matched donors (n = 8) were collected using the Trima apheresis platform and resuspended in either 40% plasma/60% PAS-E or 40% plasma/60% PAS-F. In a secondary study, platelets in PAS-F were supplemented with sodium citrate, to match the concentration in PAS-E. Components were refrigerated (2-6°C) and tested over 21 days., Results: Cold-stored platelets in PAS-F had a lower pH, a greater propensity to form visible (and micro-) aggregates, and higher activation markers compared to PAS-E. These differences were most pronounced during extended storage (14-21 days). While the functional capacity of cold-stored platelets was similar, the PAS-F group displayed minor improvements in ADP-induced aggregation and TEG parameters (R-time, angle). Supplementation of PAS-F with 11 mM sodium citrate improved the platelet content, maintained the pH above specifications and prevented aggregate formation., Discussion: In vitro parameters were similar during short-term cold storage of platelets in PAS-E and PAS-F. Storage in PAS-F beyond 14 days resulted in poorer metabolic and activation parameters. However, the functional capacity was maintained, or even enhanced. The presence of sodium citrate may be an important constituent in PAS for extended cold storage of platelets., (© 2023 AABB.)

Background: Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation., Results and Conclusion: Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 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. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

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(2+), 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(2+)-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-/thrombin-induced 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 TMEM16F.

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 α(IIb) β(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., (© 2012 International Society on Thrombosis and Haemostasis.)

Platelets are unique anucleated blood cells that constantly patrol the vasculature to seal and prevent injuries in a process termed haemostasis. Thereby they rapidly adhere to the subendothelial matrix and recruit further platelets, resulting in platelet aggregates. Apart from their central role in haemostasis, they also kept some of their features inherited by their evolutionary ancestor-the haemocyte, which was also involved in immune defences. Together with leukocytes, platelets fight pathogenic invaders and guide many immune processes. In addition, they rely on several signalling pathways which are also relevant to immune cells. Among these, one of the central signalling hubs is the PI3K pathway. Signalling processes in platelets are unique as they lack a nucleus and therefore transcriptional regulation is absent. As a result, PI3K subclasses fulfil distinct roles in platelets compared to other cells. In contrast to leukocytes, the central PI3K subclass in platelet signalling is PI3K class Iβ, which underlines the uniqueness of this cell type and opens new ways for potential platelet-specific pharmacologic inhibition. An overview of platelet function and signalling with emphasis on PI3K subclasses and their respective inhibitors is given in this chapter., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days., Study Design and Methods: This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics., Results: CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs., Conclusion: Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients., (© 2020 AABB.)

Background: Cryopreservation of platelets with dimethylsulfoxide (DMSO) at -80°C increases their shelf life from days to years. Once thawed, platelets are stored at room temperature (RT), and the shelf life is limited to 4-6 hours. However, refrigeration (cold storage) may facilitate a prolongation of the shelf life of thawed platelets., Study Design and Methods: ABO-matched buffy coat-derived platelets (30% plasma/70% SSP+) were cryopreserved at -80°C in 5%-6% DMSO. Paired cryopreserved platelet components were thawed, resuspended in 30% plasma/70% SSP+, and then stored at either 20°C-24°C with agitation (RT) or at 2°C-6°C (cold). In vitro platelet quality was assessed over 10 days of postthaw storage., Results: During postthaw storage, the platelet concentration of RT-stored components decreased significantly more than components in cold storage (Day 10 RT 58 ± 10 × 10 9 /unit vs Day 10 cold 142 ± 16 × 10 9 /unit; P < .0001). Cold storage reduced the metabolic rate of thawed platelets. During storage, the surface glycoprotein ([GP] Ibα, GPVI, GPIIb, GPIIIa) and activation marker (P-selectin and phosphatidylserine) profile of cold platelets was closer to freshly thawed platelets (Day 0) than those stored at RT. Thromboelastography (reaction time) demonstrated that the procoagulant nature of cryopreserved platelets was preserved during 10 days of cold storage, while RT-stored thawed platelets displayed a gradual prolongation of the time taken to initiate clot formation., Conclusion: Cold storage of thawed platelets preserves the platelet phenotype and function for up to 10 days, compared to thawed platelets stored at RT. Thus, cold storage of thawed platelets may represent a simple approach to extend the postthaw shelf life., (© 2020 AABB.)

Thrombin converts fibrinogen to fibrin and activates blood and vascular cells in thrombo-inflammatory diseases. Platelets are amplifiers of thrombin formation when activated by leukocyte- and vascular cell-derived thrombin. CD36 on platelets acts as sensitizer for molecules with damage-associated molecular patterns, thereby increasing platelet reactivity. Here, we investigated the role of CD36 in thrombin-generation on human platelets, including selected patients with advanced chronic kidney disease (CKD). Platelets deficient in CD36 or blocked by anti-CD36 antibody FA6.152 showed impaired thrombin generation triggered by thrombin in calibrated automated thrombography. Using platelets with congenital function defects, blocking antibodies, pharmacological inhibitors, and factor-depleted plasma, CD36-sensitive thrombin generation was dependent on FXI, fibrin, and platelet signaling via GPIbα and SFKs. CD36-deficiency or blocking suppressed thrombin-induced platelet αIIbβ3 activation, granule exocytosis, binding of adhesion proteins and FV, FVIII, FIX, FX, but not anionic phospholipid exposure determined by flow cytometry. CD36 ligated specifically soluble fibrin, which recruited distinct coagulation factors via thiols. Selected patients with CKD showed elevated soluble fibrin plasma levels and enhanced thrombin-induced thrombin generation, which was normalized by CD36 blocking. Thus, CD36 is an important amplifier of platelet-dependent thrombin generation when exposure of anionic phospholipids is limited. This pathway might contribute to hypercoagulability in CKD., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Platelets play a critical role in both haemostasis and thrombosis, and it is now evident that not all platelets behave the same when they are called to action. A functionally distinct subpopulation of platelets forms in response to maximal agonist stimulation: the procoagulant platelet. This platelet subpopulation is defined by its ability to expose phosphatidylserine on its surface, allowing for coagulation factor complexes to form and generate bursts of thrombin and fibrin to stabilize platelet clots. Reduced levels of procoagulant platelets have been linked to bleeding in Scott's syndrome and haemophilia A patients, and elevated levels have been demonstrated in many thrombotic disorders, including identifying patients at higher risk for stroke recurrence. One obstacle for incorporating an assay for measuring procoagulant platelets into clinical management algorithms is the lack of consensus on the exact definition and markers for this subpopulation. This review will outline the biological characteristics of procoagulant platelets and the laboratory assays currently used to identify them in research settings. It will summarize the findings of clinical research demonstrating the relevance of measuring the procoagulant platelet levels in patients and will discuss how an appropriate assay can be used to elucidate the mechanism behind the formation of this subpopulation, facilitating novel drug discovery to improve upon current outcomes in cardiovascular and other thrombotic disorders., (© 2020 John Wiley & Sons Ltd.)

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Coincubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to prefreeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction. [ABSTRACT FROM AUTHOR]

Various modifications of proteins and the resulting proteoforms of a protein can associate with many diseases and are also significantly involved in the rapid regulation of hemostasis and thrombosis. For example, the release of prostacyclin from the intact endothelium and the consequent following phosphorylation of VASP in platelets is a post-translational regulation to keep them in a quiescent state. In Alzheimer's disease, proteoforms arise from the altered cleavage of the amyloid precursor protein, which finally causes amyloid plaques in the brain. This changed processing of the amyloid precursor protein can also be detected in platelets, making them an attractive source of biomarkers for this neurodegenerative disease. Age-related or prothrombotic disorders can have multiple origins, including genomic, transcriptional, and translational factors, which together can be mapped at the proteome level. Hence, recording these dynamic protein changes under physiological and pathophysiological conditions is paramount in platelet proteomics. To effectively study diseases through platelet proteomics, it is crucial to consider platelets' primary regulatory mechanism and thoroughly evaluate the disparities between the two leading proteomics technologies, top-down and bottom-up approaches. This commentary provides insights into the differences between these two technologies, which are particularly noticeable in detecting the different proteoforms of a protein. [ABSTRACT FROM AUTHOR]

Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage. [ABSTRACT FROM AUTHOR]

F11 receptor (F11R)/Junctional Adhesion Molecule -A (JAM-A) is a transmembrane protein which belongs to the immunoglobulin superfamily of cell adhesion molecules. F11R/JAM-A is present in epithelial cells, endothelial cells, leukocytes, and blood platelets. In epithelial and endothelial cells, it takes part in the formation of tight junctions. In these structures, molecules of F11R/JAM-A located on adjacent cells form homodimers and thus take part in stabilization of cellular layer integrity. In leukocytes, F11R/JAM-A was shown to play role in their transmigration through the vascular wall. Paradoxically, the function of F11R/JAM-A in blood platelets, where it was primarily discovered, is much less understood. It has been proven to regulate downstream signaling of aIIbß3 integrin and to mediate platelet adhesion under static conditions. It was also shown to contribute to transient interactions of platelets with inflamed vascular wall. The review is aimed at summarizing the current state of knowledge of the platelet pool of F11R/JAM-A. The article also presents perspectives of the future research to better understand the role of this protein in hemostasis, thrombosis, and other processes where blood platelets are involved. [ABSTRACT FROM AUTHOR]

The anucleate human platelets contain a broad pattern of mRNAs and other RNA transcripts. The high quantitative similarity of mRNAs in megakaryocytes and platelets from different sources points to a common origin, and suggests a random redistribution of mRNA species upon proplatelet formation. A comparison of the classified platelet transcriptome (17.6k transcripts) with the identified platelet proteome (5.2k proteins) indicates an underrepresentation of: (i) nuclear but not of other organellar proteins; (ii) membrane receptors and channels with low transcript levels; (iii) transcription/translation proteins; and (iv) so far uncharacterized proteins. In this review, we discuss the technical, normalization and databasedependent possibilities and challenges to come to a complete, genome-wide platelet transcriptome and proteome. Such a reference transcriptome and proteome can serve to further elucidate intra-subject and inter-subject differences in platelets in health and disease. Applications may also lay in the aid of genetic diagnostics. [ABSTRACT FROM AUTHOR]

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets. [ABSTRACT FROM AUTHOR]

Cryopreservation of platelets, at − 80 °C with 5–6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1−/CD42b+/CD62P+) and a novel population (AnnV+/PAC1−/CD42b+/CD62P−) that did not align with the phenotype of aggregatory (AnnV−/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1−/CD42b−/CD62P−) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist. [ABSTRACT FROM AUTHOR]

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets. Platelets express two sizes of the Syk molecule with possible alternate functions in the cell. We need to understand how these two differ in their structure so that further studies can be developed by selectively deleting one of them to evaluate their function in platelets. This study shows that platelet Syk molecules differ in their structure with and without a linker region in the molecule. [ABSTRACT FROM AUTHOR]

The anucleate human platelets contain a broad pattern of mRNAs and other RNA transcripts. The high quantitative similarity of mRNAs in megakaryocytes and platelets from different sources points to a common origin, and suggests a random redistribution of mRNA species upon proplatelet formation. A comparison of the classified platelet transcriptome (17.6k transcripts) with the identified platelet proteome (5.2k proteins) indicates an under-representation of: (i) nuclear but not of other organellar proteins; (ii) membrane receptors and channels with low transcript levels; (iii) transcription/translation proteins; and (iv) so far uncharacterized proteins. In this review, we discuss the technical, normalization and database-dependent possibilities and challenges to come to a complete, genome-wide platelet transcriptome and proteome. Such a reference transcriptome and proteome can serve to further elucidate intra-subject and inter-subject differences in platelets in health and disease. Applications may also lay in the aid of genetic diagnostics. Blood platelets contain thousands types of mRNAs and proteins. The mRNA composition is similar to the mRNAs of megakaryocytes, from which platelets are derived, suggesting a random redistribution upon platelet formation. First attempts to identify all classified platelet proteins from mass spectral analysis used the genome-wide information of all mRNA types. This analysis revealed that the so far absent proteins in platelets are especially located in the megakaryocyte nucleus, or have low mRNA levels or low copy numbers. In this review, we discuss the possibilities to come to subject-dependent identification of the platelet protein and mRNA composition. Future applications may aid the genetic diagnostics. [ABSTRACT FROM AUTHOR]

The objective: to study the state of the platelet link of hemostasis in pregnant women with placental dysfunction. Materials and methods. A clinical and laboratory analysis of 54 patients with placental dysfunction was carried out. The control group included 30 practically healthy women with a physiological course of pregnancy. Venous blood of the patients was collected for testing using vacuum systems in tubes with 3.8% sodium citrate. The functional activity of platelets was studied on a photooptical aggregometer AP2110 (Solar, Belarus), thrombocytogram was performed on a hematological analyzer H18 LIGHT (SFRI SAS, France), thromboelastometric tests were determined on the ROTEM delta system (Tem Innovations GmbH, Germany). Results. Platelet hemostasis has a significant effect on maximum clot firmness (MCF) according to ROTEM results in patients with placental dysfunction. This indicator can be effective in determination of the hyperreactivity of the platelet unit in patients with placental dysfunction. Although no statically significant difference was found in the optical aggregometry indicators induced by ADP and collagen between the patients with placental dysfunction and the control group, a clear tendency to a sharp reduction in the lag-period of collagen-induced platelet aggregation in patients with placental dysfunction should be mentioned. Conclusions. Disorders in the platelet chain of hemostasis can play a significant role in the formation of a thrombophilic state in patients with placental dysfunction, as well as the damage of the endothelium and coagulation changes. The use of a test based on collagen-induced platelet aggregation may be a perspective method for effective diagnosis of platelet hyperreactivity. The study of the platelet link should become an additional element of the laboratory examination in order to resolve the issue of the need to prescribe antiplatelet agents to prevent the development of placental dysfunction. [ABSTRACT FROM AUTHOR]

Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin αIIbβ3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, the results suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype. [ABSTRACT FROM AUTHOR]

Variants of the Diaphanous-Related Formin 1 (DIAPH-1) gene have recently been reported causing inherited macrothrombocytopenia. The essential/"diagnostic" characteristics associated with the disorder are emerging; however, robust and complete criteria are not established. Here, we report the first cases of DIAPH1-related disorder in Australia caused by the autosomal dominant gain-of-function DIAPH1 R1213X variant formed by truncation of the protein within the diaphanous auto-regulatory domain (DAD) with loss of regulatory motifs responsible for autoinhibitory interactions within the DIAPH1 protein. We affirm phenotypic changes induced by the DIAPH1 R1213X variant to include macrothrombocytopenia, early-onset progressive sensorineural hearing loss, and mild asymptomatic neutropenia. High-resolution microscopy confirms perturbations of cytoskeletal dynamics caused by the DIAPH1 variant and we extend the repertoire of changes generated by this variant to include alteration of procoagulant platelet formation and possible dental anomalies. [ABSTRACT FROM AUTHOR]

Platelets are anucleate cytoplasmic fragments derived from the fragmentation of medullary megakaryocytes. Activated platelets adhere to the damaged endothelium by means of glycoproteins on their surface, forming the platelet plug. Activated platelets can also secrete the contents of their granules, notably the growth factors contained in the α-granules, which are involved in platelet aggregation and maintain endothelial activation, but also contribute to vascular repair and angiogenesis. Platelets also have a major inflammatory and immune function in antibacterial defence, essentially through their Toll-like Receptors (TLRs) and Sialic acid-binding immunoglobulin-type lectin (SIGLEC). Platelet activation also contributes to the extensive release of anti- or pro-inflammatory mediators such as IL-1β, RANTES (Regulated on Activation, Normal T Expressed and Secreted) or CD154, also known as the CD40-ligand. Platelets are involved in the direct activation of immune cells, polynuclear neutrophils (PNNs) and dendritic cells via the CD40L/CD40 complex. As a general rule, all of the studies presented in this review show that platelets are capable of covering most of the stages of inflammation, primarily through the CD40L/CD40 interaction, thus confirming their own role in this pathophysiological condition. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) remain leading causes of death worldwide. While platelet-mediated thrombus formation following the rupture of an atherosclerotic plaque is one of the key pathophysiologic events in CVDs, the role of platelets in previous or more advanced stages of atherosclerosis is less known. Interestingly, the presence of platelets has been observed at the core of the atherosclerotic plaque. In order to study the conditions necessary for platelets to migrate toward an atherosclerotic lesion, we designed an in vitro co-culture model. Platelets were co-cultured with monocytes in Transwell inserts covered with a confluent endothelium and the number of migrating platelets and/or monocytes was determined under different conditions. Platelets were also exposed to media conditioned obtained from co-cultures prior to migration assays. Here we show that coculturing platelets and monocytes increased platelet transmigration, with a considerable number of transmigrated platelets found not associated to monocytes. Interestingly, conditioned media from platelet-monocyte co-cultures also increased platelet transmigration and aggregation, suggesting the existence of soluble factors secreted by monocytes that enhance the migratory and pro-aggregating capabilities of platelets. We conclude that platelets have the machinery to migrate through an activated endothelium, a response that requires the interaction with secreted factors produce in the context of the interaction with monocytes under atherogenic conditions. [ABSTRACT FROM AUTHOR]

Platelets are damage sentinels of the intravascular compartment, initiating and coordinating the primary response to tissue injury. Severe trauma and hemorrhage induce profound alterations in platelet behavior. During the acute post-injury phase, platelets develop a state of impaired ex vivo agonist responsiveness independent of platelet count, associated with systemic coagulopathy and mortality risk. In patients surviving the initial insult, platelets become hyper-responsive, associated with increased risk of thrombotic events. Beyond coagulation, platelets constitute part of a sterile inflammatory response to injury: both directly through release of immunomodulatory molecules, and indirectly through modifying behavior of innate leukocytes. Both procoagulant and proinflammatory aspects have implications for secondary organ injury and multiple-organ dysfunction syndromes. This review details our current understanding of adaptive and maladaptive alterations in platelet biology induced by severe trauma, mechanisms underlying these alterations, potential platelet-focused therapies, and existing knowledge gaps and their research implications. [ABSTRACT FROM AUTHOR]

Platelets can respond to multiple antagonists and agonists, implying that their activation state is a consequence of past exposure to these substances. While platelets are often considered as one-time responsive cells, they likely can respond to sequential application of inhibitors and stimuli. We hypothesized that the ability of platelets to sequentially respond depends on the time and type of repeated agonist application. The present proof-of-concept data show that iloprost (cAMP elevation), tirofiban (integrin αIIbβ3 blocker) and Syk kinase inhibition subacutely modulated platelet aggregation, i.e. halted this process even when applied after agonist. In comparison to thrombin-activated receptor (PAR) stimulation, glycoprotein VI (GPVI) stimulation was less sensitive to time-dependent blockage of aggregation, with Syk inhibition as an exception. Furthermore, cytosolic Ca2+ measurements indicated that, when compared to PAR, prior GPVI stimulation induced a more persistent, priming activation state of platelets that influenced the response to a next agent. Overall, these data point to an unexpected priming memory of activated platelets in subacutely responding to another inhibitor or stimulus, with a higher versatility and faster offset after PAR stimulation than after GPVI stimulation. [ABSTRACT FROM AUTHOR]

Background: Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results: The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions: Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences. [ABSTRACT FROM AUTHOR]

Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51–91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0–18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment. [ABSTRACT FROM AUTHOR]

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow‐cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real‐time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo‐3 (Ca2+), ION NaTRIUM Green‐2 (Na+), and ION Potassium Green‐2 (K+). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR‐1 agonist), AYPGKF (PAR‐4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and scanning electron microscopy (EM) provided a foundation for our initial understanding of platelet structure and how it changes upon platelet activation. EM approaches have since been utilized to study platelets and thrombi in the context of basic, translational and clinical research, and they are instrumental in the diagnosis of multiple platelet function disorders. In this brief review, we provide a sampling of the many contributions EM based studies have made to the field, including both historical highlights and contemporary applications. We will also discuss exciting new imaging modalities based on EM and their utility for the study of platelets, hemostasis and thrombosis into the future. [ABSTRACT FROM AUTHOR]

Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives. [ABSTRACT FROM AUTHOR]

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions. We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic "leak" hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type. [ABSTRACT FROM AUTHOR]

Microparticles are carriers of signals for intracellular signal transduction. These carriers include proteins, mRNAs, microRNAs and other bioactive substances. Platelets are a major source of circulating microparticles, and microparticles are closely associated with the development of certain cardiovascular diseases. In the present study, a method for separating, extracting and identifying platelet-derived microparticles was developed and differences in the expression of surface proteins on microparticles harvested from platelets stimulated by vortexing or treatment with thrombin was investigated. The counts, composition, sizes and inner structures of microparticles were determined using flow cytometry and transmission electron microscopy. Additionally, it was demonstrated that platelets could be readily activated, and a large quantity of microparticles with varying complex compositions, structures and sizes were derived from activated platelets. High purity platelet-derived microparticles were obtained by gradient centrifugation. However, the microparticles derived from platelets stimulated by thrombin treatment or vortexing differed significantly in the levels of CD63. The present study aimed to provide improved options for the extraction and identification of microparticles. [ABSTRACT FROM AUTHOR]

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) “pearling” of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, “podiasomes” and PLT “ghosts”. Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency. [ABSTRACT FROM AUTHOR]

Background: Collagen- and thrombin-activated (COAT) platelets (PLTs), generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. There is evidence that higher amounts of procoagulant COAT PLTs are associated with stroke, while a decreased ability to generate them is associated with bleeding diathesis. Our aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates (PCs) from buffy coat. Thus, we investigated the effect of processing, pathogen inactivation treatment (amotosalen-UVA), and PC storage.Study Design and Methods: Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated (n = 5). Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC-1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxin (agonist of the collagen receptor GPVI) and THR.Results: Preparation of PCs resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 5'-diphosphate sodium (ADP). Storage further decreased ADP response. Minor differences were observed between untreated or amotosalen-UVA-treated PCs.Conclusion: Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP. [ABSTRACT FROM AUTHOR]

Tissue factor (TF), the key activator of the blood coagulation cascade and of thrombus formation, is also expressed by circulating human platelets. Despite the documented in-depth characterization of platelet TF carried out in the past 15 years, some authors still fail to identify TF in platelets, especially when assessment in platelet-rich plasma (PRP) or washed platelets is carried out. This study aims to extend the characterization of the subset of TF-positive platelets in PRP from healthy subjects and to verify how different centrifugation forces, used to prepare the PRP, could affect the analysis of TF-positive platelets. Data indicate that large-size platelets express significantly higher amount of TF compared to small-size cells, in terms of both TF protein and TF mRNA. Upon stimulation, large platelets readily expose on the cell membrane TF, which is functionally active, i.e., able to generate factor Xa (FXa) as well as thrombin. By contrast, TF activity in small platelets is almost completely quenched by tissue factor pathway inhibitor (TFPI), becoming indeed detectable only after treatment with an anti-TFPI antibody. Our data highlight that particular attention must be paid to the preparation and collection of the PRP since such preanalytical variables may influence the platelet recovery and in turn affect subsequent analysis, whether it is flow cytometry, functional activity tests, proteome, or transcriptome analysis. Indeed, the TF-positive subset of large platelets can easily be lost if centrifugation protocols are not optimized, thus erroneously leading to a false-negative result. [ABSTRACT FROM AUTHOR]

Background: Pathogen reduction technologies (PRTs) are considered for the implementation of safer platelet (PLT) transfusion. PRT treatment involves the addition of a photosensitizer to a blood component followed by ultraviolet (UV) light irradiation. However, the effects of PRT treatment on PLT thrombus formation and thrombus stability have not been satisfactorily clarified.Study Design and Methods: Leukoreduced PLT concentrates (PCs) were treated with riboflavin and UV light (Mirasol PRT). PLT thrombus formation on collagen was evaluated by the microchannel method, by which the total amount of PLTs deposited was measured as indices of thrombus formation and thrombus stability. Using a cone-plate shear-induced PLT aggregometer, PLT reactivity in blood flow was examined in a wide range of shear stresses of 6 to 108 dyn/cm2 .Results: There was no significant difference in surface coverage between PRT-treated PLTs and control PLTs on collagen. On the other hand, the total amount of PRT-treated PLTs deposited was higher than that of control PLTs. The promotive effect of PRT treatment on PLT deposition completely disappeared in the presence of tirofiban, a potent integrin αIIbβ3 inhibitor. The percentage of the dissociation of PRT-treated PLTs on collagen was lower than that of control PLTs after flushing with phosphate-buffered saline. PRT treatment significantly inhibited PLT aggregation under high-shear-stress conditions.Conclusion: Riboflavin-based PRT treatment of PCs leads to the enhancement of PLT thrombus formation and thrombus stability on collagen. However, it does not enhance the reactivity of PLTs not in contact with collagen under high-shear-stress conditions. [ABSTRACT FROM AUTHOR]

Platelets are crucial to the hemostatic response. Their role in coagulation is well documented and they have been considered for some time to promote resistance of thrombi to fibrinolysis. Platelets confer resistance to lysis by promoting clot retraction of the immediate fibrin network and through release of plasminogen activator inhibitor-1 from their α-granules. However, recent developments in the field indicate that the role of platelets in fibrinolysis is much more diverse. Indeed, novel studies suggest that platelets form different subpopulations upon activation that play varied roles in regulating hemostasis. Likewise the developments in our understanding of thrombus formation, architecture, and changes in fibrin deposition and composition suggest that these different subpopulations of platelets may populate distinct areas within thrombi and potentially dictate the local hemostatic balance in these areas. This review will discuss the diverse roles of platelets in fibrinolysis and highlight the recent developments in the field and the contribution of both the intracellular pool of modulators as well as the membrane surface in regulating these processes. [ABSTRACT FROM AUTHOR]

Whilst significant effort has been focused on development of tools and approaches to clinically modulate activation processes that consume platelets, the platelet receptors that initiate activation processes remain untargeted. The modulation of receptor levels is also linked to underlying platelet aging processes which influence normal platelet lifespan and also the functionality and survival of stored platelets that are used in transfusion. In this review, we will focus on platelet adhesion receptors initiating thrombus formation, and discuss how regulation of levels of these receptors impact platelet function and platelet survival. [ABSTRACT FROM PUBLISHER]

The lifespan of platelets in circulation is brief, close to 10 days in humans and 5 days in mice. Bone marrow residing megakaryocytes produce around 100 billion platelets per day. In a healthy individual, the majority of platelets are not consumed by hemostatic processes, but rather their lifespan is controlled by programmed cell death, a canonical intrinsic apoptosis program. In the last decade, insights from genetically manipulated mouse models and pharmacological developments have helped to define the components of the intrinsic, or mitochondrial, apoptosis pathway that controls platelet lifespan. This review focuses on the molecular regulation of apoptosis in platelet survival, reviews thrombocytopenic conditions linked to enhanced platelet death, examines implications of chemotherapy-induced thrombocytopenia through apoptosis-inducing drugs in cancer therapy as well as discusses ex vivo aging of platelets. [ABSTRACT FROM AUTHOR]

Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time. [ABSTRACT FROM AUTHOR]

The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl3-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl3-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs. [ABSTRACT FROM AUTHOR]