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Platelets are the "guardians" of the blood circulatory system. At sites of vessel injury, they ensure hemostasis and promote immunity and vessel repair. However, their uncontrolled activation is one of the main drivers of thrombosis. To keep circulating platelets in a quiescent state, the endothelium releases platelet antagonists including nitric oxide (NO) that acts by stimulating the intracellular receptor guanylyl cyclase (GC). The latter produces the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) that inhibits platelet activation by stimulating protein kinase G, which phosphorylates hundreds of intracellular targets. Intracellular cGMP pools are tightly regulated by a fine balance between GC and phosphodiesterases (PDEs) that are responsible for the hydrolysis of cyclic nucleotides. Phosphodiesterase type 5 (PDE5) is a cGMP-specific PDE, broadly expressed in most tissues in humans and rodents. In clinical practice, PDE5 inhibitors (PDE5i) are used as first-line therapy for erectile dysfunction, pulmonary artery hypertension, and lower urinary tract symptoms. However, several studies have shown that PDE5i may ameliorate the outcome of various other conditions, like heart failure and stroke. Interestingly, NO donors and cGMP analogs increase the capacity of anti-platelet drugs targeting the purinergic receptor type Y, subtype 12 (P2Y12) receptor to block platelet aggregation, and preclinical studies have shown that PDE5i inhibits platelet functions. This review summarizes the molecular mechanisms underlying the effect of PDE5i on platelet activation and aggregation focusing on the therapeutic potential of PDE5i in platelet disorders, and the outcomes of a combined therapy with PDE5i and NO donors to inhibit platelet activation.

Platelets are multifunctional cells that ensure the integrity of the vascular wall and modulate the immune response at the blood/vascular interface. Their pathological activation results in both thrombosis and inflammation and implicates them in the pathogenesis of vascular disease. Vascular diseases are sexually dimorphic in terms of incidence, clinical presentation, outcome, and efficacy of anti-platelet therapy. We here provide an overview of what is known about the role of platelets in the initiation and progression of vascular diseases and summarize what is known about the sex differences in platelet reactivity and in the thromboinflammatory mechanisms that drive these diseases, with a particular focus on atherosclerosis, obstructive and non-obstructive coronary artery disease, and ischemic stroke. Understanding the sex differences at the platelet–vascular interface is clinically relevant as it will enable: (1) to design new therapeutic strategies that prevent the detrimental effects of the immune-modulatory function of platelets taking sex into account, and (2) to evaluate if sex-specific anti-platelet drug regimens should be used to reduce the risk not only of thrombosis but also of vascular disease progression.

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Background and purposeAcute ischemic stroke (AIS) is a fearful complication of Coronavirus Disease-2019 (COVID-19). Aims of this study were to compare clinical/radiological characteristics, endothelial and coagulation dysfunction between acute ischemic stroke (AIS) patients with and without COVID-19 and to investigate if and how the SARS-CoV-2 spike protein (SP) was implicated in triggering platelet activation.MethodsWe enrolled AIS patients with COVID-19 within 12 h from onset and compared them with an age- and sex-matched cohort of AIS controls without COVID-19. Neuroimaging studies were performed within 24 h. Blood samples were collected in a subset of 10 patients.ResultsOf 39 AIS patients, 22 had COVID-19 and 17 did not. Admission levels of Factor VIII and von Willebrand factor antigen were significantly higher in COVID-19 patients and positively correlated with the infarct volume. In multivariate linear regression analyses, COVID-19 was an independent predictor of infarct volume (B 20.318, Beta 0.576, 95%CI 6.077–34.559; p = 0.011). SP was found in serum of 2 of the 10 examined COVID-19 patients. Platelets from healthy donors showed a similar degree of procoagulant activation induced by COVID-19 and non-COVID-19 patients' sera. The anti-SP and anti-FcγRIIA blocking antibodies had no effect in modulating platelet activity in both groups.ConclusionsSARS-CoV-2 infection seems to play a major role in endothelium activation and infarct volume extension during AIS.

Studies on the safety and immunogenicity of SARS-CoV-2 vaccination in patients with inflammatory rheumatic diseases have so far not included mixed cryoglobulinaemia (MC) vasculitis.1–3 We report a prospective observational multicentre study on this disorder. Participants were followed at four tertiary referral centres and were instructed to promptly inform the attending physicians about unusual events felt as possibly related to vaccination. Seventy-one patients were recruited: they had infection-cured hepatitis C virus (HCV)-related MC, either uncomplicated (HCV-MC, n=50) or complicated by low-grade non-Hodgkin’s lymphoma (MC-NHL, n=8), or essential MC (EMC, n=13). The characteristics of the patients, exclusion criteria and definition of bona fide vaccination-related flare are described in online supplemental methods.### Supplementary data [annrheumdis-2021-221248supp001.pdf] Overall, 9 of 71 (12.7%) patients had postvaccination MC vasculitis flare. However, 8 of 71 patients had experienced within 12 months before vaccination spontaneous flares, where 7 cases required rituximab and 3 of them (37.5%) had postvaccination flare (see online supplemental information). Thus, to exclude the confounding effects of high proneness to spontaneous flare as the facilitator and of rituximab as the preventor, we further restricted the evaluation of postvaccination flare rate to 63 patients off-therapy and without spontaneous flares for 20–48 months before …

Background. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome of unclear aetiology occurring after DNA-based vaccinations against COVID-19. The aim of this study was to investigate the DNA vaccine-encoded Sars-cov-2 soluble spike protein (SP). as a potential trigger of platelet activation in VITT. Methods. We studied three VITT patients and seven healthy controls (HCs) within 3 weeks from the first dose of ChAdOx1 nCoV-19, and one non vaccinated HC. Serum levels of SP and soluble angiotensin-converting enzyme 2 (sACE2), ACE2 expression in platelets and platelet response to VITT serum stimulation were studied. A thrombus retrieved during mechanical thrombectomy from one VITT patients, was analysed by immunohistochemistry for SP and ACE2. Neutrophil extracellular traps (NETs) markers and coagulation parameters were also measured. Results. We detected serum SP (up to 35 days post-vaccination) and sACE2 in all VITT patients, and respectively in two and three out of 7 vaccinated HCs. Only platelets from one non-vaccinated HC expressed ACE2. VITT sera markedly activated platelets and this activation was inhibited by both anti-SP and anti-FcγRIIA blocking antibodies. The thrombus showed positive immunohistochemical labelling of platelets using an anti-SP antibody with reduced ACE2 expression, compared to a thrombus from a pre-pandemic stroke patient. Markers of endothelial dysfunction, NETs and hypercoagulability state were present in all VITT sera. Conclusions. The present data provides first evidence that DNA vaccine-encoded Sars-cov-2 SP is detectable in VITT sera (several weeks post-vaccination) and in a platelet-rich thrombus, and that may contribute to the initial platelet stimulation in VITT patients.

Background Thrombocytopenia is a manifestation associated with primary antiphospholipid syndrome (PAPS), and many studies have stressed the leading role played by platelets in the pathogenesis of antiphospholipid syndrome (APS). Platelets are highly specialized cells, and their activation involves a series of rapid rearrangements of the actin cytoskeleton. Recently, we described the presence of autoantibodies against D4GDI (Rho GDP dissociation inhibitor beta, ARHGDIB) in the serum of a large subset of SLE patients, and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1. Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets, and small GTPases of the RHO family are critical regulators of actin dynamics in platelets. Methods We enrolled 38 PAPS patients, 15 patients carrying only antiphospholipid antibodies without clinical criteria of APS (aPL carriers) and 20 normal healthy subjects. Sera were stored at − 20 °C to perform an ELISA test to evaluate the presence of anti-D4GDI antibodies. Then, we purified autoantibodies anti-D4GDI from patient sera. These antibodies were used to conduct in vitro studies on platelet activation. Results We identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, in sera from 2/15(13%) aPL carriers, but in no sera from normal healthy subjects. Our in vitro results showed a significant 30% increase in the activation of integrin αIIbβ3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients. Conclusions In conclusion, we show here that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS.

Factor Xa (FXa) has been reported to activate platelet via interaction with glycoprotein (GP) VI but the underlying mechanism has not been fully elucidated. We investigated if Nox2-derived oxidative stress is implicated in FXa-induced platelet aggregation (PA), and the effect of a FXa inhibitor, namely rivaroxaban, with or without aspirin (ASA), on PA. We performed an in vitro study measuring convulxin-induced PA, thromboxane (Tx) B2 and isoprostanes biosynthesis, soluble Nox2-dp (sNox2-dp), a marker of Nox2 activation, soluble GPVI (sGPVI) and PLA2 activation in platelets from healthy subjects (n = 5) added with and without a Nox2 inhibitor. The same variables were also examined in platelets treated with rivaroxaban (15–60 ng/ml), combined or less with ASA (25 µM). Convulxin-stimulated platelets increased sGPVI, sNox2-dp, H2O2, eicosanoid biosynthesis and PLA2 phosphorylation, which were all inhibited by a Nox2 inhibitor. Rivaroxaban alone significantly reduced PA, sGPVI, TxB2 and isoprostanes biosynthesis, concomitantly with Syk, sNox2-dp and PLA2 activation in a dose-dependent fashion; a significant effect was achieved with 30 ng/ml rivaroxaban. Docking simulation analysis showed that rivaroxaban interacts with GPVI. In platelets co-incubated with ASA, rivaroxaban amplified the ASA antiplatelet effect, which was achieved with 30 ng/ml and prevalently attributable to Nox2 inhibition and impaired isoprostane biosynthesis. Here we show that rivaroxaban, at concentrations achievable in human circulation, inhibits PA via GPVI interaction and eventually Nox2-mediated isoprostanes biosynthesis and amplifies the ASA antiplatelet effect.

BACKGROUND AND AIMS Congenital analbuminemia is a rare autosomal recessive inherited disorder characterized by strongly decreased concentration, or complete absence, of serum albumin (SA). Several lines of evidence indicate that SA has anti-thrombotic effect. In vivo platelet function and the role of oxidative stress (OS) in platelet aggregation promotion have never been studied in analbuminaemic patients. PATIENTS AND METHODS We report two cases of congenital analbuminemia in a 38-year-old male and in a 67-year-old woman. We analyzed platelet activation (PA) and OS at baseline and 2 h after 40 g human albumin infusion. PA was evaluated as platelet aggregation, sCD40L and surface αIIbβ3 integrin and P-selectin expression. OS was evaluated measuring serum sNOX2dp, and 8-iso-PGF2α. FINDINGS Analbuminemic patients displayed higher platelet aggregation, markers of PA and of OS. Albumin infusion reduced platelet activation by reducing oxidative stress.

G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y(12) antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA(2), and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10(−/−) platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18(−/−) mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18(−/−) and RGS10(−/−)18(−/−) mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling\ud molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we conditionally deleted Rap1a and/or Rap1b in the megakaryocytic lineage (mKO). Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired pro- platelet formation by megakaryocytes. In platelets, RAP isoforms had both redundant and\ud isoform-ˇspecific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α- granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation.\ud In summary, our studies demonstrate an essential role for RAP1 signaling in platelet\ud integrin activation and a critical role in platelet production. While important for\ud hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Objective— Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The G s -coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results— We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions— Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

Platelets are highly specialized cells that continuously patrol the vasculature to ensure its integrity (hemostasis). At sites of vascular injury, they are able to respond to trace amounts of agonists and to rapidly transition from an anti-adhesive/patrolling to an adhesive state (integrin inside-out activation) required for hemostatic plug formation. Pathological conditions that disturb the balance in the underlying signaling processes can lead to unwanted platelet activation (thrombosis) or to an increased bleeding risk. The small GTPases of the RAP subfamily, highly expressed in platelets, are critical regulators of cell adhesion, cytoskeleton remodeling, and MAP kinase signaling. Studies by our group and others demonstrate that RAP GTPases, in particular RAP1A and RAP1B, are the key molecular switches that turn on platelet activation/adhesiveness at sites of injury. In this review, we will summarize major findings on the role of RAP GTPases in platelet biology with a focus on the signaling pathways leading to the conversion of integrins to a high-affinity state.

Platelets are small anucleated cells that constantly patrol the cardiovascular system to preserve its integrity and prevent excessive blood loss where the vessel lining is breached. Their key challenge is to form a hemostatic plug under conditions of high shear forces. To do so, platelets have evolved a molecular machinery that enables them to sense trace amounts of signals at the site of damage and to rapidly shift from a non-adhesive to a pro-adhesive state. However, this highly efficient molecular machinery can also lead to unintended platelet activation and cause clinical complications such as thrombocytopenia and thrombosis. Thus, several checkpoints are in place to tightly control platelet activation and adhesiveness in space and time. In this review, we will discuss select negative regulators of platelet activation, which are critical to maintain patrolling platelets in a quiescent, non-adhesive state and/or to limit platelet adhesion to sites of injury.

Essentials Part 1 of this review summarizes recent findings on the exquisite balance between activating and inhibitory mechanisms that control platelet adhesiveness in the circulation and at sites of vascular injury.Part 2 of this review discusses molecular differences and similarities for how platelets secure vascular integrity at sites of inflammation and at sites of mechanical trauma. In this brief review paper, we will summarize the State‐of‐the‐Art on how platelet reactivity is regulated in circulation and at sites of vascular injury. Our review discusses recent and ongoing work presented at this year's International Society on Thrombosis and Haemostasis (ISTH) meeting, on the role of platelets in (i) classical hemostasis at sites of mechanical injury, and (ii) the maintenance of vascular integrity at sites of inflammation.

International audience; Background The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. Objective Here, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. Methods CDGI interaction with phosphoinositides was studied by lipid co-sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. Results Lipid co-sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)-biphosphate (PIP2) and phosphatidylinositol (3,4,5)-triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co-sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. Conclusion Our studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)-biphosphate and phosphatidylinositol (3,4,5)-triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.

Background: Thrombocytopenia is a manifestation associated with Primary Antiphospholipid Syndrome (PAPS) and many studies have stressed the leading role played by platelets in the pathogenesis of Antiphospholipid Syndrome (APS) (1). Platelets are highly specialized cells and their activation involves a series of rapid rearrangements of the actin cytoskeleton (2). Recently, we described the presence of autoantibodies against D4GDI (Rho GDP Dissociation Inhibitor Beta, ARHGDIB) in the serum of a large subset of SLE patients and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1(3). Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets and small GTPases of the RHO family are critical regulators of actin dynamics in platelets (4). Objectives: The aim of the present study was to evaluate the presence of anti-D4GDI antibodies in PAPS sera and whether they can affect platelet activation, contributing to the thrombotic events and the thrombocytopenia of PAPS patients. Methods: 38 PAPS patients diagnosed according to the 2006 Sydney classification criteria were enrolled from the Lupus Clinic of the Sapienza University of Rome. 20 normal healthy subjects (NHS) served as controls. Sera were stored at −20 °C to performed an ELISA test using commercial D4GDI protein. Anti-D4GDI antibodies were purified from PAPS sera and used for in vitro treatment of platelets purified from NHS. Flow cytometry analysis was performed to determination of integrin αIIbβ3 activation, a well-established marker of platelet activation and adhesion. Results: We identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, but in no sera from NHS. Dividing the patients with PAPS according to the presence or absence of thrombocytopenia, we found a significant association between this hematologic manifestation and a higher titer of anti-D4GDI antibodies. Our in vitro results show a significant 30% increase in the activation of integrin αIIbβ3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients. Interestingly the antibody does not only increase the overall integrin activation but also the rate/speed of integrin activation. Conclusion: We demonstrated that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation. Thus, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS. References: [1] Shechter Y, Tal Y, Greenberg A, Brenner B. Platelet activation in patients with antiphospholipid syndrome. Blood Coagul Fibrinolysis. 1998 Oct;9(7):653-7. [2] Seong-Hoon Yun, Eun-Hye Sim, Ri-Young Goh, Joo-In Park, and Jin-Yeong Han. Platelet Activation: The Mechanisms and Potential Biomarkers. BioMed Research International,Volume 2016, Article ID 9060143. Review [3] Barbati C, Alessandri C, Vomero M, Vona R, Colasanti T, Vacirca D, Camerini S, Crescenzi M, Pendolino M, Truglia S, Conti F, Garofalo T, Sorice M, Pierdominici M, Valesini G, Malorni W, Ortona E.Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes. J Autoimmun. 2015 Apr;58:78-89. [4] Rowley JW, et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood. 2011;118(14). Disclosure of Interests: None declared

Platelets express a specific repertoire of surface receptors and signaling molecules, which work in concert to mediate rapid cellular responses, allowing for platelet plug formation and hemostasis to occur under the high shear environment of flowing blood. Here, we lay out the signaling pathways, both activating and inhibitory, utilized by platelets to transform extracellular signals into cellular responses, focusing mainly on platelet integrin activation. We first review how these signals are directed through platelet surface receptors, mainly G protein coupled receptors (GPCRs) and immunoreceptor tyrosine-based activation motif (ITAM) receptors. We then discuss the formation and/or regulation of second messengers, including calcium, diacylglycerol and phosphoinositides. Finally, we discuss some key signal integrators, including RAP GTPases and protein kinases, which translate second messenger signals into cellular responses leading to integrin activation and platelet adhesion at sites of vessel injury.

Background: A recent study demonstrated that a combination of rivaroxaban, an inhibitor of factor Xa, plus aspirin (100mg/day) reduces cardiovascular events in patients with stable vascular disease, compared to aspirin alone but the underlying mechanism is still unknown. Methods: In vitro study was performed consisting in measuring agonist-induced platelet aggregation, thromboxane (Tx)B 2 and isoprostane biosynthesis, soluble Nox 2 -dp, a marker of Nox2 activation, and PLA 2 activation in platelets from healthy subjects (n=5) added with or without scalar doses of aspirin (25-100 uM) and/or rivaroxaban (15-120 ng/ml). Furthermore, we measured urinary excretion of isoprostanes, and soluble Nox2 before and after 3 months of 20 mg rivaroxaban (n=25) or warfarin (n=25) treatment in atrial fibrillation(AF) patients. Results: In vitro study showed that rivaroxaban significantly reduced platelet aggregation, TxB 2 and isoprostane biosynthesis and Nox2 and PLA 2 activation in a dose-dependent fashion. Also, rivaroxaban dose-dependently inhibited aggregation of platelets treated with low-(25uM) as well as high-(100uM) aspirin doses. In low-dose aspirin-treated samples an incremental inhibition of platelet TxB 2 biosynthesis was achieved; conversely, in platelets treated with high doses of aspirin a significant inhibition of isoprostanes and Nox2 was observed. Inhibition of platelet aggregation by rivaroxaban was detected also in platelets stimulated with convulxin suggesting that its antiplatelet effect was GPVI-dependent. In addition, convulxin elicited PLA 2 activation, that was inhibited by rivaroxaban as well as by a Nox2 inhibitor. Finally, in AF patients treated with rivaroxaban a significant reduction of urinary isoprostanes and soluble Nox2 and GPVI as compared to warfarin-treated ones was detected. Conclusion: Here we show that rivaroxaban inhibits platelet aggregation via GPVI interaction and eventually Nox2-mediated thromboxane and isoprostane biosynthesis in aspirin-treated platelets so providing a novel mechanistic insight into the beneficial effects of combining rivaroxaban with aspirin.

Antioxidant status has been shown to be associated with cardiovascular events (CVEs). The aim of the study was to develop an assay measuring serum hydrogen peroxide (HWe developed the HBA assay in 121 HS and validated it in 842 AF patients. The occurrence of CVEs was registered and correlated with HBA in AF during a median follow-up of 30.6 months (3226 patient-years). A combined endpoint of CVEs included fatal/non-fatal ischemic stroke and myocardial infarction, cardiovascular death and transient ischemic attack.In HS, median HBA was 61.2% [IQR: 52.9-69.4]. AF patients disclosed lower HBA than 30 HS balanced for age and sex (48.6% [IQR: 24.7-65.1] vs. 59.4% [IQR: 49.2-66.2], p0.001). During a mean follow-up of 30.6 months (3226 patient-years), 168 CVEs occurred (5.2%/year). A multivariable Cox's proportional hazards regression analysis showed that age group 3 (71-80 years, HR:5.419, p = 0.020), age group 4 (80 years, HR:9.783, p = 0.002), diabetes (HR:1.464, p = 0.049), previous cardiac events (HR:1.887, p = 0.001) and HBA (below median, HR:2.313, p0.001) predicted CVEs.We developed an easy assay to measure serum HBA, which was associated with CVEs in AF patients. This assay may represent an additional useful tool for cardiovascular risk stratification and should be validated in other high-risk populations.

Objectives Extra virgin olive oil (EVOO) supplementation is associated with a significant reduction in cardiovascular disease but the underlying mechanism is still unclear. Methods In platelets that were taken from healthy subjects (n = 5), agonist-induced hydrogen peroxide (H 2 O 2 ) production and NADPH oxidase 2 (NOX2) activation in the presence of or without catalase, which catabolizes H 2 O 2 , were investigated. Platelet H 2 O 2 production, NOX2 activation, EVOO vitamin E, and total polyphenols as well as EVOO's ability to scavenge H 2 O 2 were also measured. Results Platelet NOX2 activation and H 2 O 2 production were significantly inhibited in catalase-treated platelets and platelets that were incubated with five different EVOOs. The EVOO content of vitamin E was 53 to 223 mg/kg and total polyphenols 145 to 392 mg/L Gallic acid equivalent. EVOOs quenched in vitro H 2 O 2 by 39 to 62%, which is an effect that is significantly correlated with vitamin E and total polyphenol concentrations ( R = 0.688; P R = 0.541; P Conclusions This in vitro study provides the first evidence that EVOO downregulates platelet H 2 O 2 and in turn NOX2 activity via H 2 O 2 scavenging.

Platelets are critical for hemostasis, i.e., the body's ability to prevent blood loss at sites of vascular injury. They patrol the vasculature in a quiescent, non-adhesive state for approximately 10 days, after which they are removed from circulation by phagocytic cells of the reticulo-endothelial system. At sites of vascular injury, they promptly shift to an activated, adhesive state required for the formation of a hemostatic plug. The small GTPase RAP1 is a critical regulator of platelet adhesiveness. Our recent studies demonstrate that the antagonistic balance between the RAP1 regulators, CalDAG-GEFI and RASA3, is critical for the modulation of platelet adhesiveness, both in circulation and at sites of vascular injury. The RAP1 activator CalDAG-GEFI responds to small changes in the cytoplasmic calcium concentration and thus provides sensitivity and speed to the activation response, essential for efficient platelet adhesion under conditions of hemodynamic shear stress. The RAP1 inhibitor RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI-dependent RAP1 activation. Upon cellular stimulation, it is turned off by P2Y12 signaling to enable sustained RAP1 activation, required for the formation of a stable hemostatic plug. This review will summarize important studies that elucidated the signaling pathways that control RAP1 activation in platelets.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Background and Objective: The cardioprotective properties of Mediterranean Diet were demonstrated for the first time from the Seven Country Study. In the last few decades, numerous epidemiological studies, as well as intervention trial, confirmed this observation, pointing out the close relationship between the Mediterranean diet and cardiovascular diseases. In this context, extra virgin olive oil (EVOO), the most representative component of this diet, seems to be relevant in lowering the incidence of cardiovascular events, including myocardial infarction and stroke. From a chemical point of view, 98-99% of the total weight of EVOO is represented by fatty acids, especially monounsaturated fatty acids such as oleic acid. Tocopherols, polyphenols and other minor constituents represent the remaining 1-2%. All these components may potentially contribute to ”health maintenance” with their beneficial effects by EVOOO. Methods: Studies that examined the effect of EVOO supplementation in healthy subjects and in individuals at cardiovascular risk were included. Conclusion: The studies analyzed demonstrated the role of EVOO as anti-inflammatory, antioxidant and vasodilatory nutrient that may contribute to lower the atherosclerotic burden.

Purpose of review G protein-coupled receptors (GPCRs) like PAR1/4 and P2Y12 have long been known for their critical role in hemostasis. In contrast, deficiency in the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors glycoprotein (GP)VI or C-type lectin-like receptor (CLEC)-2 is associated with only a mild bleeding diathesis in humans and mice. This review summarizes recent developments on the physiological importance of platelet ITAM signaling as well as the molecular mechanisms facilitating this signaling pathway. Recent findings Genetic experiments identified a critical role for platelet CLEC-2 signaling in the formation of lymphatic vessels during development. Similarly, signaling by both GPVI and CLEC-2, but not GPCRs, is required for the maintenance of vascular integrity at sites of inflammation in the adult. The molecular mechanisms underlying ITAM signaling in platelets continue to be refined. Summary Platelet ITAM signaling plays a key role for the maintenance of vascular integrity in development and the adult. This novel form of hemostasis differs from hemostasis at sites of vascular injury in that it does not depend on major platelet adhesion receptors or GPCR signaling.

The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y12 deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking Gq. We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and Gq deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from Gq/Ca2+ signaling. Moreover, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-induced thromboxane A2 synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A2 production in wild-type and P2Y12 deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin αIIbβ3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or LY294002. These data indicate that Ca2+/SFKs/PI3K and PKC represent two alternative signaling pathways mediating Gq-dependent platelet activation.

Objective— The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. Methods and Results— We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI– and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. Conclusion— We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI– and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250−1) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca2+, morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by αIIbβ3 blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.

Rapid activation of platelets at sites of vascular injury is a critical event in thrombosis and hemostasis. Here, we review recent findings, which (a) identified CalDAG-GEFI (RasGRP2) at the nexus of the rapid Ca(2+)-dependent platelet activation, (b) demonstrated a complex synergy between signaling provided by CalDAG-GEFI, protein kinase C and the Gi-coupled receptor for ADP, P2Y12, and (c) suggested CalDAG-GEFI as a novel target for anti-platelet therapy.

Platelets play an essential role in the body's ability to respond to vascular injury (hemostasis). To form a hemostatic plug and prevent bleeding from the damaged blood vessel, these cells need to be equipped with a powerful signaling machinery that allows them to respond to trace amounts of agonists produced at injury sites, and to rapidly transition from an anti-adhesive/patrolling state to an adhesive state (integrin inside-out activation) required for plug formation. However, this highly sensitive signaling machinery can also pose a significant risk, as premature platelet activation can lead to thrombocytopenia (impaired platelet homeostasis) and/or thrombosis. Thus, negative feedback within the signaling machinery is crucial to balancing sensitivity and speed. Circulating platelets are also important in various aspects of cancer [1]. They support tumor growth by providing necessary growth and angiogenic factors. They facilitate tumor metastasis by multiple mechanisms, including protection of tumor cells from immune surveillance while in circulation and adherence to the microvasculature at sites of extravasation. To engage platelets for these tasks, tumor cells have developed ways to activate and aggregate these cells. Consistent with these findings, cancer has emerged as a major risk factor for thrombotic disease, and a high peripheral platelet count correlates with poor survival in various cancers [1]. A better understanding of the signaling machinery that regulates platelet reactivity is required to determine who is at an increased risk for thrombotic disease, both in the absence and presence of cancer. We and others identified the small GTPase RAP1 as a critical regulator of platelet adhesiveness [2]. RAP1 serves as a cellular switch that cycles between a GDP-bound “off-state” and a GTP-bound “on-state”, the latter being required for integrin activation. In platelets, RAP1 activation strongly depends on the guanine nucleotide exchange factor, CalDAG-GEFI (RasGRP2). CalDAG-GEFI activity is regulated by a pair of highly sensitive EF hand domains (KD~80nM) [3, 4]. These regulatory domains provide remarkable sensitivity towards minor changes in the cytoplasmic calcium concentration, which in resting platelets was measured at ~20-50nM. Consistent with this model, platelets lacking functional CalDAG-GEFI show markedly impaired sensitivity towards threshold concentrations of agonists, both in vitro and in vivo. In addition to sensitivity, CalDAG-GEFI is also critical for the near-immediate activation of RAP1, triggered by rapid calcium mobilization from stores in the endoplasmic reticulum. Both RAP1 activation and integrin inside-out activation are delayed in platelets from mice and humans lacking functional CalDAG-GEFI [3, 5, 6]. As outlined above, a highly sensitive signaling machinery needs to be tightly controlled to prevent premature platelet activation. In recent work, we identified the RAP-GAP, RASA3 (GAP1IP4bp, R-RAS-GAP) as a critical negative regulator of RAP1 signaling in platelets [7]. RASA3 is perfectly positioned to prevent premature integrin activation as it is anchored to the plasma membrane of resting platelets by a unique PH/Btk domain. Deletion of functional RASA3 in mice led to premature activation and clearance of platelets from circulation. Concomitant deletion of CalDAG-GEFI reversed this phenotype in Rasa3 mutant mice, strongly suggesting that RASA3 is critical to restrain the highly sensitive CalDAG-GEFI/RAP1 signaling pathway. Thus, one can imagine RASA3 as a “hand brake”, which is pulled in circulating platelets to maintain the cells in a resting state. At sites of vascular injury, however, this brake would impair RAP1 activation and firm platelet adhesion and thus needs to be released to allow for hemostatic plug formation. Our studies strongly suggest that RASA3 inactivation is dependent on signaling by phosphoinositide 3 kinase (PI3K), downstream of the platelet receptor for ADP, P2Y12. More specifically, we observed that Rasa3 mutant platelets did not require feedback signaling via ADP and P2Y12, and that RAP1-dependent integrin activation in these cells was insensitive to inhibitors of P2Y12 or PI3K. Consistent with this data, platelet adhesion at sites of injury in Rasa3 mutant mice was insensitive to inhibitors of P2Y12. Together, these studies suggest that the antagonistic balance between CalDAG-GEFI and RASA3 signaling is critical for the fine-tuning of platelet adhesiveness, both in the circulation and at sites of vascular injury. Genetic and environmental factors that shift the balance towards RAP1 activation may lead to thrombocytopenia and thrombosis, while those that lead to impaired RAP1 activation may cause bleeding in patients (Figure ​(Figure1).1). Future efforts should be directed towards the testing of this hypothesis in humans, studies that will greatly benefit from the development of novel assays that monitor the expression and activity of CalDAG-GEFI, RASA3, and RAP1. The long-term benefit of this work could be a more personalized approach to thrombosis prevention, which is based on inter-individual differences in the regulation of this important platelet signaling pathway. Figure 1 The antagonistic balance between the RAP GTPase regulators, CalDAG-GEFI and RASA3, controls platelet adhesiveness both in circulation and at sites of vascular injury

The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin β cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the β3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbβ3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to β3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbβ3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-β3 integrin binding affinity results in decelerated αIIbβ3 integrin activation and protection from arterial thrombosis without pathological bleeding.

Tight regulation of integrin affinity is a critical component of hemostasis. The final step of integrin inside-out activation is talin binding to two distinct sites within the integrin β cytoplasmic tail, an NPLY sequence and a membrane-proximal region (MPR). Selective deletion of talin1 from platelets (Tln1fl/flPf4-Cre+) completely prevents integrin activation and thrombus formation, and results in profound defects in hemostasis. Interestingly, platelet-specific expression of a talin1 mutant (L325R) that lacks the ability to bind the MPR, but can still bind the NPLY sequence, phenocopies talin1-knockout platelets. In this study we sought to analyze the effects on thrombosis and hemostasis of a platelet-specific talin1 mutation (W359A) that dramatically impairs binding to the NPLY sequence and does not rescue the talin1 deficiency in transfected cells. Homozygous knock-in of talin(W359A) is embryonic lethal in mice. Thus, Tln1W359A/wt mice were crossed with Tln1fl/flPf4-Cre+ mice to generate Tln1W359A/flPf4-Cre+ (TlnWA) and Tln1wt/flPf4-Cre+ (Tlnwt) control mice. Expression of talin(W359A) in platelets partially rescued talin1 deficiency. Compared to Tlnwt controls, agonist-induced αIIbβ3 activation was reduced by ~50% and spreading on fibrinogen was only moderately impaired in TlnWA platelets. However, kinetic studies demonstrated decelerated αIIbβ3 activation in TlnWA platelets, which resulted in delayed aggregation under static conditions and reduced thrombus size at low shear rates. Interestingly, adhesion of TlnWA platelets to collagen at high shear rates was not significantly better than that of talin-deficient cells and TlnWA mice were completely protected from FeCl3-induced carotid artery occlusion. Lastly, in contrast to platelet-specific talin-knockout or TlnLR mice, TlnWA mice showed no detectable gastrointestinal bleeding and only modestly increased tail-bleeding times compared to littermate controls. In conclusion, we demonstrate that the talin(W359A) mutation decelerates, but does not abolish, talin-dependent integrin activation in platelets. Our studies further suggest that this delay in integrin activation is protective from pathological vessel occlusion while it does not affect hemostasis in mice.

Platelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI−/− (hFcR/CDGI−/−) were generated. Compared with controls, aggregation of hFcR/CDGI−/− platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI−/− platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT.