Platelet Signaling Research Articles

PB2252 HEREDITARY PLATELET FUNCTION DISORDERS INVESTIGATION. TEN YEAR REFERRALS IN A TERTIARY HOSPITAL OF NORTHERN GREECE

Background:Hereditary platelet function disorders constitute a rare cause of symptomatic bleeding. These disorders are heterogeneous in clinical expression and also in laboratory evaluation.The accurate measurement of the platelet function and the identification of a congenital platelet disorder is complex, includes different assays and the repetition of testing.Aims:371 patients (241 women/ 130 men) from the region of Macedonia and Thrace, Northern Greece, were referred for platelet function evaluation due to personal and/ or family bleeding tendency. They all manifested symptoms of excessive mucocutaneous bleeding such as bruising or excessive bleeding following surgical or dental procedures. Patients with prolongation of PT, aPTT and TT were excluded and they were investigated for coagulation factors deficiencies. All patients were tested negative for Von Willebrand's Disease.Methods:The platelet function investigation consisted of the 1. the platelet number and size using an automated cell counter 2. blood film report for platelet size and white cell inclusions 3. aggregation testing in platelet rich plasma with light transmission aggregometry (LTG) by the use of 5 agonists (arachidonic acid, ADP, adrenaline, collagen, ristocetin). No medications and strict dietary conditions were given prior testing. In cases of abnormal responses the test was repeated after 1 month and it was considered as abnormal when the deficit sustained on repetition. A clinical assessment of bleeding history (BAT SCORE) and family history for potential inheritance pattern was done.Results:A percentage of 55.4% (223 patients) had abnormal LTG and were negative for coagulation factors deficiencies and thrombopenia. 53,8% (120 patients) of them were women, since these disorders are more commonly diagnosed in women because of the menstrual cycle and the haemostatic challenge of childbirths. The hemorrhagic history of the patients was serious menhorhagia (female), bruises and the excessive bleeding following surgical or dental procedures (male and female) patients. 110 patients were diagnosed as disorders of platelet secretion and signal transduction (WARD), 51 as aspirin like defect (ALD), 27 had abnormal aggregation to collagen, 8 abnormal aggregation to several agonists, 2 as thrombasthenia Glanzmann and 2 cases of MYH9 related disorders. Abnormal platelet morphology was found in 58 out of the 223 patients. 30 family members from 14 families that were investigated and detected with ALD (7 families), WARD (3 families) and collagen receptor defect (3 families) and MYH9 related (1 family).Summary/Conclusion:Thought platelet disorders are mostly undiagnosed causes of symptomatic bleeding a remarkable percentage was found in our patients. The exact prevalence in the general population is not yet established. Aggregation tests give a lot of information about various platelet disorders as seen in our patients and seem to remain the gold standard in the evaluation of platelet disorders. Families’ studies are reasonable in order to detect such abnormalities.

Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors.

Antiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced platelet aggregation, integrin αIIbβ3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-β2GPI activity. Anti-β2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-β2GPI in monocytes and endothelial cells. In conclusion, anti-β2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.

Platelet HIF-2α promotes thrombogenicity through PAI-1 synthesis and extracellular vesicle release.

Oxygen-compromised environments, such as high altitude, are associated with platelet hyperactivity. Platelets confined within the relatively impervious core of an aggregate/thrombus have restricted access to oxygen, yet they continue to perform energy-intensive procoagulant activities that sustain the thrombus. Studying platelet signaling under hypoxia is, therefore, critical to our understanding of the mechanistic basis of thrombus stability. We report here that hypoxia-inducible factor (HIF)-2α is translated from pre-existing mRNA and stabilized against proteolytic degradation in enucleate platelets exposed to hypoxia. Hypoxic stress, too, stimulates platelets to synthesize plasminogen-activator inhibitor-1 (PAI-1) and shed extracellular vesicles, both of which potentially contribute to the prothrombotic phenotype associated with hypoxia. Stabilization of HIF-α by administering hypoxia-mimetics to mice accelerates thrombus formation in mesenteric arterioles. In agreement, platelets from patients with chronic obstructive pulmonary disease and high altitude residents exhibiting thrombogenic attributes have abundant expression of HIF-2α and PAI- 1. Thus, targeting platelet hypoxia signaling could be an effective anti-thrombotic strategy.

Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib

AbstractThe third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Amyloid Peptide β1-42 Induces Integrin αIIbβ3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner.

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβ peptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients.

Transferrin Saturation Inversely Correlates with Platelet Function.

The association between iron overload (IO) and risk of cardiovascular disease is controversial. Epidemiological studies have found a significant negative association of transferrin (Tf) saturation and cardiovascular events suggesting that higher body iron possibly confer a protective effect towards developing cardiovascular events. The biological mechanisms of this phenomenon are unknown. This article investigates the role of IO on platelet reactivity. This study was a prospective case-control study comparing 45 patients with IO, mostly characterized by the HFE gene mutations C282Y and/or H63D, with 32 healthy controls. We evaluated: (1) platelet aggregation in both platelet-rich plasma and whole blood, (2) platelet membrane expression of the activation marker CD62P, (3) activation of platelet signalling phosphoinositide 3-kinase /Akt and mitogen-activated protein kinase/extracellular signal-regulated kinases (Erk)-1/2 pathways, (4) a pattern of in vivo platelet activation markers, and (5) iron biomarker predictors of platelet reactivity. IO patients had significantly lower platelet aggregability, expression of CD62P and phosphorylation amounts of pAkt and pErk-2 in response to agonists. Furthermore, patients with higher Tf saturation levels were characterized by lower circulating levels of sCD40L, PDGF-BB and thromboxane B2. Platelet aggregation and activation parameters inversely correlated with Tf saturation and the stepwise multivariate regression analysis underlined the role of Tf saturation in predicting platelet reactivity. We also found that in vitro platelet exposure to diferric Tf, but not to iron-depleted TF, dose-dependently inhibited platelet function in all investigated subjects. Tf saturation is inversely associated with platelet reactivity and this could explain, at least in part, the association of high Tf and lower risk of cardiovascular diseases in IO.

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.

Review article: shared disease mechanisms between non-alcoholic fatty liver disease and metabolic syndrome - translating knowledge from systems biology to the bedside.

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Characterised by abnormal fat accumulation in the liver, NAFLD presents high degree of comorbidity with disorders of the metabolic syndrome, including type 2 diabetes, obesity and cardiovascular disease. These comorbidities have strong negative impact on the natural course of NAFLD and vice versa, whereby the presence of NAFLD substantially modifies the course and prognosis of metabolic syndrome-associated diseases. To use systems biology strategies to interrogate disease mechanisms that are common to NAFLD and metabolic syndrome. We mapped shared gene/protein-disease interaction networks, we performed gene-disease enrichment analysis to assess pleiotropy, and we created a gene-drug connectivity network. We found that a shared network of genes/proteins is overrepresented by immune response-related pathways, post-translational modifications of nuclear receptors, and platelet-related processes, including activation and platelet signalling. Likewise, gene-based disease-enrichment analysis suggested underlying molecular effectors that are shared with major systemic disorders, including diverse autoimmune diseases, kidney, respiratory and nervous system disorders, cancer and infectious diseases. The shared list of genes/proteins was enriched in drug targets for anti-inflammatory therapy, drugs used to treat cardiovascular diseases, antimicrobial agents and phytochemicals, among many other approved pharmaceutical compounds. By leveraging on publicly available OMICs data, we were able to show that shared loci are not necessarily affected by reverse causality. We provide evidence indicating that NAFLD treatment, including severe histological traits, cannot be limited to the use of a single drug, as it rather requires a multi-target therapeutic approach.

Platelet dysfunction during trauma involves diverse signaling pathways and an inhibitory activity in patient-derived plasma.

Trauma-induced coagulopathy occurs in about 25% of injured patients and accounts for about 10% of deaths worldwide. Upon injury, hemostatic function may decline due to vascular dysfunction, clotting factor deficiencies, hyperfibrinolysis, and/or platelet dysfunction. We investigated agonist-induced calcium signaling in platelets obtained over time from trauma patients. Platelets from trauma patients and healthy donors were monitored via intracellular calcium mobilization and flow cytometry markers (α2bβ3 activation, P-selectin display, and phosphatidylserine exposure) following stimulation with a panel of agonists (adenosine 5'-diphosphate sodium salt, U46619, convulxin, PAR-1/4 activating peptides, iloprost) used in isolation or in pairwise tests. Furthermore, healthy donor platelets were tested in heterologous plasma isolated from healthy subjects and trauma patients. When exposed to agonists over the first 24 hours postinjury, trauma patient platelets mobilized less calcium in comparison to healthy platelets. Partial recovery of platelet activity was observed in about a third of patients after 120 hours, although not fully obtaining healthy baseline function. Flow cytometry markers of trauma platelets were similar to healthy platelets prior to stimulation, but were depressed in trauma platelets stimulated with adenosine 5'-diphosphate sodium salt or convulxin. Also, washed healthy platelets showed a significant reduction in calcium mobilization when reconstituted in plasma from trauma patients, relative to healthy plasma, at all plasma doses tested. Platelet dysfunction in trauma patients included poor response to multiple agonists relevant to hemostatic function. Furthermore, the inhibitor effect of patient plasma on healthy platelets suggests that soluble plasma species may downregulate endogenous or transfused platelets during trauma.

CLEC-2-Induced Signaling in Blood Platelets

CLEC-2-Induced Signaling in Blood Platelets

Micromolar concentrations of citalopram or escitalopram inhibit glycoprotein VI-mediated and integrin αIIbβ3-mediated signaling in human platelets

Collagen and convulxin induce platelet aggregation through glycoprotein VI (GPVI)-FcRγ-Syk signaling pathway. In addition, fibrinogen induces platelet activation through integrin αIIbβ3-FcγRIIa-Syk signaling pathway. We previously reported that high concentrations of selective serotonin reuptake inhibitors (SSRI) reduce platelet aggregation induced by collagen. We further investigated the effects of SSRI on GPVI- and αIIbβ3-mediated signaling pathway. Citalopram and escitalopram, two relatively pure SSRI, were used in this study. Both citalopram and escitalopram concentration-dependently inhibited convulxin-induced platelet aggregation, serotonin (5-HT) release and the activation of αIIbβ3. 5-HT concentration in washed platelets was unchanged after short-term treatment with citalopram. The additional 5-HT failed to fully rescue the inhibitory effect of citalopram on convulxin-induced aggregation. Convulxin-induced phosphorylation of Syk, LAT, and Akt was inhibited by citalopram and escitalopram. Citalopram inhibited the interaction between FcRγ and Syk, whereas the phosphorylation of FcRγ in response to convulxin remained unaltered. Further, citalopram inhibited the increase of the interaction between serotonin transporter and Syk induced by convulxin. In the presence of Mn2+, escitalopram inhibited the formation of lamellipodia on immobilized fibrinogen. Escitalopram did not influence the binding of fibrinogen to platelets. It inhibited the phosphorylation of Syk and PAK triggered by the adhesion on fibrinogen. Our data demonstrate that micromolar concentrations of citalopram and escitalopram inhibit GPVI- and αIIbβ3-mediated platelet functions. The mechanism of the inhibitory effect of citalopram or escitalopram is not the influence on the activation of GPVI or the interaction between fibrinogen and αIIbβ3, but the interaction between Syk and its upstream molecules.

Therapeutic Vaccine Against S100A9 (S100 Calcium-Binding Protein A9) Inhibits Thrombosis Without Increasing the Risk of Bleeding in Ischemic Stroke in Mice.

Decreased adherence to daily ingestion of antiplatelet drugs is a critical issue, increasing mortality and morbidity in poststroke patients. As vaccination could be a promising approach to solving this, we designed an antiplatelet vaccine that inhibited S100A9 (S100 calcium-binding protein A9)/CD36 (cluster of differentiation 36) signaling in platelets, which was reported to be a key signal in arterial thrombosis, but not hemostasis. Immunization with this vaccine induced a sustainable increase in the anti-S100A9 antibody titer for >2 months and an additional booster immunization enhanced the antibody production further. The middle cerebral artery occlusion time was successfully prolonged in the vaccinated mice, which was comparable to that in mice treated with clopidogrel. The antithrombotic effect lasted for 84 days after the last vaccination, as well as after the booster immunization. Importantly, the bleeding time was not affected in the immunized mice. The antithrombotic effect was also observed in the common carotid artery, which was similar to that found in CD36-/- mice. Vascular injury increased the expression of S100A9 in the serum and phosphorylation of JNK (c-Jun N-terminal kinase) and VAV1 in the platelets, but these increases were inhibited in the immunized mice. Moreover, the S100A9 vaccine did not induce cell-mediated autoimmunity, as demonstrated by the enzyme-linked immunosorbent spot assay. Thus, immunization with the S100A9 vaccine resulted in long-term inhibition of thrombus formation through inhibition of increased S100A9/CD36 signaling without risk of bleeding or adverse autoimmune responses. Vaccination against S100A9 might be a novel therapy to prevent recurrent ischemic stroke.

Sin1 (Stress-Activated Protein Kinase-Interacting Protein) Regulates Ischemia-Induced Microthrombosis Through Integrin αIIbβ3-Mediated Outside-In Signaling and Hypoxia Responses in Platelets.

Objective- Microthrombosis as a serious consequence of myocardial infarction, impairs the microvascular environment and increases the occurrences of heart failure, arrhythmia, and death. Sin1 (stress-activated protein kinase-interacting protein) as an essential component of mTORC2 (mammalian target of rapamycin complex 2) is required for cell proliferation and metabolism in response to nutrients, stress, and reactive oxygen species and activates Akt and PKC (protein kinase C). However, the activation and function of Sin1/mTORC2 in ischemia-induced microthrombosis remain poorly understood. Approach and Results- The phosphorylation of the mTORC2 target Akt at S473 (serine 473) was significantly elevated in platelets from the distal end of left anterior descending obstructions from patients who underwent off-pump coronary artery bypass grafting compared with platelets from healthy subjects. Consistent with this finding, phosphorylation of T86 in Sin1 was also dramatically increased. Importantly, the augmented levels of phosphorylated Sin1 and Akt in platelets from 61 preoperative patients with ST-segment-elevation myocardial infarction correlated well with the no-reflow phenomena observed after revascularization. Platelet-specific Sin1 deficiency mice and Sin1 T86 phosphorylation deficiency mice were established to explore the underlying mechanisms in platelet activation. Mechanistically, Sin1 T86 phosphorylation amplifies mTORC2-mediated downstream signals; it is also required for αIIbβ3-mediated outside-in signaling and plays a role in generating hypoxia/reactive oxygen species through NAD+/Sirt3 (sirtuin 3)/SOD2 (superoxide dismutase 2) pathway. Importantly, Sin1 deletion in platelets protected mice from ischemia-induced microvascular embolization and subsequent heart dysfunction in a mouse model of myocardial infarction. Conclusions- Together, the results of our study reveal a novel role for Sin1 in platelet activation. Thus, Sin1 may be a valuable therapeutic target for interventions for ischemia-induced myocardial infarction deterioration.

Decoding Gq Signaling in Platelets

Most platelet agonists work through G protein coupled receptors (GPCRs), activating pathways that involve members of the Gq, Gi, and G12 families of heterotrimeric G proteins. Gq is critical for most functional responses during platelet activation. A defect in Gq expression in patients leads to impaired platelet secretion and aggregation, which is associated with bleeding diathesis. We have previously shown that the Regulators of G protein Signaling (RGS) proteins function as negative regulators for platelet activation by limiting G protein-dependent signaling, but much remains to be learned about the mechanisms by which G proteins can be regulated during platelet activation.Here we took advantage of an RGS-insensitive Gq(G188S) mutant mouse line to decipher the regulatory complex of Gq-dependent signaling in platelets. The G188S mutation disrupts RGS18 and Gq interaction in a way similar to what we have observed in an analogous Gi2(G184S) mutation. However, in contrast to increased platelet activation in Gi2(G184S) expressing platelets, G188S expressing platelets have decreased activation in response to thrombin, TxA2 and ADP, but not to collagen. The hemostatic thrombi formed in G188S mice are significantly reduced with constant embolization as compared with WT controls. Underlying these changes is a decrease in Gq-dependent signaling events, whereas shape change, which is G13-mediated, is unaffected. Our data further reveal that PLCβ3 and GRK2 complex with activated Gq, but not to Gi2. The G188S mutation in Gq not only prevents the binding of RGS proteins to Gq but also impairs the Gq/PLCβ3 interaction, though it does not affect the GRK2 binding to Gq. Instead, there an increase of GRK2 binding to Gq. Finally, the structural analysis shows that PLCβ3 shares a large overlapping binding area with RGS18 to Gq. Computational alanine scanning to predict binding interfaces indicates that the G188S mutation resides in a region important for the binding of both PLC and RGS. The PLC binding interface also overlaps with that of GRK2, which suggests that when PLC is in complex with Gq, it precludes the binding of both RGS proteins and GRK2. In the G188S mutant, however, PLC binding is reduced, which may reduce competition and allow more GRK2 to bind. The binding interfaces of RGS and GRK2 are not predicted to be overlapping, raising the possibility that the two proteins could bind simultaneously to activated WT Gq.Collectively, these observations 1) indicate that the feedback mechanism of Gq inactivation in platelets is different from that of Gi2, 2) reveal, for the first time, that a Gq/PLCβ/RGS/GRK2 signaling node is present in platelets and plays an important role in Gq-dependent signaling during platelet activation, and 3) show that the G188S mutation affect Gq-mediated signaling by disrupting Gq/PLCβ/RGS interaction. DisclosuresNo relevant conflicts of interest to declare.

Severely Impaired Platelet Function Due to a Novel Mutation of P2Y12 from a Human Subject with No History of Bleeding

Platelets mediate hemostasis through amplifying an initial stimulus and aggregating at a site of injury. The identification of bleeding phenotypes in humans has the potential to reveal new insight into the mechanisms behind platelet signaling, as well as pinpoint new potential therapeutic targets. In fact, the study of bleeding disorders in human subjects has led to the identification of a number of disease states and proteins important to platelet functional responsiveness.We have recently identified a human subject (PDS25) with a platelet functional disorder that has a normal CBC, and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to even high concentrations of 2-MeSADP, even though P2Y12 protein expression appears unaltered (Figure 1). Further, platelet reactivity to high doses of agonist for other receptors, such as PAR-4 and GPVI, is normal. Low concentrations of these agonists, which typically rely on feedback for platelet reactivity, elicit an inhibited response using platelets from PDS25 compared to platelets from a healthy control. Consistently, Rap1b activity (Figure 2) was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To determine whether it is the receptor or a downstream signaling component that is dysfunctional and because shape change in response to 2-MeSADP is normal in platelets from PDS25, we co-stimulated 2-MeSADP treated platelets with epinephrine to initiate signaling through Gz, which is very similar to Gi. We found that the response of platelets from PDS25 to 2-MeSADP can be rescued with the addition of epinephrine, suggesting that the signaling components downstream of Gi/zare intact. These data suggest that the aberrant reactivity observed in platelets from PDS25 is due to the P2Y12 receptor. Therefore, we performed a genetic analysis of P2Y12 from PDS25, which revealed a heterozygous mutation of D121N within the conserved DRY motif (Figure 3). We are currently evaluating how this heterozygous mutation confers such a strong phenotype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dysfunctional CLEC-2 and GPVI-Mediated Signaling in Syk Y342F Knock-in Mouse Platelets

Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors like GPVI, the protease activated receptors (PARs), and C-type lectin-like receptor 2 (CLEC-2). GPVI is an ITAM-bearing receptor, while Clec-2 is a HemITAM-bearing receptor. Both ITAM and hemITAM-bearing receptors signal through spleen tyrosine kinase (Syk). Syk activity is dependent on phosphorylation of several residues including Y342, Y346, and Y519/520 (Y348, Y352, and Y525/526 in human). However, the importance of each residue on either phosphorylation of other residues or activation of Syk is unknown. Therefore, we produced a Syk Y342F knock-in mouse line via CRISPR/Cas9 to investigate the importance of Y342 on Syk-mediated signaling using a platelet system. Syk Y342F mice are viable, have no gross abnormalities, and their blood cell counts are normal. We were unable to detect any Syk Y342 phosphorylation following stimulation of Y342F platelets with the GPVI agonist collagen-related peptide (CRP) (Figure 1). Further, Platelet reactivity is reduced in response to the GPVI agonists CRP (Figure 2) and collagen, as well as the CLEC-2 agonist rhodocytin. When using CLEC-2 antibody as an agonist no response is observed in Syk Y342F mouse platelets even though WT littermate control mouse platelets responded well (Figure 3). Signaling initiated by either GPVI or CLEC-2 is also inhibited, as Syk Y519/520 phosphorylation is impaired. Furthermore, PLCg2 and SLP-76 phosphorylation are reduced in Syk Y342F platelets compared to platelets from littermate control mice following stimulation of either GPVI or CLEC-2. Although reactivity of Y342F platelets is clearly reduced compared to WT platelets, there was no difference in occlusion time following ferric chloride injury of the carotid artery. Similarly, there was no difference in mortality rate following pulmonary thromboembolism using Syk Y342F mice compared to WT littermate control mice. Nevertheless, these data demonstrate that phosphorylation of Y342 on Syk following stimulation of either ITAM or hemITAM-bearing receptors is important for Syk activation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Insights into Platelet Signalling Pathways by Functional and Proteomic Approaches.

As circulating sentinels of vascular integrity, platelets act as crucial haemostatic cells as well as important inflammatory and immune cells, whereas under pathological conditions platelets drive thrombotic as well as non-thrombotic diseases related to chronic inflammation. In addition, platelets serve as an important cellular model to study the biology and pharmacology of signal transduction pathways. Platelet inhibition and activation responses are mediated by multiple signalling networks, which are tightly regulated by balanced catalysis of protein phosphorylation and dephosphorylation through protein kinases and protein phosphatases, respectively. However, we are only at the beginning of understanding the complexity of interacting signalling pathways and their impact on platelet function. Here, we review current functional and proteomic approaches that lead to novel concepts of understanding the proteome, kinome and phosphatome of human platelets. A more in-depth understanding of both protein kinases and protein phosphatases using human platelets will contribute to evaluate their further diagnostic and therapeutic potential in inflammation- and immune-mediated diseases.

Platelet CD36 signaling through ERK5 promotes caspase-dependent procoagulant activity and fibrin deposition in vivo

AbstractDyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction–induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI–mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet–fed conditions comparable to that seen in chow diet–fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.

A mutation of the human EPHB2 gene leads to a major platelet functional defect

AbstractThe ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 2.

Platelet signal transduction is the focus of this review. While 'classic' platelet signaling through G protein-coupled receptors in response to fluid-phase agonists has been extensively studied, signaling mechanisms linking platelet adhesion receptors such as GPIb-IX-V, GPVI and α2β1 to the activation of αIIbβ3 are less well established. Moreover, 'non-haemostatic' pathways can also activate platelets in various settings, including platelet-immune or platelet-tumour cell interactions, platelet responses to neutrophil extracellular traps, or stimulation by microbial pathogens. Genetically determined integrin variants can modulate platelet function and increase thrombogenicity. A typical example is the Pro33 (HPA-1b) variant of αIIbβ3. Recent advances in the genotype-phenotype relation of this prothrombotic variant and its impact on outside-in signaling will be reviewed.