Determination of PAR4 numbers on the surface of human platelets: no effect of the single nucleotide polymorphism rs773902.

The thrombin receptor, protease-activated receptor 4 (PAR4), is important for platelet activation and is the target of emerging anti-thrombotic drugs. A frequently occurring single nucleotide polymorphism (SNP; rs773902) causes a function-altering PAR4 sequence variant (NC_000019.10:p.Ala120Thr), whereby platelets from Thr120-expressing individuals are hyper-responsive to PAR4 agonists and hypo-responsive to some PAR4 antagonists than platelets from Ala120-expressing individuals. This altered pharmacology may impact PAR4 inhibitor development, yet the underlying mechanism(s) remain unknown. We tested whether PAR4 surface expression contributes to the altered receptor function. Quantitative flow cytometry was used to determine the absolute number of PAR4 on platelets from individuals subsequently genotyped at rs773902. We detected 539 ± 311 PAR4 per platelet (mean ± SD, n = 84). This number was not different across rs773902 genotypes. This first determination of cellular PAR4 numbers indicates variations in platelet surface expression do not explain the altered pharmacology of the rs773902 PAR4 sequence variant.

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

Antithrombotic Effects of Combined PAR (Protease-Activated Receptor)-4 Antagonism and Factor Xa Inhibition.

OBJECTIVE: PAR (protease-activated receptor)-4 antagonism has antiplatelet effects under conditions of high shear stress. We aimed to establish whether PAR4 antagonism had additive antithrombotic activity in the presence of factor Xa inhibition in an ex vivo model of acute arterial injury. Approach and Results: Fifteen healthy volunteers (29±6 years, 7 women) completed a phase zero double-blind randomized controlled crossover trial. Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured following blood perfusion of low shear and high shear stress chambers. Upstream of the chambers, extracorporeal blood was admixed with (1) vehicle, (2) low-dose apixaban (20 ng/mL), (3) high-dose apixaban (80 ng/mL), (4) BMS-986141 (400 ng/mL), (5) BMS-968141 and low-dose apixaban, or (6) BMS-968141 and high-dose apixaban in 6 sequential studies performed in random order. Compared with vehicle, BMS-986141 demonstrated selective inhibition of PAR4-AP (agonist peptide)-stimulated platelet aggregation, platelet-monocyte aggregates, and P-selectin expression (P≤0.01 for all). Total thrombus area was reduced under both low shear and high shear stress conditions for all drug infusions (P<0.0001 for all versus vehicle). BMS-968141 reduced total (≤44.4%) and platelet-rich (≤39.3%) thrombus area, whereas apixaban reduced total (≤42.9%) and fibrin-rich (≤31.6%) thrombus area. Combination of BMS-986141 with apixaban caused a further modest reduction in total thrombus area (9.6%-12.4%), especially under conditions of high shear stress (P≤0.027). CONCLUSIONS: In the presence of factor Xa inhibition, PAR4 antagonism with BMS-986141 further reduces thrombus formation, especially under conditions of high shear stress. This suggests the potential for additive efficacy of combination PAR4 antagonism and factor Xa inhibition in the prevention of atherothrombotic events.

Probing the epigenetic signatures in subjects with coronary artery disease.

Depletion of S-adenosyl methionine and 5-methyltetrahydrofolate; and elevation of total plasma homocysteine were documented in CAD patients, which might modulate the gene-specific methylation status and alter their expression. In this study, we have aimed to delineate CAD-specific epigenetic signatures by investigating the methylation and expression of 11 candidate genes i.e. ABCG1, LIPC, PLTP, IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66 and TGFBR3. The methylation-specific PCR and qRT-PCR were used to assess the methylation status and the expression of candidate genes, respectively. CAD patients showed the upregulation of IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66, and TGFBR3. Hypomethylation of CDKN2A loci was shown to increase risk for CAD by 1.79-folds (95% CI 1.22-2.63). Classification and regression tree (CART) model of gene expression showed increased risk for CAD with F2RL3 > 3.4-fold, while demonstrating risk reduction with F2RL3 < 3.4-fold and IL-6 < 7.7-folds. This CAD prediction model showed the excellent sensitivity (0.98, 95% CI 0.88-1.00), specificity (0.91, 95% CI 0.86-0.92), positive predictive value (0.82, 95% CI 0.75-0.84), and negative predictive value (0.99, 95% CI 0.94-1.00) with an overall accuracy of 92.8% (95% CI 87.0-94.1%). Folate and B12 deficiencies were observed in CAD cases, which were shown to contribute to hypomethylation and upregulation of the prime candidate genes i.e. CDKN2A and F2RL3. Early onset diabetes was associated with IL-6 and TNF-α hypomethylation and upregulation of CDKN2A. The expression of F2RL3 and IL-6 (or) hypomethylation status at CDKN2A locus are potential biomarkers in CAD risk prediction. Early epigenetic imprints of CAD were observed in early onset diabetes. Folate and B12 deficiencies are the contributing factors to these changes in CAD-specific epigenetic signatures.

Genetic Variant in Human PAR (Protease-Activated Receptor) 4 Enhances Thrombus Formation Resulting in Resistance to Antiplatelet Therapeutics.

OBJECTIVE: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. APPROACH AND RESULTS: Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. CONCLUSIONS: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.

Protease-activated receptor-4 (PAR4) variant influences on platelet reactivity induced by PAR4-activating peptide through altered Ca2+ mobilization and ERK phosphorylation in healthy Japanese subjects.

BACKGROUND: Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIbα and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear. OBJECTIVES: We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype. METHODS: Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin αIIbß3 activation, granule release, Ca2+ mobilization, and activation of ERK and MLC were evaluated. The specificity of effects observed in platelets was confirmed in 293T cells transfected PAR4-Thr120 or Ala120. RESULTS: The frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, αIIbß3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher Ca2+ mobilization and ERK activation in platelets with Thr/Thr120 than Ala/Ala120. Ca2+ mobilization and ERK activation were also increased in 293T cells transfected with PAR4-Thr120 compared to Ala120. CONCLUSION: Our data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca2+ mobilization and ERK activation.

Perinatal lethality of Par4-/- mice delivered by primiparous dams reveals spontaneous bleeding in mice without platelet thrombin receptor function.

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4-/- mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4-/- mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2-/-), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4-/- pups. We observed significant postpartum loss of Par4-/- pups derived from Par4+/- intercrosses that was restricted to a dam's first litter; only 9% of surviving pups genotyped as Par4-/- in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2-/-; 10%) but not in those lacking fibrinogen (Fga-/-; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4-/- mice, suggest that a dam's first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets-and more specifically thrombin-induced platelet activation-in hemostasis during these more traumatic births.

PAR4 (Protease-Activated Receptor 4) Antagonism With BMS-986120 Inhibits Human Ex Vivo Thrombus Formation.

OBJECTIVE: BMS-986120 is a novel first-in-class oral PAR4 (protease-activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation. APPROACH AND RESULTS: Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 µM)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (P<0.001 for all). Compared with pretreatment, total thrombus area (µm2/mm) at high shear was reduced by 29.2% (95% confidence interval, 18.3%-38.7%; P<0.001) at 2 hours and by 21.4% (9.3%-32.0%; P=0.002) at 24 hours. Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus deposition: 34.8% (19.3%-47.3%; P<0.001) at 2 hours and 23.3% (5.1%-38.0%; P=0.016) at 24 hours. In contrast to aspirin alone, or in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (P=nonsignificant). BMS-986120 administration was not associated with an increase in coagulation times or serious adverse events. CONCLUSIONS: BMS-986120 is a highly selective and reversible oral PAR4 antagonist that substantially reduces platelet-rich thrombus formation under conditions of high shear stress. Our results suggest PAR4 antagonism has major potential as a therapeutic antiplatelet strategy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Performance of urine cotinine and hypomethylation of AHRR and F2RL3 as biomarkers for smoking exposure in a population-based cohort.

There is a growing body of evidence demonstrating an association between smoking and DNA methylation. Accordingly, DNA methylation is now considered a promising biomarker of smoking exposure. We evaluated the relationship between methylation markers (AHRR and F2RL3) and urine cotinine as well as self-reported smoking status. DNA methylation levels of AHRR and F2RL3 in blood as well as urine cotinine were measured in 330 adults (46 to 87 years of age). Pyrosequencing was performed to measure DNA methylation of AHRR and F2RL3 associated with smoking exposure. The lung cancer risk associated with DNA methylation and urine cotinine was analyzed using logistic regression analysis. The AHRR and F2RL3 genes were significantly hypomethylated in current smokers compared to in individuals who have never smoked. An inverse relationship was observed between urine cotinine and methylation levels. Methylation of AHRR and F2RL3 distinguished current smokers from never-smokers with high accuracy. Logistic multivariate analysis showed that AHRR methylation is significantly associated with the risk of lung cancer (OR = 0.96, P = 0.011). Our study validated the smoking-associated DNA methylation markers reported in a Korean population-based cohort. In conclusion, DNA methylation of AHRR and F2RL3 provided accurate measures for smoking exposure. Methylation markers reflecting the long-term effect of smoking on the risk of lung cancer showed better performance in distinguishing former smokers from never-smokers.

DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression.

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling.

Essentials Roles of the two thrombin receptors in platelet signaling are poorly understood. Computational systems biology modeling was used together with continuous flow cytometry. Dual-receptor system has wide-range sensitivity to thrombin and optimal response dynamics. Procoagulant platelet formation is determined by donor-specific activities of the two receptors. SUMMARY: Background Activation of human platelets with thrombin proceeds via two protease-activated receptors (PARs), PAR1 and PAR4, that have identical main intracellular signaling responses. Although there is evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear. Methods We developed a multicompartmental stochastic computational systems biology model of dual-receptor thrombin signaling in platelets to gain insight into the mechanisms and roles of PAR1 and PAR4 functioning. Experiments employing continuous flow cytometry of washed human platelets were used to validate the model and test its predictions. Activity of PAR receptors in donors was evaluated by mRNA measurement and by polymorphism sequencing. Results Although PAR1 activation produced rapid and short-lived response, signaling via PAR4 developed slowly and propagated in time. Response of the dual-receptor system was both rapid and prolonged in time. Inclusion of PAR1/PAR4 heterodimer formation promoted PAR4 signaling in the medium range of thrombin concentration (about 10 nm), with little contribution at high and low thrombin. Different dynamics and dose-dependence of procoagulant platelet formation in healthy donors was associated with individual variations in PAR1 and PAR4 activities and particularly by the Ala120Thr polymorphism in the F2RL3 gene encoding PAR4. Conclusions The dual-receptor combination is critical to produce a response combining three critical advantages: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease-activated receptors do not allow combination of all three in a single receptor.

Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.

OBJECTIVE: Platelets express a functional ubiquitin-proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown. APPROACH AND RESULTS: Platelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function. CONCLUSIONS: Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.

Exposure to welding fumes is associated with hypomethylation of the F2RL3 gene: a cardiovascular disease marker.

BACKGROUND: Welders are at risk for cardiovascular disease. Recent studies linked tobacco smoke exposure to hypomethylation of the F2RL3 (coagulation factor II (thrombin) receptor-like 3) gene, a marker for cardiovascular disease prognosis and mortality. However, whether welding fumes cause hypomethylation of F2RL3 remains unknown. METHODS: We investigated 101 welders (median span of working as a welder: 7 years) and 127 unexposed controls (non-welders with no obvious exposure to respirable dust at work), age range 23-60 years, all currently non-smoking, in Sweden. The participants were interviewed about their work history, lifestyle factors and diseases. Personal sampling of respirable dust was performed for the welders. DNA methylation of F2RL3 in blood was assessed by pyrosequencing of four CpG sites, CpG_2 (corresponds to cg03636183) to CpG_5, in F2RL3. Multivariable linear regression analysis was used to assess the association between exposure to welding fumes and F2RL3 methylation. RESULTS: Welders had 2.6% lower methylation of CpG_5 than controls (p<0.001). Higher concentrations of measured respirable dust among the welders were associated with hypomethylation of CpG_2, CpG_4 and CpG_5 (ß=-0.49 to -1.4, p<0.012); p<0.029 adjusted for age, previous smoking, passive smoking, education, current residence and respirator use. Increasing the number of years working as a welder was associated with hypomethylation of CpG_4 (linear regression analysis, ß=-0.11, p=0.039, adjusted for previous smoking). Previous tobacco smokers had 1.5-4.7% (p<0.014) lower methylation of 3 of the 4 CpG sites in F2RL3 (CpG_2, CpG_4 and CpG_5) compared to never-smokers. A non-significant lower risk of cardiovascular disease with more methylation was observed for all CpG sites. CONCLUSIONS: Welding fumes exposure and previous smoking were associated with F2RL3 hypomethylation. This finding links low-to-moderate exposure to welding fumes to adverse effects on the cardiovascular system, and suggests a potential mechanistic pathway for this link, via epigenetic effects on F2RL3 expression.

Platelet Lipidomic Profiling: Novel Insight into Cytosolic Phospholipase A2α Activity and Its Role in Human Platelet Activation.

With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.

Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice.

OBJECTIVE: Current antiplatelet strategies to prevent myocardial infarction and stroke are limited by bleeding risk. A better understanding of the roles of distinct platelet-activating pathways is needed. We determined whether platelet activation by 2 key primary activators, thrombin and collagen, plays distinct, redundant, or interacting roles in tail bleeding and carotid thrombosis in mice. APPROACH AND RESULTS: Platelets from mice deficient for the thrombin receptor protease-activated receptor-4 (Par4) and the collagen receptor glycoprotein VI protein (GPVI) lack responses to thrombin and collagen, respectively. We examined tail bleeding and FeCl3-induced carotid artery occlusion in mice lacking Par4, GPVI, or both. We also examined a series of Par mutants with increasing impairment of thrombin signaling in platelets. Ablation of thrombin signaling alone by Par4 deficiency increased blood loss in the tail bleeding assay and impaired occlusive thrombus formation in the carotid occlusion assay. GPVI deficiency alone had no effect. Superimposing GPVI deficiency on Par4 deficiency markedly increased effect size in both assays. In contrast to complete ablation of thrombin signaling, 9- and 19-fold increases in EC50 for thrombin-induced platelet activation had only modest effects. CONCLUSIONS: The observation that loss of Par4 uncovered large effects of GPVI deficiency implies that Par4 and GPVI made independent, partially redundant contributions to occlusive thrombus formation in the carotid and to hemostatic clot formation in the tail under the experimental conditions examined. At face value, these results suggest that thrombin- and collagen-induced platelet activation can play partially redundant roles, despite important differences in how these agonists are made available to platelets.

Mechanism of race-dependent platelet activation through the protease-activated receptor-4 and Gq signaling axis.

OBJECTIVE: Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. APPROACH AND RESULTS: Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca(2+) mobilization, Rap1 (Ras-related protein 1) activation, and integrin αIIbß3 activation with no observed difference in platelet protein expression between the groups tested. CONCLUSIONS: Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.

Changes in response to antiaggregatory treatment in patients with myeloproliferative neoplasms: a sequential study using multiple electrode aggregometry.

In the present study, we used multiple electrode aggregometry (MEA) to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with myeloproliferative neoplasms (MPNs). arachidonic acid (ASPI), ADP, and thrombin receptor activating peptide (TRAP) tests were performed at two timepoints between 32-50 months in 21 patients with MPN and 1-46 months in 29 controls. We further checked the medical records of the participants to identify a potential correlation of changes in the treatment response with clinical events. In MPN, four out of 13 patients treated with 100 mg of aspirin, no patients receiving 50 mg of aspirin, and one out of five clopidogrel-treated patients showed a therapeutic antiplatelet effect. In the subsequent examinations, five patients changed from response to nonresponse or vice versa. Initial nonresponse and changes from an initial response to nonresponse were observed in six patients with thrombotic events. In the controls, 25 out of 26 aspirin-treated patients and two out of three clopidogrel-treated patients showed an initially adequate in-vitro response. Except from one patient changing from initial aspirin nonresponse to response, all controls showed a stable response state. One control with two ischemic strokes showed a nonresponse to clopidogrel. In conclusion, MEA detects the response to antiaggregatory treatment, as well as its changes during the disease course in patients with MPN. An initial or subsequent nonresponse was observed in patients with thrombotic events.

Effects of prolactin on platelet activation and blood clotting.

Increased levels of prolactin often coincide with an increased risk for thromboembolic events, but it is unclear whether a direct causal relation exists. Our aim was to examine the effect of prolactin on platelet function. In addition to using recombinant prolactin for experiments in vitro, we analyzed platelet function by flow cytometry in a group of 13 females with hyperprolactinaemia and 18 healthy female controls. Platelet activation was measured by P-selectin expression and by the amount of platelet-bound fibrinogen after stimulation with adenosine di phosphate (ADP), collagen-related peptide and the protease activated receptor (thrombin receptor) (PAR)-activating peptides PAR4-AP and PAR1-AP. Free oscillation rheometry was used to measure clotting time in whole blood. No significant effect on platelet activation or clotting time could be seen in in vitro experiments by adding recombinant prolactin. However, significantly lower P-selectin expression was found in the hyperprolactinemic group when platelets were activated by ADP (5 and 10 µM) or PAR4-AP. The expression of fibrinogen did not differ between the two groups for any of the activators used. For all samples, inverse significant correlations between P-selectin expression and prolactin concentration were found for both 5 µM ADP (r = - 0.61, p < 0.01), 10 µM ADP (r = - 0.62, p < 0.001) and PAR4-AP (r = - 0.69, p < 0.001). Thrombin cleavage of recombinant prolactin resulting in a 16 kDa C-terminal fragment did not alter the P-selectin expression upon activation. We found an indirect inhibitory effect of prolactin on platelets in hyperprolactinemic patients, suggesting that prolactin might have a protective role in thromboembolic disease.