Identification of a Racially Dimorphic Variant in the Human Platelet PAR4 Thrombin Receptor Altering Platelet Function and Pharmacologic Inhibition

Abstract Compared to whites, blacks have a 2-fold higher incidence of coronary heart disease (CHD), and black race is an independent predictor of worse survival after CHD events after accounting for other confounding variables (e.g., socioeconomic, demographic, etc.). However, there has been a paucity of literature considering racial differences in platelet function. We recently reported results from the Platelet RNA And eXpression-1 (PRAX1) study of platelets in healthy black (n=70) and white (n=84) subjects (Edelstein et al, Nat. Med 2013). Human platelets express two thrombin receptors, protease activated receptor (PAR) 1 and PAR4. We demonstrated 1) a 3.7-fold increased PAR4-mediated aggregation kinetics and greater calcium mobilization in platelets from black subjects compared to whites, and 2) phosphatidylcholine transfer protein (PC-TP) was a mediator of this racial difference. These findings have potential clinical significance because greater platelet-mediated thrombosis could contribute to the worse outcomes in blacks than whites after coronary events. Additionally, in the presence of vorapaxar (an FDA-approved PAR1 inhibitor for patients with coronary and peripheral vascular disease) PAR4 is the primary means by which thrombin activates platelets, and the risks and benefits of vorapaxar and other PAR inhibitors by race are unknown. Because PC-TP expression only accounted for 18% of the observed variance in PAR4 function, we have considered additional mechanisms to explain the racial difference in thrombin-induced PAR4-mediated platelet reactivity. Although platelets from blacks express 14% more PAR4 protein than whites, this difference does not explain the variance in platelet PAR4 function. Genome-wide quantitative trait locus analysis identified common 3 SNPs in the PAR4 gene (F2RL3) as associated with PAR4-induced platelet aggregation (P = 4.79 x 10-11). Importantly, the allele frequency of these SNPs differs by race in both the 1000 Genomes dataset and in PRAX1 (P= 4.31 x 10-16). One of these SNPs, rs773902, determines if residue 120 in PAR4 is an alanine (81% in whites vs. 37% in blacks) or a threonine (63% in blacks vs. 19% in whites). A second less frequent, non-synonymous F2RL3SNP, Phe296Val, was only observed in blacks and was associated with loss of PAR4 function. This variant appears to have a dominant negative effect since only one copy of PAR4-296Val abolished the enhanced PAR4-AP induced platelet aggregation associated with PAR4-Thr120. A proximal step in PAR4-induced platelet signal transduction is Gq activation leading to hydrolysis of phosphatidylinositol 4,5-biphosphate to IP3 and diacylglycerol, followed by increased cytoplasmic calcium. Platelets with the PAR4-Thr120 variant exhibited a 65% greater Ca2+ flux than PAR4-Ala120 homozygotes (P = 0.03). To eliminate other sources of inter-individual variation we cloned and expressed each variant in 293 cells, which lack endogenous PAR4 expression, and measured the generation of IP3 in response to PAR4-AP treatment. Compared to PAR4-Ala120 expressing cells, PAR4-Thr120 expressing cells generated 41% higher IP3 levels (P = 0.03). Because PAR inhibitors are either currently approved (vorapaxar) or in development, we examined platelet function in the presence of PAR1 and PAR4 antagonists. PAR4 genotype had no effect on vorapaxar inhibition of PAR1 signaling or PAR4 signaling in the presence of vorapaxar. YD-3, a selective inhibitor of PAR4 and not PAR1, was also tested. In stark contrast to vorapaxar, PAR4 genotype had a significant effect of the efficacy of PAR4 inhibition by the compound YD-3. Platelets from subjects homozygous for the PAR4-Thr120 were not inhibited by any tested concentration of YD-3 while PAR4-Ala120 homozygotes were inhibited in a dose-dependent manner (P = 6x10-8). In summary, we have identified variation in the PAR4 gene that alters PAR4 function and accounts for 48% of the observed racial difference in platelet PAR4 signaling. These results, couple with other racial data (Tourdot et al., submitted), have two critical clinical implications for anti-platelet therapy: (1) Patients expressing the PAR4-Thr120 variant (mostly black) may not be adequately protected on current anti-platelet drugs, and (2) PAR4 antagonists currently in development may not be effective in PAR4-Thr120 expressing platelets. This suggests a critical need for new and effective therapies for these patients. Disclosures No relevant conflicts of interest to declare.

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Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

Blood ◽

10.1182/blood.v122.21.1054.1054 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1054-1054

Author(s):

Leonard C. Edelstein ◽

Lukas M Simon ◽

Raul Teruel Montoya ◽

Michael Holinstat ◽

Edward Chen ◽

...

Keyword(s):

Racial Differences ◽

Platelet Activation ◽

Platelet Function ◽

Racial Difference ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Wild Type Mouse ◽

Induced Aggregation ◽

Megakaryocytic Cell

Abstract Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10-23; q = 10-20) and with PAR4 reactivity (p = 3.4x10-8; q = 3.5x10-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca+ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca+ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10-4; q = 1.38 x10-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34+ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3’UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy. Disclosures: No relevant conflicts of interest to declare.

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The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽

10.1182/blood.v108.11.3904.3904 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3904-3904

Author(s):

Samantha Baldassarri ◽

Alessandra Bertoni ◽

Paolo Lova ◽

Stefania Reineri ◽

Chiara Sarasso ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Cannabinoid Receptors ◽

Thromboxane A2 ◽

Human Platelets ◽

Dependent Manner ◽

Cb2 Receptor ◽

Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

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Platelet-Like-Particles Sheared From Myosin-II-Inhibited Megakaryocytes Highlights The Elevated Thrombocrit Of May-Hegglin Anomaly

Blood ◽

10.1182/blood.v122.21.2426.2426 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2426-2426

Author(s):

Kyle R Spinler ◽

Jae-Won Shin ◽

Dennis E Discher

Keyword(s):

Conflicts Of Interest ◽

Fragment Size ◽

Myosin Ii ◽

Clinical Importance ◽

Normal Activity ◽

Human Platelets ◽

Micropipette Aspiration ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type

Abstract Megakaryocytes (MKs) in the marrow extend projections into blood flow and generate platelets under shear. Understanding MK differentiation and platelet production is of broad clinical importance and extends a need to augment platelet numbers in patients. Reversible but sustained inhibition of non-muscle myosin-II (NMM-II) with the drug blebbistatin increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation under shear. Using a cone and plate rheometer to apply fluid shear to drug-treated MKs in bulk, platelet-like-particles (PLPs) that are collagen-I responsive can be generated with intermediate shear. The MKs naturally down-regulate NMM-IIA activity through phosphorylation of S1943, but this site proves shear sensitive, consistent with results for human platelets. Using micropipette aspiration of MKs, inhibition of NMM-IIA is found necessary to generate CD41+ fragments that approximate the size of human platelets. Localization of NMM-IIA to the fragments is modulated by S1943 as seen by unique distribution patterns resulting from specific S1943 mutations that can be abrogated by addition of blebbistatin. The approach is extended to clinically relevant mutations associated with May-Hegglin anomaly (MHA) co-expressed with wild type protein to mimic heterozygotes. As with blebbistatin inhibition of myosin, May-Hegglin mutants result in a higher frequency of fragmentation during micropipette aspiration, indicating a dominant negative effect. Immunofluorescence documents abnormal myosin aggregation in cells transfected with May-Hegglin myosin mutations compared to wild type constructs. Finally, peripheral blood from a patient with a D1414N May-Hegglin mutation is cultured to produce megakaryocytes used to support both the micropipette and immunofluorescence results. These findings reveal a phospho-switch in NMM-II, from inactive to active in the terminal stages of platelet-poiesis, and that proper myosin activity is critical to fragment size and number. Disruption of normal activity enhances fragment generation suggesting a novel mechanism in MHA: in particular, MHA thrombocytopenia results in an increased thrombocrit due to abnormally large platelets, which overcompensates for the reduction in platelet number. Disclosures: No relevant conflicts of interest to declare.

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Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3513-3513

Author(s):

Wenxiu Yi ◽

Wei Li ◽

Lijie Ren ◽

Xinliang Mao ◽

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Venous Blood ◽

Pi3k Pathway ◽

Dependent Manner ◽

Clot Retraction ◽

Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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A Large Cluster of Micrornas At 14q32 Defines an RNA Expression Module That Accounts for Racial Differences in Protease Activated Receptor 4-Mediated Platelet Reactivity

Blood ◽

10.1182/blood.v120.21.380.380 ◽

2012 ◽

Vol 120 (21) ◽

pp. 380-380

Author(s):

Leonard C. Edelstein ◽

Lukas M. Simon ◽

Edward Chen ◽

Angela Bergeron ◽

Xiango Kong ◽

...

Keyword(s):

Platelet Aggregation ◽

Racial Differences ◽

Individual Variation ◽

Racial Difference ◽

Platelet Reactivity ◽

Differentially Expressed ◽

Lower Sensitivity ◽

Rna Expression ◽

Term Survival ◽

Target Mrnas

Abstract Abstract 380 Myocardial infarction and stroke typically result from an occlusive platelet thrombus formed at the site of atherosclerotic plaques. There is reproducible variation in platelet reactivity among different individuals – a variation that likely contributes to thrombosis risk. The inter-individual variation in platelet reactivity is highly heritable, and this heritability is greater in African Americans (AAs) than European Americans (EAs), but there is limited understanding of the genetic mechanisms responsible for this variability. The Platelet RNA And eXpression-1 (PRAX1) study was designed to identify novel genes and miRNAs responsible for inter-individual variation in platelet reactivity. We assessed platelet aggregation in 70 AA and 84 EA healthy subjects and profiled miRNA and mRNA from highly purified blood platelets. The richness of these data provided unique opportunities to establish miRNA-mRNA-physiology relationships that led to a number of novel and unexpected results. Although 607 miRNAs were detected above background, some were virtually absent in some samples. Using highly stringent criteria based on a biphasic distribution of expression in the 154 samples, we defined 178 platelet miRNAs present in at least 65% of subjects. Statistical analysis identified miRNAs that were differentially expressed (DE) by age, gender and race. A miRNA-miRNA correlation matrix uncovered a cluster of 52 miRNAs located at 14q32 that were highly co-regulated and that were expressed at higher levels in platelets from EAs than AAs. Unexpectedly, platelet aggregation in response to PAR4-AP was significantly higher in AAs than in EAs, whereas no racial difference was observed for the platelet aggregation response to arachidonic acid, ADP or PAR1-AP. Having established that 14q32 miRNAs were DE by race and that PAR4-mediated platelet reactivity was different by race, we sought to determine whether there were mRNAs that linked these two discoveries. Using a simple linear regression with an FDR q<.25, we found 119 mRNAs that were positively correlated with PAR4 reactivity. Notably, the gene encoding PAR4 (F2RL3) was not DE by race. A substantially higher proportion of 14q32 miRNAs were represented among the miRNAs enriched for targets among PAR4-positively-correlated mRNAs than would be expected by chance (p = 0.0092, OR=3.71). In other words, the racially-differentially expressed miRNAs located on 14q32 target mRNAs that are positively associated with PAR4 reactivity. A conditional correlation analysis showed that the significant association between PAR4 reactivity and race vanished after accounting for RNA expression, indicating that these two traits are highly collinear. We were especially intrigued with the gene, PCTP, which encodes phosphatidylcholine (PC) transfer protein (TP), because of the well-established importance of fatty acids, phospholipids and PC in platelet reactivity and because PC-TP catalyzes the transfer of PC between membranes. PCTP was DE by race (q value=10−19) and by PAR4 reactivity (p=10−6), and PC-TP protein was higher in AAs than EAs. PCTP is predicted to be targeted by 14q32 miRNA, hsa-miR-376c, and we validated that PC-TP mRNA and protein were knocked down by hsa-miR-376c. In summary, we have discovered racial differences in PAR4-mediated platelet activation and uncovered a genetic regulatory module in which racial differences in expression of miRNAs located at 14q32 and their target mRNAs account for this racial difference in platelet function. High levels of 14q32 miRNAs in EAs specifically target mRNAs that positively regulate PAR4 activity, resulting in lower sensitivity to PAR4-mediated activation. The inverse is true of AAs. Compared to EAs, AAs have a 2-fold increase in the incidence of CHD and a lower long-term survival. In addition, it is unknown whether the racial difference in platelet reactivity impacts the benefits and risks of anti-platelet therapies. Because novel inhibitors of PAR1 and 4 are currently in clinical development, it is critical to know whether dosing of such agents should be adjusted by race to maximize benefit and avoid toxicity. Lastly, since PAR4 is expressed in tissues other than platelets (e.g., heart, brain, liver), if 14q32 miRNA targets are DE by race in non-platelet tissues, it is possible that the consequent altered gene expression could contribute to non-thrombotic diseases known to show racial differences. Disclosures: No relevant conflicts of interest to declare.

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Blockade of Lipid Receptor GPR31 Suppresses Platelet Reactivity and Thrombosis with Minimal Effect on Hemostasis

Blood ◽

10.1182/blood-2019-126111 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1064-1064

Author(s):

Layla Van Doren ◽

Nga Nguyen ◽

Chris Garzia ◽

Elizabeth Fletcher ◽

Ryan Stevenson ◽

...

Keyword(s):

Platelet Aggregation ◽

Calcium Release ◽

Conflicts Of Interest ◽

Human Platelet ◽

Platelet Reactivity ◽

Human Platelets ◽

P2y12 Inhibitor ◽

Occlusion Time ◽

Genetic Ablation ◽

Human Platelet Aggregation

Objective: Platelet agonist-activated 12-lipoxygenase (12-LOX)/12-Hydroxyeicosatetraenoic acid (12-HETE)/G protein-coupled receptor 31 (GPR31) signaling has been proposed to regulate platelet reactivity. While inhibition or genetic ablation of 12-LOX supports an important role of 12-HETE in response to platelet agonists thrombin and collagen, the participation of GPR31 in platelet lipid signaling has not been examined. We developed a potent pepducin inhibitor, GPR-310, to test the downstream involvement of GPR31 in thrombin and collagen mediated platelet activation and thrombosis. Approach and Results: Treatment of mice with GPR-310 reversibly inhibited ex vivo platelet aggregation in response to thrombin and the PAR4 agonist, AYPGKF. There was significant protection (P<0.002) against FeCl3-induced carotid artery injury in mice by extending occlusion time from 100% occlusion at 27 min in the vehicle cohort to 20% occluded at 45 min in the GPR-310 cohort. GPR-310 treatment did not affect tail bleeding time. In human platelets, GPR-310 significantly (P<0.001) inhibited PAR4 agonist and collagen-mediated platelet aggregation and PAR4 calcium release. GPR-310 inhibited 12(S)-HETE- and PAR4-mediated RAP1 activation, with no effect on the PAR1-RAP1 signal. Accordingly, PAR1-mediated aggregation of human platelets was not affected by either GPR-310 or the 12-LOX inhibitor, ML355. GPR-310 caused a 5-fold shift in thrombin-mediated human platelet aggregation, comparable to a direct P2Y12 inhibitor, AZD1283. Dual GPR31 and P2Y12 inhibition showed synergy and protected against thrombin-mediated human platelet aggregation with a 19-fold shift. Blockade of GPR31 was more effective than the P2Y12 inhibitor in a thrombin-mediated clot retraction assay. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: GPR31 may serve as a new therapeutic target in platelet-dependent arterial thrombosis and aggregation in humans. Disclosures No relevant conflicts of interest to declare.

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Adenosine Regulation of cAMP through Phosphodiesterases

Blood ◽

10.1182/blood-2018-99-114929 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2424-2424

Author(s):

Bunyan Teng ◽

Daniel N Darlington ◽

Andrew P Cap

Keyword(s):

Platelet Aggregation ◽

Adenylate Cyclase ◽

G Protein ◽

Adenosine Receptors ◽

Conflicts Of Interest ◽

Human Platelets ◽

Intracellular Camp ◽

Dependent Manner ◽

Adenosine Agonist ◽

Sch 58261

Abstract Introduction: Adenosine, an autacoid and metabolite of ATP, has been known to have anti-platelet properties. Of the 4 adenosine receptors (ARs), only A2A AR have been implicated in adenosines anti-platelet properties in human. A2A AR is a G-Protein Coupled Receptors associated with a stimulatory G-Protein (Gs) that can activate adenylyl cyclase (AC) and increase intracellular cAMP. An elevation of cAMP has been shown to inhibit platelet aggregation to natural stimuli. Regulation of intracellular cAMP is balanced between synthesis by adenylate cyclase and degradation by phosphdiesterases (PDE). There are 3 PDE subtypes found in platelets: PDE2, PDE3, and PDE5. However, it is not know which subtype(s) is (are) responsible for regulating cAMP level in human platelets after adenosine stimulation. Materials and Methods: Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed after stimulation of platelets with 100uM ADP, with or without NECA (non-specific AR agonist), DPCPX (A1 AR antagonist), and Sch 58261 (A2A AR antagonist). PRP treated with NECA, DPCPX, Sch 58261, and PDE inhibitors (EHNA, E in figures, for PDE2, Trequinsin, T in figures, for PDE3, and 4-{[3'4'-(methylenedioxy) benzyl]amino}-6-methoxyqunazolin, 4 in figures, for PDE 5). Cyclic AMP was measured in platelets after treatment by liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher) after treated with these drugs. Results: ADP-induced platelet aggregation was inhibited in a dose dependent manner by the non-specific adenosine agonist, NECA (Figure 1) and the effect was blocked by A2A specific antagonist Sch 58261, not by the A1 AR antagonist, DPCPX (Figure 2). NECA inhibition of platelet aggregation was likely due to an elevation of intracellular cAMP (1 uM, 5min incubation, Figure 3). Inhibition of PDE3 alone, significantly increased intracellular cAMP, suggesting that basal PDE3 activity is present. PDE 3 inhibition combined with NECA elevated cAMP even higher than PDE inhibition or NECA alone (Figure 3), suggesting that NECA (A2A stimulation) effects PDE activity. Inhibition of PDE2 or 5 had no effect on basal or NECA stimulated cAMP (Figure 3). Inhibition of all 3 PDE (2,3,5) combined with NECA elevated cAMP to levels higher then NECA+ PDE3 inhibition, again suggesting that NECA maybe effecting the activity of the PDEs (Figure 3). The potentiation of cAMP by PDE3 inhibition + NECA was block by A2A, but not A1 antagonist (Figure 4) suggesting that the nonspecific adenosine agonist is elevating cAMP through A2A. Conclusion: 1. In human platelets, NECA stimulates cAMP through A2A receptors and this elevation is likely due to an elevation in adenylate cyclase via Gs coupled to A2A. PDE3 is basally active and likely regulated by adenosine receptors. Disclosures No relevant conflicts of interest to declare.

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Identification Of a Novel Small Molecule Inhibitor Of ASK1 As a Potent Anti-Platelet Agent

Blood ◽

10.1182/blood.v122.21.20.20 ◽

2013 ◽

Vol 122 (21) ◽

pp. 20-20

Author(s):

Meghna Ulhas Naik ◽

Maloney David ◽

Ramya Turaga ◽

Hidinori Ichijo ◽

Ulhas P Naik

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Receptor Activation ◽

Human Platelets ◽

Dependent Manner ◽

Genetic Ablation ◽

Fibrinogen Receptor ◽

Molecule Inhibitor

Abstract Apoptosis signal-regulating kinase (ASK1) is a serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its presence and role in platelets are not known. We found that ASK1 is expressed in platelets and is rapidly activated during platelet stimulation by various agonists in a dose-dependent manner. In addition, we found that TRAF2/6, known endogenous activators of ASK1, are expressed in platelets and associate with ASK1 upon platelet activation with agonists. Furthermore, genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2x10-9). While WT mice showed an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by a mixture of collagen and epinephrine as determined by increased survival and lack of large occlusive thrombi in the lung. We also found that ADP- and AYPGKF (PAR4 receptor peptide) -induced platelet aggregation was diminished in Ask1 null mice compared to WT mice. Furthermore, PAR4 peptide-induced alpha- and dense-granular secretion was also reduced in Ask1 null platelets compared to WT. Interestingly, we also found that Ask1 null platelets bind less FITC-fibrinogen compared to the WT upon activation by PAR4 peptide. Furthermore, thrombin failed to activate MKK6 and p38 in Ask1 knockout platelets, showing that Ask1 is indispensable for p38 activation by thrombin. These results indicated that ASK1 regulates platelet function by augmenting platelet secretion as well as fibrinogen receptor activation, making it an important target for combating thrombosis. We therefore synthesized a novel and highly specific ASK1 inhibitor, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) based on the published report. IPTB has been found to be a very potent inhibitor that inhibits ASK1 activity at nM concentrations. IPTB is also highly specific to ASK1 and does not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1. We found that in human platelets, IPTB dose-dependently inhibits p38 activation induced by a variety of platelet agonists. Furthermore, IPTB dose-dependently inhibited ADP and PAR4 peptide-induced platelet aggregation. Interestingly, IPTB also dose-dependently inhibited platelet spreading on immobilized fibrinogen. Our results strongly suggest that the dose of IPTB could be adjusted so that it attenuates thrombosis without affecting hemostasis. This development would make IPTB a novel potential therapeutic agent to be used to combat thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

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Fucoidan Is a Novel Platelet Agonist for CLEC-2 Receptor

Blood ◽

10.1182/blood.v120.21.94.94 ◽

2012 ◽

Vol 120 (21) ◽

pp. 94-94

Author(s):

Bhanukanth Manne ◽

Todd M Getz ◽

Craig Hughes ◽

Carol T Dangelmaier ◽

Steve P Watson ◽

...

Keyword(s):

Platelet Aggregation ◽

Animal Models ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Human Platelets ◽

Lag Phase ◽

Dependent Manner ◽

Wild Type ◽

Concentration Dependent Manner

Abstract Abstract 94 Fucoidan, a sulphated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor (TFPI) (Prasad et al., Blood 111:672, 2008). The decrease in bleeding times in the hemophilia animal models by in vivo administration of fucoidan suggests the beneficial effect of fucoidan as a novel treatment. Furthermore, in vitro studies using platelet poor plasma from hemophilia animal models and human patients has shown that fucoidan inhibits TFPI thereby contributing to an increase in the extrinsic coagulation pathway activity. The effect of fucoidan on platelets however has not been studied. As it is known that the platelet count remains unaffected in hemophilia A patients and bleeding times are primarily measured to assess normal platelet function, we hypothesize that the decrease in bleeding times in the hemophilia animal models may be due to platelet activation by fucoidan. In this study, we demonstrate for the first time that fucoidan induces platelet activation in a concentration dependent manner. Fucoidan-induced platelet activation is completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, and in the absence of Syk and PLC-g2. Furthermore, fucoidan-induced platelet activation has a lag phase, which is reminiscent of platelet activation by collagen and by CLEC-2 receptor agonists. Platelet activation by fucoidan however was only slightly inhibited in FcRg-chain null mice indicating that fucoidan is not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in CLEC-2 deficient mouse platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data shows fucoidan as a novel CLEC-2 receptor agonist that activates platelets through an SFK-dependent signaling pathway. Further, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. . / B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. . / C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. Disclosures: No relevant conflicts of interest to declare.

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An Antibody Targeted To The Anionic Region Of Human Protease Activated Receptor 4 Inhibits Thrombosis Without Influencing Bleeding

Blood ◽

10.1182/blood.v122.21.2376.2376 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2376-2376

Author(s):

Michele M. Mumaw ◽

Maria de la Fuente ◽

Amal Arachiche ◽

Daniel N. Nobel ◽

Marvin T. Nieman

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Preliminary Evidence ◽

Human Platelets ◽

Control Treatment ◽

Dependent Manner ◽

Protease Activated Receptors

Abstract Protease activated receptors (PARs) are G-protein coupled receptors which are activated by cleavage of their N-terminus by thrombin. This generates a tethered ligand which is then able to activate the corresponding receptor. Human platelets express PAR1 and PAR4, which both have crucial roles in mediating the response of platelets to injury. Our hypothesis is that PAR4 is an ideal target for new anti-platelet therapies because it is required for stable clot formation and has limited tissue distribution. We have previously determined a region on PAR4 that is required for efficient activation by thrombin. A polyclonal antibody (CAN12) targeted to this region of the PAR4 exodomain does not cross react to PAR1. Initial studies determined that CAN12 is able to block thrombin-induced human platelet aggregation with an IC50 of 10 ng/ml. Control IgG does not inhibit aggregation at 2 mg/ml. In mouse platelets, CAN12 inhibits P-selectin expression and integrin activation. In the Rose-Bengal mouse model of carotid artery thrombosis, CAN12 (1 mg/kg) administered 10 minutes prior to injury was able to completely inhibit the formation of a thrombus in a dose dependent manner. The antibody delayed thrombosis to greater than 90 min; the experiment was terminated at 90 minutes. In contrast, control treatment (2 mg/kg IgG or saline) resulted in complete occlusion at ∼40 minutes. Further, the minimal dose of CAN12 required for complete inhibition of thrombosis (0.5 mg/kg) administered fifteen minutes after injury also delayed thrombosis from ∼50 minutes to ∼80 minutes. This indicates that CAN12 is able to disrupt a thrombus after it has been initiated. Preliminary evidence indicates that CAN12 is able to delay the cleavage of PAR4. Importantly, CAN12 (2 mg/kg) treatment does not increase bleeding time or blood loss in the tail clip assay compared to control IgG (2 mg/kg) treatment. There was also no significant increase in bleeding in the saphenous vein assay. The mice treated with CAN12 (2 mg/kg) had an average bleeding time of 102 seconds for 12 clot formations in 20 minutes compared to the control mice (IgG 2 mg/kg) which had an average bleeding time of 143 seconds for 11 clot formations. These data demonstrate that we are able to inhibit platelet aggregation in vitro and thrombosis in vivo without influencing bleeding time. Overall, these studies provide insight towards the development of new anti-platelet therapies and, specifically, PAR4 as an antiplatelet therapy target. Disclosures: No relevant conflicts of interest to declare.

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