Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

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

Blood ◽

10.1182/blood.v124.21.1434.1434 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1434-1434

Author(s):

Leonard C. Edelstein ◽

Lukas M Simon ◽

Cory R Lindsay ◽

Xiango Kong ◽

Raul Teruel Montoya ◽

...

Keyword(s):

Platelet Aggregation ◽

Racial Differences ◽

Platelet Function ◽

Racial Difference ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Human Platelets ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Dependent Manner

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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In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽

10.1182/blood-2011-11-393900 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4066-4072 ◽

Cited By ~ 55

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

Bracken Babula ◽

Youfu Li ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Immune Thrombocytopenia ◽

Ex Vivo ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

High Dose ◽

Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

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Platelet reactivity in patients with aortic stenosis depends on LV-AO angle

European Heart Journal ◽

10.1093/ehjci/ehaa946.1866 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

P Mourikis ◽

S Zako ◽

L Dannenberg ◽

R M'Pembele ◽

T Hohlfeld ◽

...

Keyword(s):

Aortic Stenosis ◽

Platelet Activation ◽

Platelet Function ◽

Cox Regression ◽

Light Transmission ◽

Platelet Reactivity ◽

Funding Source ◽

Local Inflammation ◽

German Research Foundation ◽

Induced Aggregation

Abstract Background The pathogenesis of aortic stenosis (AS) is not fully understood. However, local inflammation of the valve appears to be a main player. Except haemostasis, platelets contribute to inflammatory processes in various ways. Furthermore, platelet function is altered in patients with AS. Moreover, a steep angle between the left ventricle and the aorta (LV-AO-angle) leads to turbulent blood flow. However, it is not known if platelet reactivity is associated with steep LV_AO angle in patients with AS. Methods We included 289 patients with severe AS and performed cardiac computertomography to assess the LV-AO-angle. Platelet function was evaluated by light transmission aggregometry by using collagen and adenosine-diphosphate to induce platelet activation Results ADP- and collagen induced aggregation showed a significant negative correlation with LV-AO-angle (ADP: r=−0.19, p=0.0009, R2=0.022; collagen: r=−0.21, p=0.0004, R2=0.027). ADP-induced MoA was significant higher in patients with a LV-AO-angle <160° in comparison to patients with an angle ≥160° (<160°: 66.99±20.72% vs. ≥160°: 60.66±19.85%, p=0.009). Collagen-induced platelet reactivity was significant higher in patients with a LV-AO-angle <160° in comparison to patients with an angle ≥160° (<160°: 78.67±13.19% vs. ≥160°: 73.85±14.44%, p=0.003). Multivariate cox-regression revealed that LV-AO angle <160 was a robust predictor of ADP- and collagen-induced platelet aggregation Conclusion A steep LV-AO-angle is associated with enhanced platelet reactivity in patients with AS. Platelet activation is known to lead to local inflammation. Therefore, enhanced platelet reactivity could play crucial in the progression of AS. The clinical significance of a steep LV-AO-angle needs be evaluated in further trials. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Research Foundation

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In vivo platelet activation and platelet hyperreactivity in abacavir-treated HIV-infected patients

Thrombosis and Haemostasis ◽

10.1160/th12-07-0504 ◽

2013 ◽

Vol 110 (08) ◽

pp. 349-357 ◽

Cited By ~ 35

Author(s):

Barbara Belfiori ◽

Eleonora Petito ◽

Giuseppe Guglielmini ◽

Lisa Malincarne ◽

AnnaMaria Mezzasoma ◽

...

Keyword(s):

Platelet Activation ◽

Cardiovascular Events ◽

Light Transmission ◽

Ex Vivo ◽

Platelet Reactivity ◽

Retrospective Case ◽

Activation Markers ◽

Induced Aggregation

SummaryAbacavir (ABC) has been associated with ischaemic cardiovascular events in HIV-infected patients, but the pathogenic mechanisms are unknown. Aim of our study was to assess whether ABC induces in vivo platelet activation and ex vivo platelet hyper-reactivity. In a retrospective, case-control study, in vivo platelet activation markers were measured in 69 HIV-infected patients, before starting therapy and after 6–12 months of either ABC (n=35) or tenofovir (TDF) (n=34), and compared with those from 20 untreated HIV-infected patients. A subgroup of patients was restudied after 28–34 months for ex vivo platelet reactivity. In vivo platelet activation markers were assessed by ELISA or flow cytometry, ex vivo platelet reactivity by light transmission aggregometry (LTA) and PFA-100®. The in vitro effects of the ABC metabolite, carbovir triphosphate, on aggregation and intra-platelet cGMP were also studied. sPLA2, sPsel and sGPV increased significantly 6–12 months after the beginning of ABC, but not of TDF or of no treatment. Ex vivo platelet function studies showed enhanced LTA, shorter PFA-100® C/ADP closure time and enhanced platelet expression of P-sel and CD40L in the ABC group. The intake of ABC blunted the increase of intraplatelet cGMP induced by nitric oxide (NO) and acutely enhanced collagen-induced aggregation. Preincubation of control platelets with carbovir triphosphate in vitro enhanced platelet aggregation and blunted NO-induced cGMP elevation. In conclusion, treatment with ABC enhances in vivo platelet activation and induces platelet hyperreactivity by blunting the inhibitory effects of NO on platelets. These effects may lead to an increase of ischaemic cardiovascular events.

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Apoptosis Signal-Regulating Kinase (ASK1) Regulates Thrombosis in Part By Regulating cPLA2 phosphorylation-Dependent TxA2 Generation

Blood ◽

10.1182/blood.v128.22.3719.3719 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3719-3719

Author(s):

Pravin Patel ◽

Meghna U. Naik ◽

Ulhas Naik

Keyword(s):

Map Kinase ◽

Platelet Activation ◽

Platelet Function ◽

Map Kinases ◽

Conflicts Of Interest ◽

Cellular Stress ◽

Substantial Reduction ◽

Genetic Ablation ◽

Independent Mechanism

Abstract When vascular endothelium is injured, circulating platelets are activated by primary agonists. Activation causes platelets to change shape, aggregate, and release secondary agonists which reinforce initial platelet activation as well as help recruit additional platelets to the site of vascular injury. MAP kinases have been shown to be important regulators of platelet function and secondary agonist production. One important secondary agonist released by activated platelets is TxA2. TxA2 is generated by metabolism of Arachidonic acid (AA). AA is released from platelet membrane phospholipids via the activity of PLAs. In platelets cPLA2 activity has been shown to be regulated by MAP kinases, however, the mechanisms which regulate platelet MAP kinase activity are not well understood. Our laboratory has identified that ASK1 (a Ser/Thr kinase of the MAP3K family) is present in both human and murine platelets and is activated by physiological agonists. ASK1 is known to be activated by a number of cellular stress response pathways. When challenged by cellular stress, ASK1 auto phosphorylates Thr845 on its activation loop, which is required for its ability to phosphorylate its substrates. Here we show that ASK1 regulates platelet function in part by regulating agonist-induced TxA2 generation. To determine the role of Ask1 in hemostasis and thrombosis, we evaluated in vivo thrombosis using carotid artery injury induced by 10% FeCl3 or pulmonary thromboembolism induced by injecting mixture of collagen/epinephrine. We found that genetic ablation of Ask1 renders mice significant protection from thrombosis. To determine the mechanism by which Ask1 regulates platelet activation leading to thrombosis, we evaluated the MAP kinase cascade using Ask1 null platelets. We found that genetic ablation of Ask1 blocked agonist-induced activation of the MAP2Ks (MKK3 and MKK4) in murine platelets. Since MKK3 can activate p38 and MKK4 can activate both p38 and JNK, we assessed MAPKs activation in murine platelets. When stimulated by various agonists, activation of p38 was entirely lost in Ask1 null platelets while activation of ERK1/2 and JNK remained unaffected indicating that Ask1 solely regulates p38 activity in platelets. Activity of p38 has been linked to agonist-induced generation of TxA2, an important contributing factor to thrombosis. We therefore evaluated agonist-induced production of TxA2 by measuring TxB2 (a stable metabolite of TxA2). We saw a substantial reduction (~50% in thrombin- and ~70% in convulxin-induced) production of TxA2 in Ask1 null platelets suggesting a separate Ask1 independent mechanism for TxA2 generation. Since TxA2 is a metabolite of AA, whose production in platelets is caused by cPLA2 enzymatic activity and cPLA2 activity is regulated by phosphorylation of its Ser505 residue by p38, we evaluated phosphorylation of cPLA2 (p-Ser505). We found that agonist-induced phosphorylation of cPLA2 (Ser505) was completely lost in Ask1 null platelets. Although in Ask1 null platelets cPLA2 phosphorylation (Ser505) is completely abolished, substantial amount (~50%) of TxA2 was generated in response to thrombin suggesting that there exists an Ask1 independent mechanism of activation of cPLA2. To rule out the possibility that an alternative PLA2 is responsible for the residual TxA2 production found in Ask1 null platelets, we evaluated agonist-induced TxA2 production in the presence of pyrrophenone, a cPLA2 specific inhibitor. Pretreatment with pyrrophenone completely abolished agonist-induced TxA2 production in murine as well as human platelets, suggesting that cPLA2 is solely responsible for the majority of agonist induced AA/TxA2 in platelets. In addition to its phosphorylation, it is documented that cPLA2 activity is also dependent on intracellular Ca2+, which facilitates translocation of cPLA2 to AA containing membranes. It is therefore possible that the remainder of TxA2 formed is dependent on Ca2+-dependent activity of cPLA2. Taken together these in vivo and in vitro results strongly suggest that ASK1 plays a key role in regulating thrombosis, in part, by regulating the signaling mechanisms involved in agonist-induced production of TxA2. Disclosures No relevant conflicts of interest to declare.

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Apoptosis Signal-Regulating Kinase 1 Regulates Platelet Function through Integrin aIIbb3 Outside-In Signaling

Blood ◽

10.1182/blood.v116.21.328.328 ◽

2010 ◽

Vol 116 (21) ◽

pp. 328-328

Author(s):

Meghna Ulhas Naik ◽

Hidinori Ichijo ◽

Ulhas P Naik

Keyword(s):

Map Kinase ◽

Platelet Function ◽

Stress Responses ◽

Intracellular Signaling ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Phosphorylation Site ◽

Mapk Cascades ◽

Induced Aggregation

Abstract Abstract 328 Atherosclerosis tends to develop in areas of the circulation where the blood flow is either low or turbulent, such as regions where the arteries bifurcate. Studies have shown that shear stress stimulates endothelial cells and modulates platelet function. However, the intracellular signaling events that coordinate these effects are not completely understood. The mitogen-activated protein kinase (MAPK) cascades are crucial in regulating cellular stress responses. It is known that a number of diseases, including cardiovascular diseases, are intimately related to stress related mechanisms mediated by MAPK cascades. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family which responses to diverse array of stresses. Here, we show for the first time that ASK1, a 155 kDa protein, is present in human and murine platelets and its role as a novel regulator of platelet function. In human platelets, when stimulated with thrombin, ASK1 is rapidly activated within 30 sec, as indicated by phosphorylation of Thr 845 on its activation loop. Interestingly, we found that ASK1 is rapidly inactivated after 3 min by dephosphorylation of phospho-Thr 845. Concomitantly, ASK1 is phosphorylated on Ser 967, a known inhibitory phosphorylation site. The activation of ASK1 coincided well with its downstream effector, p38 Map kinase. These results suggested that ASK1 may play a role in platelet function. We therefore tested the hemostatic functions in congenic Ask-1 knockout mice and compared it to C57/BL6 wild type (Wt) mice. We found a significant delay in tail bleeding time (P=0.2×10−9) in Ask1 null mice. While all Wt mice stopped bleeding with an average bleeding time of 100 sec, the Ask1 null mice had an average bleeding time of 576 sec, with 5 out of 9 mice that did not stop bleeding. A 10% FeCl3-induced carotid artery injury, a well-established in vivo thrombosis model, 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 intravenous injection of a mixture of collagen and epinephrine. This finding was further supported by the histological examination of the lung tissue of mice surviving after 3 min of injection, which showed very few thrombi in Ask1 null mice compared to Wt mice. We next asked if the platelet function is affected in Ask1 null mice. We found that low dose of PAR-4 peptide-induced aggregation is significantly less in Ask1 null mice compared to Wt mice. When analyzed for the ability of Ask1 null platelets to retract clots, we found that while clot retraction was completed within 2h in Wt platelets, Ask1 null platelets failed to retract clots even after 18h. When assessed for phosphorylation of the integrin b3 subunit as an indicator of outside-in signaling, we found that Wt platelet exposure to immobilized fibrinogen showed a robust phosphorylation of b3 subunit. Interestingly, Ask1 null platelets failed to show any induction of b3 phosphorylation. These in vitro and in vivo results strongly suggest that ASK1 plays a significant role in the regulation of platelet function and hemostasis. Disclosures: No relevant conflicts of interest to declare.

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12-HETrE, An Endogenous Inhibitor of Platelet Activation,

Blood ◽

10.1182/blood.v118.21.3254.3254 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3254-3254

Author(s):

Jennifer Yeung ◽

Kenneth Ikei ◽

Joanne Vesci ◽

Alex Arnouk ◽

Theodore R Holman ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Fatty Acid Content ◽

Pharmacological Intervention ◽

Direct Role ◽

Granule Secretion

Abstract Abstract 3254 Platelet activation plays a pivotal role in thrombosis and hemostasis. Understanding the signaling events mediating this process is essential in preventing unwanted clot formation, which can lead to the development of heart attack, stroke, and venous thromboembolism. Fatty acids may play an important role in determining the level of platelet reactivity, however the mechanism(s) by which this occurs are not entirely clear. 12-lipoxygenase (12-LOX) has been shown to oxidize the fatty acid, arachidonic acid (AA), in order to produce the bioactive eicosanoid 12-HETE, which has recently been shown to play a role in tissue factor activation and subsequent thrombin generation in the platelet (Thomas et al., 2010, J Biol Chem; 285:6891–903). 12-HETE has also been shown to signal to cells, in part, through the G protein-coupled receptor GPR31 (Guo et al, 2011, J Biol Chem; epub). While research on 12-LOX-mediated eicosanoid regulation of platelets has primarily focused on 12-HETE, the potential for regulation of platelets by eicosanoids derived from other fatty acids has been overlooked. As the fatty acid content on the platelet membrane is extremely dynamic and fatty acid supplementation is correlated with a reduced risk for cardiovascular disease, we hypothesized that 12-LOX oxidation of another fatty acid may play a direct role in regulating platelet function. To test this hypothesis, the Ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) as well as its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12-HETrE), were applied to washed platelets followed by stimulation with thrombin, PAR1-AP, or PAR4-AP. Platelets treated with either DGLA or 12-HETrE showed significant attenuation in platelet aggregation following stimulation with thrombin or PAR-AP. A number of biochemical intermediates were also tested in the presence of 12-HETrE or DGLA including Rap1 activation, αIIbβ3 integrin activation, α-granule secretion, and dense granule secretion. All endpoints tested were attenuated in the presence of DGLA or 12-HETrE relative to control. To confirm the regulation was unique to DGLA and its metabolite, the same endpoints were measured in the presence of AA or 12-HETE. Pre-treatment with either AA or 12-HETE did not attenuate any of the agonist-mediated platelet activation endpoints. Thus, our data supports a unique role for 12-HETE and 12-HETrE and that fatty acid regulation of platelet function may be highly dependent on the lipid content of the platelet. Shifting the ratios of fatty acids in the platelet through dietary supplementation or pharmacological intervention may be sufficient to induce a cardio-protective state, in part, through increasing 12-HETrE formation and subsequently inhibiting platelet activation. Disclosures: No relevant conflicts of interest to declare.

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Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

Blood ◽

10.1182/blood-2019-128250 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4883-4883

Author(s):

Stephanie Renna ◽

Leonard C. Edelstein ◽

Steven Edward McKenzie

Keyword(s):

Amino Acid ◽

Platelet Activation ◽

Knockout Mice ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Wild Type Mouse ◽

Human Platelets ◽

Amino Acid Sequences ◽

Wild Type

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

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Cathepsin G Cleavage of PAR4 Generates a Novel Tethered Ligand That Induces Platelet Activation

Blood ◽

10.1182/blood-2020-134931 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 2-2

Author(s):

Michelle L. Stoller ◽

Indranil Basak ◽

James Alsobrooks ◽

Paul F. Bray ◽

Robert A. Campbell

Keyword(s):

Platelet Activation ◽

Cleavage Site ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Cathepsin G ◽

Cleavage Sites ◽

Tethered Ligand ◽

Tethered Ligands

Atherosclerotic vessel injury induces recruitment of both platelets and neutrophils where multiple proteases induce platelet activation and aggregation. Platelets contain two protease activated receptors, PAR1 and PAR4, the cleavage of which results in exposure of a new amino terminus to serve as a tethered ligand. Released neutrophil cathepsin G (CatG) has been shown to be a physiologic modulator of platelet thrombus formation in mice. CatG activates PAR4 and not PAR1, presumably because CatG cleaves PAR1 by removing its tethered ligand. However, neither the CatG biochemical cleavage of PAR4 nor the resulting tethered ligands have been reported. The goals of the current study are to (1) identify the CatG-PAR4 cleavage sites and resulting tethered ligands and (2) determine how CatG-stimulated PAR4 signaling is altered by the PAR4 Ala120Thr variant. We synthesized two portions of the PAR4 extracellular N-terminus: amino acids Asp38-Ser58 (PAR4-B) and Asp57-Arg78 (PAR4-C) and exposed each peptide to purified CatG. Mass spectrometry identified two major cleavage sites for PAR4-B: the previously documented CatG and thrombin site Arg47-Gly48 and a novel Cys54-Ala55. Analysis of PAR4-C digestion yielded an additional three novel CatG cleavage sites, two major: Arg68-Ala69 and Leu71-Leu72, and one minor: Leu70-Leu71. Neither concentration or time of exposure appeared to alter the CatG cleavage sites. To assess functionality, we generated peptides based on the novel cleavage sites produced by CatG cleavage. Human washed platelets were treated with each peptide, and platelet activation was assessed by PAC-1 binding. As expected, the known tethered ligand sequence GYPGQV showed a statistically significant increase in PAC-1 binding (p=.02) compared to resting platelets. Three of the remaining four novel peptides generated no significant change in PAC-1 binding compared to baseline. However, peptide 3, representing novel tethered ligand ALLLGW, induced a substantial increase (462%) in PAC-1 binding compared to resting platelets. To assess the effect of the PAR4Ala120Thr variant on CatG-stimulated platelet reactivity, human washed platelets were collected from donors homozygous for Ala120 or Thr120 and stimulated with CatG. Platelets expressing the Thr120 variant displayed a significant increase in PAC-1 compared to Ala120 platelets (54%; p=.004). Addition of a CatG inhibitor caused a significant decrease in platelet activation triggered by CatG for both groups (Thr: -81%, p=.000001; Ala: -78%, p=.00017), and abrogated the significant increase in platelet activation displayed by Thr120 platelets (p=.99). To further examine the relationship between the PAR4 Ala120Thr variant and CatG, platelets from each variant were subjected to increasing amounts of CatG. Platelet activation was measured by PAC-1 binding and P-selectin expression. Both PAC-1 binding and P-selectin expression were significantly increased in platelets from Thr120 donors compared to Ala120 platelets (PAC-1, p=.026; P-selectin, p=.025). Overall, our study identified a previously unidentified CatG cleavage site in PAR4, which produced a novel tethered ligand capable of activating platelets. Because cleavage resulting in the ALLLGW ligand is downstream of the thrombin cleavage site, CatG may modulate in vivo thrombin-induced signaling in platelets or other cell types. Our findings also indicate the hyperactive response of 120Thr platelets is not dependent on a specific PAR4 protease. These new insights into PAR4 biology may provide targets for future antithrombotic therapies. Disclosures No relevant conflicts of interest to declare.

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Pharmacogenocis of PAR4: PAR4 Polymorphism Determines Platelet Response in the Presence of Dual Anti-Platelet Therapy

Blood ◽

10.1182/blood.v126.23.3446.3446 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3446-3446

Author(s):

Benjamin Tourdot ◽

Paul F. Bray ◽

Leonard C. Edelstein ◽

Michael Holinstat

Keyword(s):

Platelet Activation ◽

Racial Difference ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Platelet Reactivity ◽

Day Treatment ◽

Platelet Response ◽

Platelet Activity ◽

P2y12 Inhibitors ◽

Cox 1

Abstract Aberrant platelet activation contributes to the mortality associated with coronary heart disease (CHD). Our lab group has recently shown that platelets from black donors were hyper-responsive to activation of the thrombin receptor Protease-Activated Receptor-4 (PAR4) compared to platelets from white donors. The racial difference observed through PAR4 persists in platelets treated ex vivo with dual antiplatelet inhibitors (COX-1 and P2Y12 receptor inhibitors), a standard of care for many CHD patients. The racial difference in PAR4 signaling is largely attributable to one PAR4 variant more frequently expressed in blacks than whites, PAR4 T120A. Independent of race, T120 is associated with greater PAR4-mediated platelet reactivity compared to the A120 variant. The role PAR4 variation plays in regulating thrombin-mediated platelet activation in CHD has not been elucidated to date. We hypothesize that people homozygous for the T120 will have an increase in PAR4-mediated platelet reactivity compared to patients homozygous for A120. To investigate this hypothesis, we studied PAR4 signaling in platelets obtained from healthy donors. Platelet activity was assessed in each subject at the beginning of the study. Subsequently subjects were placed on a 7-day treatment of either COX-1 or P2Y12 inhibitors and on day 7 their blood was again drawn and tested for PAR4-AP-mediated platelet activation. Subjects who are homozygous for T120, have an increase in platelet reactivity as measured by aggregation, αIIbβ3 activation and granule secretion compared to individuals who are homozygous for A120. Further distal signaling nodes such as calcium and Rap1 are hyperactive in individuals who are homozygous for T120. This study reinforces the personalized medicine approach to therapeutic intervention and challenges the one size fits all approach which often leaves at risk populations without adequate protection from thrombotic events and stroke. This study was supported in part by grants MD007880, GM105671, and HL 114405 (MH) from the NIH. Disclosures No relevant conflicts of interest to declare.

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