title: PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation.
abstract: Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Holinstat
authors.forename: Bryan
authors.initials: B
authors.lastname: Voss
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Bilodeau
authors.forename: Joseph N
authors.initials: JN
authors.lastname: McLaughlin
authors.forename: John
authors.initials: J
authors.lastname: Cleator
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002614:Chelating Agents / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D015539:Platelet Activation / Q000502:physiology; D010974:Platelet Aggregation / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D002614:Chelating Agents; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C548576:P2RY12 protein, human; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D058919:Purinergic P2 Receptor Antagonists; D044463:Receptor, PAR-1; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D013917:Thrombin; C112908:protease-activated receptor 4; D002118:Calcium