title: Role of protease-activated and ADP receptor subtypes in thrombin generation on human platelets.
abstract: The activated platelet surface serves as an integral part of the prothrombinase complex upon activation by potent platelet agonists such as thrombin and collagen. We determined the receptor specificity through which thrombin was enhancing collagen-induced thrombin generation. Whereas SFLLRN or AYPGKF alone produced minimal thrombin generation or phosphatidylserine exposure through protease activated receptor (PAR) stimulation, they caused a leftward shift in the collagen-induced thrombin generation dose-response curve. Although SFLLRN or AYPGKF potentiated collagen-induced thrombin generation, neither of them potentiated to the same extent as thrombin. However, SFLLRN and AYPGKF together potentiated collagen-induced thrombin generation to the same extent as thrombin. We conclude that thrombin mediates its procoagulant activity through activation of both PAR1 and PAR4 receptors. Similarly, neither PAR1 nor PAR4 stimulation alone mimicked the annexin V-binding response caused by thrombin stimulation. The combination of PAR activating peptides caused minimal increases in annexin V binding, but caused significant thrombin generation, suggesting that events other than phosphatidylserine exposure may play a role in platelet prothrombinase complex formation. We also investigated the ability of ADP to potentiate agonist-induced thrombin generation. Whereas P2Y(1) antagonism did not affect collagen or thrombin-induced thrombin generation, P2Y(12) antagonism did decrease both collagen- and thrombin-induced thrombin generation, suggesting that ADP potentiates thrombin generation primarily through the P2Y(12) receptor. Collectively, these results suggest that stimulation of both the PAR1 and PAR4 receptors are necessary for thrombin-induced procoagulant activity, and that the P2Y(12) receptor, but not the P2Y(1) receptor, is responsible for the potentiation of agonist-induced platelet procoagulant activity.
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Dorsam
authors.forename: M
authors.initials: M
authors.lastname: Tuluc
authors.forename: S P
authors.initials: SP
authors.lastname: Kunapuli
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin / Q000378:metabolism; D013917:Thrombin / Q000096:biosynthesis*; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D017304:Annexin A5; D008565:Membrane Proteins; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; D044463:Receptor, PAR-1; D044462:Receptors, Proteinase-Activated; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; D003094:Collagen; D013925:Thromboplastin; D013917:Thrombin; C112908:protease-activated receptor 4