title: GPV is a marker of in vivo platelet activation--study in a rat thrombosis model.
abstract: Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.
authors.affiliation: INSERM U.311, Biologie et Pharmacologie de l'Hémostase et de la Thrombose, Etablissement de Transfusion Sanguine de Strasbourg, France. catherine.ravant@etss.u-strasbg.fr
authors.forename: C
authors.initials: C
authors.lastname: Ravanat
authors.forename: M
authors.initials: M
authors.lastname: Freund
authors.forename: P
authors.initials: P
authors.lastname: Mangin
authors.forename: D O
authors.initials: DO
authors.lastname: Azorsa
authors.forename: C
authors.initials: C
authors.lastname: Schwartz
authors.forename: S
authors.initials: S
authors.lastname: Moog
authors.forename: S
authors.initials: S
authors.lastname: Schuhler
authors.forename: J
authors.initials: J
authors.lastname: Dambach
authors.forename: J P
authors.initials: JP
authors.lastname: Cazenave
authors.forename: F
authors.initials: F
authors.lastname: Lanza
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000990:Antithrombin III / Q000096:biosynthesis / Q000187:drug effects; D015415:Biomarkers / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects; D003029:Coagulants / Q000378:metabolism; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005434:Flow Cytometry; D010196:Pancreatic Elastase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000187:drug effects; D015539:Platelet Activation; D010978:Platelet Factor 4 / Q000096:biosynthesis / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D012680:Sensitivity and Specificity; D012995:Solubility; D013917:Thrombin / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013925:Thromboplastin / Q000008:administration & dosage / Q000494:pharmacology; D013927:Thrombosis / Q000097:blood* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D003029:Coagulants; D000939:Epitopes; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C046193:antithrombin III-protease complex; D010978:Platelet Factor 4; D000990:Antithrombin III; D013925:Thromboplastin; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D013917:Thrombin