title: Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V.
abstract: Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibalpha of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca(2+) mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbalpha by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbalpha binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbalpha proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbalpha cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbalpha.
authors.affiliation: Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Lova
authors.forename: Ilaria
authors.initials: I
authors.lastname: Canobbio
authors.forename: Gianni F
authors.initials: GF
authors.lastname: Guidetti
authors.forename: Cesare
authors.initials: C
authors.lastname: Balduini
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
mesh_terms: D000199:Actins / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D006801:Humans; D008666:Metalloendopeptidases / Q000494:pharmacology; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism; D020668:rap GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D019038:Platelet Glycoprotein GPIb-IX Complex; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D010738:Type C Phospholipases; D013917:Thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin; D008666:Metalloendopeptidases; C099092:mocarhagin; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins; C112908:protease-activated receptor 4