Early intraplatelet signaling enhances the release of human platelet PAR-1 and -4 amino-terminal peptides in response to thrombin.
Activation of washed human platelets initiated with alpha-thrombin, SFLLRN, or AYPGKF invariably results in the generation of PAR-1-(1-41) and PAR-4-(1-47). PAR-1-(1-41) and PAR-4-(1-47) are amino-terminal peptides generated when PAR-1 and -4 are cleaved in their first extracellular domains after R(41) and R(47), respectively, to expose the tethered ligand domains of PAR-1 and -4. Since soybean trypsin inhibitor decreases generation of PAR-1-(1-41) and PAR-4-(1-47) and other platelet aggregation-related responses to these three agonists, but does not inactivate alpha-thrombin, a platelet trypsin-like proteinase apparently activates PAR-1 and -4 to propagate PAR-dependent platelet responses. This study identified the signaling pathways implicated in the generation of the platelet proteinase that in turn produces PAR-1-(1-41) and PAR-4-(1-47), to thereby drive the subsequent PAR-dependent platelet aggregation-related responses to alpha-thrombin, SFLLRN, or AYPGKF. Only inhibitors of signaling enzymes that prevented ATP release (forskolin, PGE(1), or BIMI-1) prevented or delayed the generation of PAR-1-(1-41) and PAR-4-(1-47) in response to all three agonists. SBTI prevented platelet aggregation initiated by alpha-thrombin, SFLLRN, or AYPGKF but did so less effectively when it was added 10 s after each agonist. Thus, the platelet-derived proteinase acts within 10 s of each agonist addition to generate PAR-1-(1-41) and PAR-4-(1-47). Furthermore, alpha-thrombin may not effectively catalyze PAR-1-(1-41) and PAR-4-(1-47) generation. We propose that unidentified ATP-dependent phosphorylation reactions catalyzed by PKC help to generate the platelet-derived proteinase that propagates human platelet PAR-1 and -4 activation by the three agonists.
Ofosu
Frederick A
FA
Department of Pathology and Molecular Medicine, McMaster University, and Canadian Blood Services, Hamilton, Ontario L8N 3Z5, Canada. ofosuf@mcmaster.ca
Dewar
Lori
L
Song
Yingqi
Y
Cedrone
Aisha C
AC
Hortelano
Gonzalo
G
Craven
Sharon J
SJ
D055162:Biocatalysis; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010766:Phosphorylation; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000378:metabolism; D018179:Receptors, Thrombin / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000494:pharmacology*
D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; C112908:protease-activated receptor 4