Coordinate activation of human platelet protease-activated receptor-1 and -4 in response to subnanomolar alpha-thrombin.
We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after <or=1.0 nm alpha-thrombin, 10 microm SFLLRN, or 100 microm AYPGKF were added to platelets. When SFLLRN or AYGPKF were added to platelets, generation of PAR-1 and -4 activation peptides was complete at 10 s. Generation of both PAR-1 and -4 activation peptides in response to 1 nm alpha-thrombin was significantly inhibited by affinity-purified anti-PAR-1-(35-62) IgY, anti-PAR-4-(34-54) IgY, and by the specific PAR-1 antagonist BMS 200261, but not by the PAR-4 antagonist YD3. Effective inhibition of platelet aggregation in response to 1.0 nm alpha-thrombin occurred only in the presence of both anti-PAR span antibodies. We conclude that platelet activation initiated with <or=1.0 nm alpha-thrombin proceeds via simultaneous PAR-1 and -4 activation. Inhibiting the activation of either PAR inhibits activation of the other. Both PAR-1 and -4 activation must be inhibited to prevent platelet activation subsequent to thrombin binding to platelets. The more efficient generation of PAR activation peptides by platelets activated with SFLLRN or AYGPKF, compared with alpha-thrombin, indicates that a platelet-derived serine protease that is inactivated by soybean trypsin inhibitor propagates PAR-1 and -4 activation.
Ofosu
Frederick A
FA
Canadian Blood Services, Hamilton, Ontario, Canada. ofosuf@mcmaster.ca
Dewar
Lori
L
Craven
Sharon J
SJ
Song
Yingqi
Y
Cedrone
Aisha
A
Freedman
John
J
Fenton
John W
JW
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D006629:Hirudins / Q000494:pharmacology; D007136:Immunoglobulins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D006629:Hirudins; C016512:IgY; D007136:Immunoglobulins; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D014361:Trypsin Inhibitors; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; C082835:thrombin receptor peptide (42-47); D012697:Serine Endopeptidases; D013917:Thrombin; C112908:protease-activated receptor 4