Regulation of postaggregation events induced by protease-activated receptor 1 ligation in human platelets: evidence of differential signaling pathways.
In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 microM) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin alpha(IIb)beta(3) to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin alpha(IIb)beta(3), Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.
Ramars
Amanchy S S
AS
Department of Biochemistry, Banaras Hindu University, Varanasi, 221005, India.
Mukhopadhyay
Saikat
S
Dash
Debabrata
D
D001792:Blood Platelets / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D010974:Platelet Aggregation / Q000502:physiology*; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000502:physiology; D013997:Time Factors
D008024:Ligands; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin