title: A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation.
abstract: BACKGROUND
  The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A2 (TXA2 ) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown.
  
  
  OBJECTIVE
  We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation.
  
  
  RESULTS
  ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of αII b β3 -associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from αII b β3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence.
  
  
  CONCLUSIONS
  A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.
authors: Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou.
authors: L
authors: L
authors: Jiang
authors: C
authors: C
authors: Xu
authors: S
authors: S
authors: Yu
authors: P
authors: P
authors: Liu
authors: D
authors: D
authors: Luo
authors: Q
authors: Q
authors: Zhou
authors: C
authors: C
authors: Gao
authors: H
authors: H
authors: Hu
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D010974:Platelet Aggregation / Q000502:physiology*; D044463:Receptor, PAR-1 / Q000502:physiology*; D013917:Thrombin / Q000502:physiology*
chemical_list: D044463:Receptor, PAR-1; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D013917:Thrombin; D002118:Calcium