title: Antiplatelet agents can promote two-peaked thrombin generation in platelet rich plasma: mechanism and possible applications.
abstract: BACKGROUND
  Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks.
  
  
  OBJECTIVE
  We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve.
  
  
  METHODS
  Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers.
  
  
  RESULTS
  The addition of the P(2)Y(12) receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E(1) (PGE(1)), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE(1) or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE(1) mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one.
  
  
  CONCLUSIONS
  Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types--plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.
authors: The Laboratory of the Molecular Mechanisms of Hemostasis, the Center for Theoretical Problems of Physicochemical Pharmacology RAS, Moscow, Russia. taran_@list.ru
authors: Ivan D
authors: ID
authors: Tarandovskiy
authors: Elena O
authors: EO
authors: Artemenko
authors: Mikhail A
authors: MA
authors: Panteleev
authors: Elena I
authors: EI
authors: Sinauridze
authors: Fazoil I
authors: FI
authors: Ataullakhanov
mesh_terms: D000527:Alprostadil / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D005165:Factor V / Q000187:drug effects*; D015951:Factor Xa / Q000187:drug effects*; D005434:Flow Cytometry; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D053657:Platelet-Rich Plasma / Q000187:drug effects / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000494:pharmacology
chemical_list: D003374:Coumarins; D006490:Hemostatics; D009842:Oligopeptides; D019007:P-Selectin; D010718:Phosphatidylserines; D010975:Platelet Aggregation Inhibitors; C411346:benzyloxycarbonyl-glycyl--glycyl-arginine-4-methylcoumaryl-7-amide; C022475:prothrombinase complex; D005165:Factor V; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa; D000527:Alprostadil