Abstract
BackgroundThrombin 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.ObjectiveWe sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve.MethodsTissue 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.ResultsThe addition of the P2Y12 receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E1 (PGE1), 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, PGE1 or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE1 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.ConclusionsProcoagulant 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.
Highlights
The thrombin generation test (TGT) is one of the most informative and popular blood coagulation assays used for both research and diagnostic purposes
The addition of the P2Y12 receptor antagonist MeS-AMP (160 mM), 83 nM prostaglandin E1 (PGE1), or 1.6% DMSO to platelet-rich plasma (PRP) caused the appearance of two peaks in the Thrombin generation curve (TGC)
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
Summary
The thrombin generation test (TGT) is one of the most informative and popular blood coagulation assays used for both research and diagnostic purposes. This simple technique for measuring thrombin concentration in clotting plasma was presented by H.C. Hemker [1,2,3]. Thrombin generation curve (TGC) is usually bell-shaped and has four main features. The second and the third features are described by the time and amplitude of the thrombin peak. 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. TGC in platelet-rich plasma (PRP) can sometimes have two peaks
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