VPB0901 - Effects of platelets activated by different agonists on fibrin formation and thrombin generation.

Background: Platelet procoagulant function is usually assessed by exposure of phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes, and/or by the activity of these complexes. However, the direct effects of platelets on fibrin formation have not been thoroughly studied so far.

Aims: We investigated the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with PS expression.

Methods: A modified version of a recalcification assay was developed for studying the effects of platelets on fibrin formation. Washed human platelets were not activated or activated by ionophore A23187, collagen, arachidonic acid, ADP, and TRAP (Thrombin Receptor Activating Peptide), and spin down on the bottom of 96-well plates. Plasma was added to platelets, recalcified, and fibrin formation was followed by changes in light absorption. Platelets prepared in the same way were tested in the thrombin generation test. PS expression was evaluated by annexin V staining using flow cytometry.

Results: Platelets significantly accelerated both fibrin formation and thrombin generation. They shortened the lag phase and increased the maximal rate of plasma clotting, increased the peak and the maximal rate of thrombin generation. In both tests, platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment of platelets with exogenous agonists additionally increased their procoagulant activity. Maximal activity was observed after pretreatment with A23187, lesser with collagen and arachidonic acid, and minimal with ADP and TRAP (the latter might be ineffective due to the competition with endogenous thrombin). Effects of differently activated platelets on fibrin formation and thrombin generation in general corresponded with the levels of PS expression.

Conclusion(s): The ability of platelets activated by different agonists to accelerate fibrin formation correlates with their action on thrombin generation and is determined by the level of PS expression.
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