Background: Platelet procoagulant activity is usually assessed by expression of phosphatidylserine (PS) which appeared on the surface of activated platelets and served as a substrate for assembling of coagulation complexes. However, direct effects of activated platelets on fibrin formation are not thoroughly studied so far.

Aims: To develop the method for evaluation of the effects of activated platelets on fibrin formation in blood plasma.

Methods: Washed human platelets were activated by thrombin receptor activating peptide (TRAP), collagen and ionophore A23197. Platelets were spun down on the bottom of multiplate wells (0.5 x 10⁷ platelets per well). After supernatant removal, 100 µl of plasma diluted at 1/1 ratio with Owren-Koller buffer supplemented with 150 µg/ml of corn trypsin inhibitor was added into the wells. Clotting was initiated by addition of 50 µl 25 mM CaCl2 and followed in a multiplate reader at 25^oC by increase of adsorption at 450 nm. PS expression on activated platelets was measured by annexin V binding using flow cytofluorimetry.

Results: Platelets localized on the bottom of multiplate wells accelerated recalcified plasma clotting. The shortest clotting lag phases were detected in the presence of platelets activated by ionophore A23187. Significant shortage of the lag phases in comparison with not activated platelets was also observed when platelets were activated by collagen and to a less extent when they were activated by TRAP. Not activated platelets also accelerated clotting in comparison with plasma alone, presumably due to their activation by endogenous thrombin in recalcified plasma. Procoagulant effects of activated platelets correlated with the expression of PS detected by flow cytofluorimetry in parallel samples.

Conclusions: A new method based on modification of plasma recalcification assay was developed for evaluation of platelet procoagulant activity.
This work was supported by Russian Foundation for Basic Research (grant #20-015-00106).

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/modified-plasma-recalcification-assay-for-evaluation-of-platelet-procoagulant-activity/