ALBERT

Description: Microparticles (MPs) are phospholipid vesicles about 0.1-1 μm in size released into blood from vascular and blood cells upon activation and apoptosis. The mechanism of MP formation by the exocytic budding of the outer cell membrane provides them with procoagulant activity, mainly due to phosphatidylserine exposure and tissue factor expression. MPs are present in the peripheral blood of healthy subjects but their level is elevated in a variety of vascular, infectious, and immune-mediated pathologies. While there are extensive studies on multiple roles of MPs in various diseases, including thrombotic disorders, the question remains open as to whether MPs are involved in the formation and properties of fibrin, the scaffold of hemostatic clots and obstructive thrombi. To investigate this problem, we studied in vitro the effects of MPs on the kinetics of fibrin polymerization, fibrin clot structure and susceptibility to fibrinolysis. Blood was drawn from healthy donors according to an IRB protocol. MPs were removed from citrated platelet-free plasma (PFP) by filtration through 0.1-μm pores, yielding microparticle-depleted plasma (MDP). To restore the phospholipids eliminated, MDP was replenished with cephalin (MDP-C). Fresh samples of PFP, MDP, and MDP-C from the same donor were analyzed in parallel. Concentrations of MPs and expression of platelet-specific CD61 were studied by flow cytometry and confocal microscopy. A thrombin generation test in recalcified defibrinated plasma was performed using a chromogenic substrate. Fibrin polymerization induced by recalcification of PFP, MDP, and MDP-C was studied by dynamic turbidimetry. tPA-induced fibrinolysis was also followed optically as a decrease of a plasma clot turbidity. Fibrin ultrastructure was studied with scanning electron microscopy (SEM) in a FEI Quanta 250 microscope. Each test was performed with plasma samples from at least 3 donors and the results were averaged. Enumeration of MPs in PFP vs. MDP showed that about 90% of total MPs were removed by filtration, including 99.6% of CD61-positive platelet-derived MPs. The rate of thrombin generation was significantly reduced in MDP vs. PFP and was fully restored in MDP-C, confirming an essential role of MPs in the intrinsic coagulation pathway. Based on the lag times and slopes of the turbidimetric curves, fibrin formation in recalcified plasma samples was significantly delayed in the absence of MPs (MDP) and was much faster in their presence (PFP and MDP-C). The maximum optical density was consistently and significantly higher in MDP than in PFP and MDP-C, suggesting that fibrin clots formed in the absence and presence of MPs have fibers of different thickness. This presumption was confirmed by SEM of fibrin clots formed upon recalcification of the plasma samples. Clots formed in PFP were less porous and had thinner fibers (170 ± 38 nm), while clots from MDP had larger pores and were built of thicker fibers (214 ± 53 nm, p