A role of methionines in the functioning of oxidatively modified fibrinogen.

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/-OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/-OCl, 10 µM HOCl/-OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BßMet367, γMet264, and γMet94, identified in 10 µM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Adhesive properties of plasma-circulating and platelet-derived microvesicles from healthy individuals.

BACKGROUND: Microvesicles (MVs) produced by platelets upon activation possess high procoagulant activity and represent a possible thrombotic risk marker. However, direct experimental evaluation of the adhesive properties of MVs and their potential role in thrombus growth is lacking. OBJECTIVES: We investigated integrin αIIbß3 status and adhesive properties of plasma-circulating and platelet-derived MVs from healthy individuals. METHODS: MVs were isolated from whole blood or produced from activated platelets. Flow cytometry was used for quantification of fluorescently labeled PAC-1 and fibrinogen binding to MVs. Confocal microscopy was used for evaluation of MVs adhesion to fibrinogen and for estimation of their involvement in whole blood thrombus formation in a parallel-plate flow chambers under arterial shear conditions. RESULTS AND CONCLUSIONS: Neither circulating plasma MVs, nor platelet-activation-produced MVs bound PAC-1. However, both types of MVs specifically and weakly bound fibrinogen (about 400 molecules of bound fibrinogen per MV versus >100,000 per non-procoagulant activated platelet). Still, the MVs did not adhere stably to the immobilized fibrinogen. Both types of MVs were weakly incorporated into a thrombus and did not affect thrombus formation: average thrombus height in the recalcified whole blood in the presence of platelet-activation-produced MVs was 4.19 ± 1.38 µm versus 4.87 ± 1.72 µm (n = 6, p > 0.05) in the control experiments. This suggests that MVs present in plasma of healthy individuals are not likely to be directly involved in thrombus formation under arterial flow conditions.