Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles

Abstract

Activation of human platelets considerably enhanced their ability to accelerate factor Va inactivation by activated protein C (APC). The anticoagulant activity of platelet suspensions was markedly dependent on the kind of agonist used to activate platelets. APC-catalyzed factor Va inactivation in free solution was characterized by an apparent second-order rate constant of 2 x 10(5) (mol/L)-1 (seconds)-1. Nonstimulated platelets (2.4 x 10(8)/mL) and platelets stimulated with adenosine diphosphate or adrenalin accelerated factor Va inactivation fourfold. Rates of factor Va inactivation were increased 11-fold by thrombin-stimulated platelets, 29-fold after platelet stimulation with the Ca(2+)-ionophore A23187. At low platelet concentrations (3 x 10(7)/mL) only background levels of anticoagulant activity were observed in platelet suspensions that were nonstimulated or stimulated with thrombin or collagen. However, when such reaction mixtures were stirred during the activation procedure, platelet anticoagulant activity was increased more than 10-fold. Independent of platelet stimulation and stirring conditions, exogenously added purified plasma protein S increased platelet-dependent factor Va inactivation approximately twofold. Addition of a neutralizing antiprotein S antibody had little effect on the anticoagulant activity of platelets. This indicates that, under the reaction conditions tested, platelet- released protein S did not contribute to factor Va inactivation. Approximately 25% of the anticoagulant activity of stimulated platelet suspensions appeared to be associated with microparticles that were released on platelet activation. Such microparticles may provide an important source of anticoagulant activity. A similar distribution of procoagulant, ie, prothrombinase, activity between platelets and microparticles was observed for the same platelet suspensions. Because platelet stimulation and stirring also had the same overall effects on the ability of platelets and platelet microparticles to promote prothrombin activation and factor Va inactivation, it appears likely that the generation of potential platelet anticoagulant and procoagulant activities is coupled to the same platelet stimulation reactions.

Importance of Protein S and Phospholipid for Activated Protein C-mediated Cleavages in Factor Va

“…␣-Thrombin and monoclonal antibody AHV-5146 (against the heavy chain of FVa) were from Hematologics Inc. (Essex Junction, VT). Human FV was purified from plasma as described (22) with minor modifications (23). Recombinant human APC was prepared as described (24), and its concentration was determined with S2366.…”

Microparticles in sickle cell anaemia: promise and pitfalls

“…Platelet activation generally involves a robust production of PMP, executing a nearly two‐log increase in coagulation support (Sinauridze et al , ). These PMP display activation markers and are mostly positive for externalized PS, providing additional phospholipid surface area for assembly of both procoagulant enzymatic complexes, including the prothrombinase and intrinsic X'ase complexes (Sims et al , ; Gilbert et al , ) and anticoagulant complexes (Tans et al , ; Somajo et al , ). Specifically identified activators of PMP production include: ADP, thrombin, collagen, lipopolysaccharide (LPS), complement complex C5b‐9, CD40 Ligand and shear stress.…”

Role of P2Y receptor subtypes in platelet-derived microparticle generation

“…are approximately 0.1 micrometers in diameter and have been implicated in being involved in inflammation, coagulation and vascular function (1)(2)(3). These small fragments are generated under conditions of high shear stress and platelet activation (4,5).…”