Plasma Coagulation on Microparticles Is Modulated by Water Activity. Relevance to Inflammation-Mediated Disruption of the Extracellular Matrix.

The microenvironment of extravascular spaces influences the reactivities of extravasated plasma proteins. During inflammation, the structural and physicochemical characteristics of peri- and extra-vascular spaces can change widely depending on the severity of extracellular matrix disruption. Plasma extravasation after limited vascular injury results in transient changes in water activity (a_w) and fibrin deposition.

This work investigates whether changes in a_w influence either pro- or anticoagulant pathways assembled on procoagulant microparticles. Based on conclusions from in vivo models indicating that factor Xa (fXa) concentration is the limiting factor in prothrombinase assembly we modeled the extravascular coagulation process in vitro, using plasma as the matrix; controlling a_w as the independent variable; and measuring the fXa generation rate as the dependent variable. Initial rates of fXa generation were measured in human plasma (1/100 final dilution) containing procoagulant particles and 200 nM factor X. Water activity levels decreased from 0 to 0.8 atm relative to isosmolar plasma (~7 atm) with polyethylene glycol 8000 (~26 Å radius), while all reactant concentrations remained constant. Procoagulant particles were either recombinant tissue factor (TF) in PC/PS vesicles (at a mol ratio of 1/2666) or microvesicles collected from the monocytoid cell line THP-1 after 24-hour stimulation with endotoxin (1ug/ml). As the reaction environment’s a_w progressively decreased, fXa generation rate first increased, and then decreased. Rate/a_w profiles followed nearly symmetrical peaks with a maximum at ~0.2 atm and minimum at ~0.7 atm. Similar profiles were obtained with cell-derived procoagulant microvesicles. At each a_w level, the fXa generation rate increased exponentially with TF concentration (25–100 pM). Activation and inhibition indices of ~1.5 and ~2.1, calculated as the ratio between the maximal and baseline and the maximal and minimal rates did not change significantly with TF concentration. Rates increased with added factor VIII (~2 units/ml) indicating fIX activation and a contribution of the intrinsic pathway protease to fXa generation. Furthermore, reaction rates changed with hirudin suggesting that thrombin-mediated regulatory loops also affect the rate profiles. Results in this simple model of extravascular coagulation indicate that a_w modulates the rate of coagulation reactions stimulating or inhibiting them depending on whether the reaction environment’s a_w is high or low. This conclusion in turn predicts that immediately after limited plasma extravasation coagulation rates accelerate with the decrease in a_w as it tend to equilibrate with a_w in the extravascular spaces. Continued water adsorption by an intact extracellular matrix would equilibrate a_w and decrease coagulation rates. However, in a structurally damaged matrix with compromised water sorption ability, rates may remain longer at the higher level.