EFFECTIVE ELECTROSTATIC CHARGE OF COAGULATION-FACTOR-X IN SOLUTION AND ON PHOSPHOLIPID-MEMBRANES - IMPLICATIONS FOR ACTIVATION MECHANISMS AND STRUCTURE-FUNCTION-RELATIONSHIPS OF THE GLA DOMAIN

Electrostatic interactions during activation of coagulation factor X w ere analysed by comparing effects of ionic strength on reaction rates with predictions of classical electrostatic theory. Geometrical correl ations were investigated using alpha-shape-based computations on the c rystal structure of Ca-fragment 1 of prothrombin. The ionic strength o f the reaction environment was controlled with different univalent sal ts including NaCl, KCl, CsCl, LiCl, NaI, NaBr and KI. Reactions were a ssembled in three different environments: aqueous phase, cell membrane s and synthetic TF/PS/PC (tissue factor relipidated in 30% phosphatidy lserine, 70 % phosphatidylcholine) vesicles. Reaction rates were measu red at pH 7.2, 4 mM CaCl2 and 33 degrees C, using chromogenic substrat e to follow factor Xa generation. Rates decreased with increasing conc entration of univalent salt, and the magnitude of the decrease was ind ependent of salt type. On the basis of electrostatic relationships on PS/PC vesicles, the effective charge on factor X was +1.5, and the PS/ factor X stoichiometry was 2.28. Structural analysis of the gamma-carb oxy-glutamic acid (Gla) domain revealed three surface pockets, forming potential sites for Ca2+ binding, with distinct spatial orientations. Interpreted together, the results of the geometric analysis and the m easured effective charges suggest an efficient electrostatic mechanism for capture and retention of substrates by procoagulant membranes. No n-specific and delocalized interaction between the membrane and each o ne of the charged facets of the Gla domain can increase the probabilit y of substrate binding, while allowing rotational and translational mo bility of substrate for specific interaction with the enzyme.