Reactions of activated factor X-phosphatide mixtures in vitro and in vivo.

The composition, surface properties, and capacity of lipid particles to bind activated factor X can be correlated both with the influence of various phosphatide combinations on activated factor X activity in vitro and on the intensity and duration of the thrombogenic stimulus as mea- sured by a standard bioassay for thrombus formation. The measurable activity of activated factor X in vitro increased as a linear function of lipid concentration from 0 to 40 pmoles/liter. The effectiveness of the lipids examined was in the following decreasing order : phosphatidyl serine-phosphatidylcholi ne mixture, "cephalin", and phosphatidyl serine alone. An in- crease in the duration of hypercoagulability with increasing lipid concentration was also observed, but, with regard to the three lipid fractions tested, the in vivo system appeared to be more discriminatory than in vitro assays. Lipid mixtures containing phosphatidyl serine with either phosphatidyl- choline, phosphatidyl ethanolamine, or cetyltrimethylammo- nium bromide markedly enhanced the in vitro activity of activated factor X. Phosphatidic acid-phosphatidylcholine mixtures had a similar but smaller effect, and phosphatidyl ethanolamine-phosphatidylcholine mixtures were inert. The duration of the hypercoagulability was similarly related to the composition of the phosphatide infused. In mixtures containing phosphatidyl serine, the surface charge density of the lipid particles and the binding of the activated factor X activity to lipid showed some correlations with the in vitro activated factor X assay and with the intensity and duration of the thrombogenic stimulus. These data suggest that the catalytic effect of phosphatides on prothrombin activation and their role in the retardation of in vivo compensatory mechanisms directed against circulating activated factor X, are dependent on the affinity of activated factor X for the lipid surface. A STANDARDIZED TECHNIQUE for the study of hyper- coagulability and thrombosis in the large veins of ex- perimental animals has been developed (1). The thrombi which are formed are histologically similar to human thrombi, and the technique, when applied to human subjects, will produce thrombi in the large veins (2). The method has several important features. First, thrombosis is dependent upon the injection into the cir- culation of an activated species of a clotting factor, thus being in accord with one generally accepted definition of hypercoagulability (3). Second, the application of stasis as an integral part of the technique, permits the recognition of thrombi with doses of activated clotting factors that in freely flowing blood are not thrombogenic. Third, with minor modifications in technique, different plasma and serum fractions, including highly purified individual clotting factors and accessory moieties, can be evaluated for their effect on thrombogenesis. Finally, it is possible to compare the role of the various test frac- tions in vitro with their function in intravascular coagula- tion. We have recently been interested in the role of factor X (Stuart-Prower factor) in thrombosis because both the nonactivated and activated forms can be obtained relatively pure, because activation of factor X repre- sents a focal point in the intrinsic and extrinsic clotting systems, and because the activated species is directly