Relationship of vesicle curvature and PS content to inhibition of the prothrombinase complex by lactadherin or annexin V.
Sonicated vesicles (▴ and ▵), extruded vesicles (● and ○), and LMVs (▪ and ■) contained 4% PS (top row) or 15% PS (bottom row). Effective concentrations of the vesicles for supporting the prothrombinase complex were identified in phospholipid titration experiments (A,D). Subsaturating phospholipid concentrations were selected for inhibition experiments (open symbols; A,D). The indicated phospholipid composition/concentration was added to factor Xa, factor Va, prothrombin, and either lactadherin or annexin V. After 5 minutes, the reaction was quenched with EDTA, and thrombin was measured with chromogenic substrate S-2238 in a kinetic microplate reader. Lactadherin was an effective inhibitor of the prothrombinase complex regardless of membrane curvature or PS content (B,E). In panel B, × indicates Ki = 1 nM. In contrast, inhibition by annexin V was inversely related to curvature and directly related to PS content (C,F). The Ca++concentration was 1.5 mM for sonicated vesicles of 4% PS (▴; A-C) and 5 mM for all other conditions. Results displayed are from a single experiment representative of either 2 or 3 experiments for all conditions.
