Production of thrombin as a probe for mixing of phospholipids in membranes on solid supports

Phospholipid-covered solid supports have been used successfully as model membranes in studies on blood coagulation and other research fields. In order to produce such membranes, simple exposure of the support to suspensions of phospholipid vesicles was recently introduced, but questions have remained about the process of vesicle adherence to the surface and the physico-chemical properties of the resulting membranes. Using a new technique, mixing of phospholipids in such membranes was demonstrated. A rotating, hydrophilic, silicon disc was exposed in a two-step procedure to vesicles prepared from mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC). Factor Xa, factor Va and prothrombin were added and the transport-limited production rate of thrombin was measured. For low surface coverage with 40% DOPS/60% DOPC, a much higher conversion rate was found if, prior to addition of coagulation factors, excess DOPC vesicles were added to fill up vacant surface area. It is concluded that DOPS is spread over the entire surface and that confluent bilayers are formed. The presented technique may also be used to measure lateral diffusion constants.