Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.

Thrombin production by the phospholipid-bound complex of blood clotting factors Xa and Va (prothrombinase) was studied in a tubular flow reactor. The inner wall of a glass capillary was coated with a phospholipid bilayer of 25% phosphatidylserine and 75% phosphatidylcholine. Prothrombinase was assembled on this bilayer by perfusion with a mixture containing an excess of factor Va (2 nM) and a limiting amount of factor Xa (1-100 pM), either in the absence or presence of prothrombin. The rate of assembly of prothrombinase in the presence of prothrombin appeared to be limited by the transfer rate of factor Xa to the phospholipid surface. A good agreement was found between the predicted mass transfer coefficient for factor Xa and the observed pre-steady state rate of thrombin production. The eventually obtained steady state rates of thrombin production were proportional to the prothrombin concentration and independent of the surface density of prothrombinase. The observed rate of thrombin production was in excellent agreement with the predicted mass transfer rate for prothrombin. Transport-limited prothrombin conversion was observed for prothrombinase densities exceeding 1 fmol/cm2, which corresponds to 0.05% occupation of available binding sites. The kinetic parameters of the reaction were determined at low prothrombinase densities (0.02-0.04 fmol/cm2). Even in this situation the Michaelis-Menten equation had to be corrected for substrate depletion near the catalytic surface. We hereto employed an accurate approximation of the mass transfer coefficient. The kinetic parameter kcat was 60 s-1 and the intrinsic Km had a surprisingly low value of 3 nM. Both parameters were not influenced by the wall shear rate.

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipidcoated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min ؊1 ) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (<10 mol%) a linear relationship between the prothrombinase activity and the arithmetical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.