Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin.
Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.
Giesen
P L
PL
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
Willems
G M
GM
Hermens
W T
WT
D000327:Adsorption; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers; D010743:Phospholipids; D011516:Prothrombin / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism*
D008051:Lipid Bilayers; D010743:Phospholipids; D011516:Prothrombin; D013925:Thromboplastin; D013917:Thrombin