Adsorption and conversion of prothrombin on a rotating disc.
In most flow systems, the rate of protein transfer from bulk solution to a macroscopic surface is site-dependent. In studies on surface-mediated protein conversion, this hampers the comparison of a proposed expression for the conversion process, such as the Michaelis-Menten equation, which actually measured overall conversion rates. However, the rotating disc is a classical example of a uniformly accessible surface and therefore was used for a quantitative analysis of prothrombin conversion by the phospholipid-bound factor Xa/factor Va complex (prothrombinase complex). A simple design of a rotating disc, adapted for ellipsometric measurement of protein adsorption, is presented. Agreement between experiment and theory was obtained for the influence of rotation velocity on the initial, transport-limited, adsorption rates of lysozyme, prothrombin, and fibrinogen. After coverage of the disc with a 20% phosphatidylserine/80% phosphatidylcholine bilayer and preadsorption of factor Va, addition of excess factor Xa and prothrombin resulted in effective conversion of prothrombin. For high (10 fmol.cm-2) surface coverage of prothrombinase, the rate of conversion equals the transport limited adsorption rate of prothrombin. For low (0.1 to 0.5 fmol.cm-2) surface concentrations of prothrombinase, the conversion rate dropped below the transport limit and the intrinsic kinetic parameters could be estimated at Km = 7.1 +/- 1.2 nM and kcat = 25 +/- 1.0 s-1 (20 degrees C). At these low surface activities of prothrombinase, the effect of the rotation rate (6 to 225 rad.s-1) on prothrombin conversion could be explained by the rotation-rate dependent prothrombin transport. This indicates that the fluid shear rate has no drastic influence on the intrinsic kinetics of prothrombin conversion.
Willems
G M
GM
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
Giesen
P L
PL
Hermens
W T
WT
D000327:Adsorption; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005165:Factor V / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D012399:Rotation; D012996:Solutions; D013917:Thrombin / Q000378:metabolism
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D012996:Solutions; C022475:prothrombinase complex; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D005340:Fibrinogen; D009113:Muramidase; D013917:Thrombin; D015951:Factor Xa