Adsorption and conversion of prothrombin on a rotating disc
GM Willems, PL Giesen and WT Hermens
Cardiovascular Research Institute Maastricht, University of Limburg, The
Netherlands.
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.
Volume 82,
Issue 2,
pp. 497-504,
07/15/1993
Copyright © 1993 by The American Society of Hematology
