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Heparin-stimulated inhibition of factor IXa generation and factor IXa
neutralization in plasma
J Pieters, T Lindhout and G Willems
Department of Biochemistry, University of Limburg, Maastricht, The
Netherlands.
Generation and inhibition of activated factor IXa was studied in factor
XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L
phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine).
Interference of other (activated) clotting factors with the factor IXa
activity measurements could be avoided by using a highly specific and
sensitive bioassay. Factor IXa generation curves were analyzed according to
a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed
factor IXa formation and pseudo first order kinetics of inhibition of
factor XIa and factor IXa. In the absence of heparin, factor IXa activity
in plasma reached final levels that were found to increase with increasing
amounts of factor XIa used to activate the plasma. When the model was
fitted to this set of factor IXa generation curves, the analysis yielded a
rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a
kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of
factor IXa activity was observed (the estimated rate constant of inhibition
of factor IXa was 0). Thus, in the absence of heparin, the final level of
factor IXa in plasma is only dependent on the initial factor XIa
concentration. While neutralization of in situ generated factor IXa in
normal plasma was negligible, unfractionated heparin dramatically enhanced
the rate of inactivation of factor IXa (apparent second order rate constant
of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic
pentasaccharide heparin, the smallest heparin chain capable of binding
antithrombin III, stimulated the inhibition of in situ generated factor
IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per
microgram pentasaccharide/mL). We found that free calcium ions were
absolutely required to observe an unfractionated heparin and
pentasaccharide-stimulated neutralization of factor IXa activity. Factor
XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not
affected by unfractionated heparin or pentasaccharide in the range of
heparin concentrations studied.
Volume 76,
Issue 3,
pp. 549-554,
08/01/1990
Copyright © 1990 by The American Society of Hematology
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