DETECTION OF FACTOR X ACTIVATION IN HUMANS

Abstract

The activation of factor X by factor VII/VIIa-tissue factor or factor IXa plays a pivotal role in the hemostatic mechanism. This reaction results in the liberation of a peptide from the zymogen for which we have developed a sensitive and specific radioimmunoassay (RIA), The native peptide was purified from activated human factor X by hydroxylapatite chromatography and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration experiments demonstrated that the peptide was not physically associated with the enzyme. A 15 amino acid peptide with the COOH-terminal sequence of the activation fragment was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used to construct a double antibody RIA and was able to measure as little as 0.02 nM of this component. The antibody reactivity toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However because other plasma constituents contributed to a nonspecific basal signal in the RIA, we developed an extraction procedure for the native peptide utilizing perchloric acid. Plasma peptide levels in normal individuals were ∼0.1 nM, and elevations up to 0.8 nM were observed in patients with evidence of disseminated intravascular coagulation. Individuals chronically anticoagulated with coumarin derivatives had plasma levels of this peptide suppressed to ∼0.02 nM. The validity of our measurements of factor X activation in vivo is supported by the fact that the immunoreactive signal migrates on reverse-phase HPLC in a manner identical to that of the native activation peptide and can be quantitatively recovered. This assay should be useful for studying the pathophysiology of thrombotic as well as bleeding disorders in humans.