Factor V and Thrombin in the Extrinsic Clotting System ∗

Abstract

Factor V (1), whose synonyms include labile factor (2), accelerator globulin (3), and other terms (4), has evoked an extensive modern literature (5) in which serious discrepancies of both fact and interpretation invite further investigation. Particularly questionable are ideas of a conversion of factor V to an “activated” material (Va?) and its possible relation to thrombin. The present contribution introduces a restudy of these problems and will be limited to 1-stage and 2-stage assay systems that depend on tissue thromboplastin. This agent is also called factor III and is considered as an extrinsic activator system (6).

Materials and Methods. Buffers are either imidazole-buffered 0.85% NaCl (IBS) or 0.05 M Tris-0.05 M NaCl, both types adjusted to pH 7.3 ± 0.05. V-deficient substrate (7) is aged normal human oxalated plasma, stored at −20 ° and made factor V-deficient by repeated thawings and refreezings over a period of weeks. Tissue thromboplastin (Tpln) is commercial Simplastin (Warner-Chilcott). This contains ionized calcium (CA²⁺) adequate for 1-stage tests, but needing supplementation for the 2-stage tests. Factor V (V) is purified from BaSO₄-adsorbed bovine oxalated plasma by a recent method (8), which is now improved by use of an isoelectric precipitation (6.3>pH>5.2) between the prechromatographic 18-39% saturation with ammonium sulfate (SAS) and the Sephadex G-200 gel filtration. Thrombin-treated factor V (V_T) is obtained by treating 9 vol of the above SAS fraction with 1 vol of purified thrombin (Tp, see below) for 30 min at 37 ° in Tris-NaCl buffer. After subsequent centrifuging at 1500g for 10 min at 0-4 °, the filtered supernatant is subjected to the usual Sephadex G-200 chromatography. The new elution pattern and fraction test activities are restudied. Some lots of the tubes containing active treated factor V are pooled and lyophilized.