Abstract
Due to its position at the juncture of the intrinsic and extrinsic pathways, activation of factor X - Xa assumes an important role in regulation of coagulation. Factor X activators other than those of the intrinsic (factors IX and VIII) and extrinsic (factor VII and thromboplastin) pathways have been described. Among these are tumor extracts (Gordon, et al., 1975), mucus extracts (Pineo et al., 1973), a component of Russells viper venom (DiScipio et al., 1977), a procoagulant in ascites fluid (Phillips and Rodgers, 1979), and a procoagulant in amniotic fluid (Weiner, et al., 1949; Rendelstein, et al., 1951; Courtney and Allington, 1972; Phillips and Davidson, 1972; Van Arkel, et al., 1973; Yaffe et al., 1977). The distinguishing features of the latter factors is the lack of requirement for components of the intrinsic or extrinsic pathway for factor Xa generation. All do require Ca('++), while the role of PL is variable.Previous attempts at definition of the amniotic fluid factor (AFF) have used raw or filtered amniotic fluid (AF). Assays have been performed using various plasmas deficient in coagulation factors. The results have indicated that AFF requires factors II, V, X, and Ca('++), but not factors VII, VIII, IX, XI, and XII for its procoagulant activity.Our investigations into AFF activity were initially concerned with confirmation of previous results, particularly the reported lack of requirement for factor VII. We have shown that factors V and X are required, and have confirmed the lack of requirement for factors XI, IX, and VII. We have demonstrated the latter by use of congenitally VII deficient plasma, and by use of materials depleted in factor VII by either DFP treatment or by immunoadsorption with anti factor VII.AFF has been partially purified by gel filtration on Sepharose 4B. The activity is found to elute in the void volume of the column. Purification is between 35 - 70 fold over the starting material. AFF procoagulant activity is not adsorbed onto BaSO(,4), and attempts at further purification using ion exchange or other adsorption - elution techniques have been unsuccessful. The activity fails to penetrate into 5% gels upon disc gel electrophoresis.Using a two stage coagulation assay system with partially purified factors, we have demonstrated direct AFF activation of factor X. Only Ca('++) is required for initiation of the reaction. This has been verified using a more direct chromogenic assay system (chromogenic factor X substrate S-2222) and highly purified human factor X. Thus, no additional coagulation factors are required for AFF factor X activation. Using the chromogenic assay system, the Km of AFF for factor X has been determined to be .37 ('(+OR-)) .06 uM. Additionally AFF was shown to be DFP and PMSF sensitive, and thus may possess a catalytic serine site.Although about an order of magnitude less sensitive than a comparable human brain thromboplastin (hbTPN) preparation, AFF was shown to be rapidly inactivated by treatment with phospholipase C (PL-C, E.C. No. 3.1.4.3.). This demonstrates an AFF requirement for phospholipid (PL), although PL added in excess has been shown to inhibit AFF activity. Zn('++), (a required cofactor of PL-C), was shown to inhibit AFF but not hbTPN activity.Very low factor VII activity was demonstrable in ultracentrifuged AF supernatant. Treatment of AFF with PL-C, followed by assay on factor VII deficient plasma with TPN, showed the release of a component from the AFF which could shorten the prothrombin time of the factor VII deficient plasma. By definition, this factor must be identified with factor VII, and AFF is thus an active TPN: factor VII complex. It appears that this is the first reported instance of such a complex in a biological fluid.