Partial purification and characterization of contact activation cofactor.

Abstract

A B S T R A C T The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56°C; gradually loses activity at 800C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by Al (OH) s; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 560C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XI. generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated.

Purification of High Molecular Weight Kininogen and the Role of This Agent in Blood Coagulation

“…Whether activated PTA remains attached to a complex is not clear. As far as HMWkininogen is concerned, this is consistent with a previous report (41) that the amount of activated PTA was proportional to the amount of contact activation co-factor (HMW-kininogen) present. Griffin and Cochrane (40) (Table II) and in a purified system (Table III).…”

Association of factor XI and high molecular weight kininogen in human plasma.

“…We have recently demonstrated that prekallikrein and HMW kininogen normally circu-late as a complex (15) and proposed that such binding might facilitate the trimolecular interaction that takes place upon the surface. Because the activation of Factor XI, the other Hageman factor substrate, is also augmented by HMW kininogen (10)(11)(12)(13)16), we have examined the possibility that Factor XI forms a complex with HMW kininogen (17). In this paper we show that Factor XI is bound to HMW kininogen in whole plasma and that such binding is also demonstrable utilizing highly purified components.…”

Prekallikrein deficiency in a kindred with kininogen deficiency and Fitzgerald trait clotting defect. Evidence that high molecular weight kininogen and prekallikrein exist as a complex in normal human plasma.

“…Kininogens were isolated from normal human plasma at room temperature by modifications of published methods (5,(7)(8)(9)(10). Blood was drawn into silicone-coated vessels containing 0.02 vol of 0.5 M citrate buffer, pH 5.2, hexadimethrine bromide to give a final concentration of50 ,ug/ml and EDTA to give a final concentration of 0.003 M. The plasma was separated during centrifugation at room temperature and made platelet-deficient by recentrifugation.…”