THE EFFECT OF THROMBIN ON HUMAN FACTOR-VIII - CLEAVAGE OF THE FACTOR-VIII PROCOAGULANT PROTEIN DURING ACTIVATION

Abstract

Human VIII:C was partially purified by immunoadsorbent chromatography and agarose gel filtration in the presence of 2 mM DFP. The ratio of VIII:C to VIIIR:Ag in these preparations was greater than 1000:1 and the VIII:C procoagulant and immunologic (VIII:CAg) activities eluted together from Sephadex G-200 with a Kav of 0.05, a value consistent with a Stokes radius of 88 .ANG.. The MW estimated from this measurement and sucrose density-gradient centrifugation studies (8.2S) is 285,000. VIII:C activation was detected when the purified procoagulant was incubated with 2 .times. 10-5 to 10-2 U/ml highly purified human .alpha.-thrombin. Although 2 mM DFP inhibits VIII:C activation by .alpha.-thrombin, DFP added after activation did not prevent subsequent loss of activity. When VIII:C was incubated for 4 h with dilute .alpha.-thrombin, 2 .times. 10-5 to 2 .times. 10-3 U/ml, the activated procoagulant (VIII:C/VIII:CAg > 1) eluted from Sephadex G-200 with a Kav of about 0.2. This gel filtration pattern corresponds to a protein with a Stokes radius of 60 .ANG. and taken together with a preliminary estimate of sedimentation properties (5S) suggests a MW of about 116,000. Higher concentration of thrombin, 10-3 to 10-1 U/ml, inactivated VIII:C in these experiments and the nonfunctional protein was identified by VIII:CAg immunoassay. The inactivated VIII:CAg eluted from Sephadex G-200 in a broad peak of Kav .apprx. 0.3. .alpha.-Thrombin apparently activates and inactivates human VIII:C by proteolytic modification of VIII:C structure and thrombin-activated VIII:C is smaller than the unactivated procoagulant.