The effect of thrombin on human factor VIII. Cleavage of the factor VIII procoagulant protein during activation.
Human VIII: C has been partially purified by immunoabsorbent 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 A. The molecular weight 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 x 10(-5) to 10(-2) U/ml highly purified human alpha-thrombin. Although 2 mM DEP 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 hr with dilute alpha-thrombin, 2 x 10(-5) to 2 x 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 A and, taken together with a preliminary estimate of sedimentation properties (5S), suggests a molecular weight 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 approximately 0.3. THese data suggest that alpha-thrombin activates and inactivates human VIII:C by proteolytic modification of VIII:C structure and that thrombin-activated VIII:C is smaller than the unactivated procoagulant.
D001777:Blood Coagulation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D005169:Factor VIII / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D046911:Macromolecular Substances; D011498:Protein Precursors; D005169:Factor VIII; D013917:Thrombin