Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma.

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Abstract
The relationship between factor VIII coagulant antigen (VIII:CAg) and factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg were determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4 [sodium dodecyl sulfate]/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had MW 3.2 .times. 105. This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of MW 2.7 .times. 105. Minor bands were also present on autoradiograms of normal plasma corresponding to MW values of 2.5, 1.85 and 1.7 .times. 105 (free VIII:CAg related proteins with MW values of 2.0, 1.35 and 1.2 .times. 105, respectively). None of the VIII:CAg bands was present in plasma samples from 5 patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 .times. 105 and increased the intensity of a band of MW 1.7 .times. 105. Generation of this presumed VIII:CAg fragment of MW .apprxeq. 1.2 .times. 105 coincided with a thrombin-induced increase in factor VIII coagulant activity. The form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from 5 hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.