Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds.
Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.
D001550:Benzamidines; D002122:Calcium Chloride; D002850:Chromatography, Gel; D005169:Factor VIII / Q000032:analysis* / Q000302:isolation & purification; D006493:Heparin; D006629:Hirudins; D006801:Humans; D008968:Molecular Conformation; D008970:Molecular Weight; D012965:Sodium Chloride; D013329:Structure-Activity Relationship
D001550:Benzamidines; D006629:Hirudins; D012965:Sodium Chloride; D005169:Factor VIII; D006493:Heparin; D002122:Calcium Chloride