Molecular Structural Studies of Human Factor Viii*

Abstract

Neither normal nor hemophilic factor VIII protein enters a 5% sosium dodecyl sulfate gel; on reduction, however, a single 195 000-molecular-weight peptide is observed. Hemophilic and normal factor VIII contain carbohydrate and appear identical in subunit molecular weight, electrical charge, and major antigenic determinants. Thrombin activation and inactivation of factor VIII does not detectably change the subunit molecular weight. Trypsin causes similar activity changes and obviously cleaves the factor VIII subunit. Human plasmin destroys factor VIII procoagulant activity and degrades the factor VIII subunit to 103 000-, 88 000-, and 17 000-molecular-weight peptides. Both normal and hemophilic factor VIII as well as thrombin-inactivated factor VIII support ristocetin-induced platelet aggregation. Purified factor VIII chromatographed on 4% agarose in 1.0 M sodium chloride shows no dissociation of the procoagulant activity from the void volume protein. Gel chromatography on 4% agarose in 0.25 M calcium chloride results in a procoagulant activity peak removed from the void volume protein; both peaks contain protein which does not enter a 5% SDS gel, but on reduction a 195 000-molecular-weight subunit band is observed for each. Both the void volume protein peak and the procoagulant activity peak from the 0.25 M calcium chloride-agarose gel column support ristocetin-induced platelet aggregation. After removal of calcium, a small amount of procoagulant activity is present only in the void volume peak. These data suggest that both the procoagulant and von Willebrand activities are on the same molecule. Thus our previous conclusion remains the same: human factor VIII is a large glycoprotein composed of identical 195 000-molecular-weight subunits jointed by disulfide bonds and is responsible for both antihemophilic and von Willebrand activities in human plasma.

Stable Suppression of Tumorigenicity by Pin1-Targeted RNA Interference in Prostate Cancer

“…5C). Paraffin-embedded sections of five different tumors were processed and stained for factor VIII, an endothelial specific marker (29). As shown in Fig.…”

Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure.

“…In addition, when purified nonreduced Factor VIII was analyzed by SDSgel electrophoresis, only the top of the gel stained for protein and no other bands were seen; after reduction by fi-mercaptoethanol, a major protein band of 195,000 daltons and two or three very minor bands ranging from 180,000-160,000 daltons were observed. These latter bands, which were frequently observed when purified Factor VIII preparations were reduced and analyzed on overloaded SDS gels, have been identified (27) as early degradation intermediates of the 195,000-dalton subunit when Factor VIII was digested by plasmin. It is very difficult to isolate native Factor VIII without minor amounts of plasmin-degraded Factor VIII since both the native and minimally degraded forms of Factor VIII are similar in size and elute in the void volume from 4% agarose.…”

“…All of these columns gave satisfactory resolution of the protein peak from the Factor VIII procoagulant activity peak when 0. 25 (27) was prepared by incubating 1 ml of Factor VIII (5 absorbance U at 280 nm) with 0.5 CTA (Committee on Thrombolytic Agents) U of purified (28) human plasminogen (22 CTA U/mg) and 5 Ploug U of urokinase at 370C for 24 h. The reaction was monitored by Factor VIII activity assays and by SDS-gel analysis of the proteolytically degraded and p-mercaptoethanol-reduced Factor VIII.…”

Structure-Inhibitory Activity Relationship of Plasmin and Plasma Kallikrein Inhibitors.

“…Although the primary function of PL is to remove intravascularly formed thrombin by the degradation of Fn, PL has many other actions, such as the degradation of adhesive macromolecules, 10-13) the activation of growth factors, 14,15) coagulation factor modification, 16,17) the activation of t-PA 18) and u-PA, 19) and so on. As a result, PL might function in pathologic phenomena, such as inflammation 20) and tumor cells growth and metastasis.…”

“…Plasma kallikrein (PK) (EC 3.4.21.34), which is one component of the contact system, 7) probably mediates the activation of prourokinase and accelerates the PL formation, 8) while PK was reported to activate the contact phase of coagulation. 9) Although the primary function of PL is to remove intravascularly formed thrombin by the degradation of Fn, PL has many other actions, such as the degradation of adhesive macromolecules, [10][11][12][13] the activation of growth factors, 14,15) coagulation factor modification, 16,17) the activation of t-PA 18) and u-PA, 19) and so on. As a result, PL might function in pathologic phenomena, such as inflammation 20) and tumor cells growth and metastasis.…”