Human factor VIII from heparinized plasma

Abstract

Human factor VIII was purified from heparinized blood by cryoprecipitation, poly(ethyleneglyco1) precipitation, Affi-Gel blue, aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. A purification of 280000-fold over plasma with a specific activity over 5300 units/mg was achieved. Analyses of factor VlII using HPLC indicated a molecular mass of 280-340 kDa. Variation in the native mass may reflect heterogeneity of the protein due to associated lipid since structural analysis confirmed that factor VIII contained variable amounts of free fatty acids and diglycerides and triglycerides, but no phospholipids. Additional characterization by denaturing polyacrylamide gel electrophoresis under reducing conditions, followed by silver staining, showed a major single-chain polypeptide of factor VIII with a mass of approximately 260 kDa. To determine whether proteolyzed forms of factor VIII were present during fractionation, we analysed earlier steps in purification. This revealed additional species of factor VIII eluting faster than the single-chain form during chromatography on polyelectrolyte E5. Gel electrophoresis showed that these species of factor VIII consisted of multiple polypeptide chains, and partial peptide mapping using Staphylococcus aureus V8 protease indicated that they were structurally related. Monoclonal and hemophilic antibodies were used in immunoadsorption experiments to demonstrate that the purified factor VIII was composed predominantly of the 260-kDa factor VIII chain.Blood coagulation depends upon the sequential activation of a large number of plasma proteins, culminating in the formation of an insoluble net of fibrin which serves to stabilize a platelet plug at the site of blood vessel injury.One of the key steps in the coagulation process is the activation of factor X to X,. In the intrinsic pathway this reaction is catalyzed by factor IX, in concert with factor VIII, calcium ions and phospholipids [I -51. A number of lines of evidence support the view that factor VIII participates as a regulatory protein or cofactor in the activation of factor X [6, 71, and that proteolytic activation of factor VIII by factor X,, factor IX, or thrombin may be a necessary prerequisite for full expression of procoagulant activity [4, 8 -

ENDOTHELIAL CELL SURFACE ACTIN SERVES AS A BINDING SITE FOR PLASMINOGEN, TISSUE PLASMINOGEN ACTIVATOR AND LIPOPROTEIN(a)

“…Proteins were radiolabelled with 125 Iodine by the Iodobead procedure (Pierce Chemicals, Rockford, Il.) described elsewhere (Ganz et al, 1988). Protein concentration was determined by Bradford's method (1976).…”

Post-translational Modifications Required for Coagulation Factor Secretion and Function

“…In this case, secretion of heavy chains that extend to residue 1648 and secretion of light chains that extend to 1313 can be detected. In addition, some single chain factor VIII is detected in conditioned medium from transfected mammalian cells and in heparin-treated human plasma (15,138). However, all analyses to date indicate that these partially processed products of factor VIII have identical activity to fully processed factor VIII.…”

Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

“…Factor VIII is processed within the cell to yield a heterodimer comprised primarily of a heavy chain of 200 kDa containing the A1, A2, and B domains and an 80-kDa light chain containing the A3, C1, and C2 domains (17). Both the single-chain polypeptide and the heterodimer circulate in plasma as inactive precursors (18). Activation of FVIII in plasma initiates by thrombin cleavage between the A2 and B domains, which releases the B domain and results in a heavy chain consisting of the A1 and A2 domains (19).…”