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Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

Human factor VIII has been isolated from a high purity factor VIII concentrate by immunoaffmity chromatography and HPLC on Mono Q gel. Two fractions of factor VIII were obtained with a specific activity of w7000 units/mg.The major fraction contained eight peptide chains of 200, 180, 160, 150, 135, 130, 115, and 105 kDa plus one doublet chain of 80 kDa. The minor fraction contained one peptide chain of 90 kDa plus the chain of 80 kDa. Both fractions were activated by thrombin to the same extent. Amino-terminal amino acid sequence analysis was performed on the 180-kDa, 130-kDa, and 90-kDa chains and showed an identical amino-terminal sequence in these chains. Each chain from 200 kDa to 90 kDa was linked to one 80-kDa chain by a metal-ion bridge(s).Studies on factor VIII in plasma and cryoprecipitate, prepared and gel ifitered in the presence of protease inhibitors, showed that one 200-kDa plus one 80-kDa chain were the only or dominating chains in the materials and may represent native factor VIII. The results indicated that all chains from 180 kDa to 90 kDa are fragments of the 200-kDa chain. All of these more or less fragmented chains form active factor VIII complexes with the 80-kDa chain.Factor VIII (antihemophilic factor) is the protein that is deficient or absent in individuals with classic hemophilia, an X-chromosome-linked bleeding disorder. It participates in the intrinsic pathway of blood coagulation as a cofactor in the activation of factor X by factor IXa, in the presence of phospholipid and calcium (1-7). In plasma, factor VIII is noncovalently bound to von Willebrand factor (vWF), a high molecular weight protein involved in primary hemostasis. Due to the low concentration of factor VIII in plasma and to the fact that factor VIII is highly susceptible to degradation by serine proteases, it is only recently that highly purified factor VIII, free of vWF, has been obtained. Bovine factor VIII, as isolated from plasma, is composed of three peptide chains of 93 kDa, 88 kDa, and 85 kDa (8). Factor VIII purified from porcine plasma has been shown to consist of subunits of 160 kDa, 130 kDa, 82 kDa, and 76 kDa (9). Purified human factor VIII has been prepared from commercial factor VIII concentrate and had a specific activity of 2294 units/mg (10). NaDodSO4/PAGE showed several peptide chains having molecular sizes of 188-79 kDa. The majority of the material was related to factor VIII as shown by immunoblot technique with use of monoclonal antibodies against factor VIII (11). A preparation of human factor VIII from cryoprecipitate, in the presence of seine protease inhibitors, has also been reported with the specific activity of 4740 units/mg (12). Unreduced NaDodSO4/PAGE showed predominant bands at 360 kDa, 210 kDa, and 90 kDa and an 80 kDa/79 kDa doublet band.Immunoblotting using monoclonal antibodies suggested that the 360 kDa component was a precursor to the other components. Thus different results are obtained when different starting materials and procedures for purification are used.The human fac...

The procoagulant protein F.VIII:C is noncovalently bound to von Willebrand factor (vWF) to give the factor VIII macromolecular complex. New highly purified preparations of isolated human F.VIII:C, devoid of vWF and about 500,000-fold purified, were administered to hemophilia A and von Willebrand disease (vWD) dogs to determine their hemostatic effectiveness and survival in the circulation. Two preparations of F.VIII:C were used: peak 1, with active components of Mr 185,000-280,000, and peak 2, with a single component of Mr 170,000. In hemophilic dogs, with no plasma F.VIII:C but normal vWF, both preparations immediately elevated plasma F.VIII:C to expected levels, promptly stopped induced and spontaneous hemorrhages, and gave sustained plasma levels of F.VIII:C. The isolated F.VIII:C immediately complexed with endogenous vWF in hemophilic plasma and was eliminated exponentially, with a half-life (t1/2) of about 9 hr. Survival of peak 2 F.VIII:C was longer than that of peak 1 material. In contrast, F.VIII:C complexed to vWF in a therapeutic concentrate administered to hemophilic dogs was eliminated biexponentially with first-phase t1/2 of 3.2 hr and second-phase t1/2 of 9 hr. In vWD dogs with no vWF and reduced F.VIII:C levels, the isolated F.VIII:C produced supernormal levels of F.VIII:C without effect on induced bleeding. It was rapidly eliminated from plasma with a t1/2 of about 1 hr, as was the complexed F.VIII:C in the concentrate. These data indicate that isolated F.VIII:C promptly complexes with vWF and in this form is highly effective in controlling hemophilic hemorrhages with good survival in plasma. Without endogenous vWF with which to complex, the F.VIII:C is promptly eliminated.

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The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.