Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies

Abstract

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor

“…The primary structure of f k t o r VIII contains 2332 amino acids and exhibits a triplicated region of 330 amino acids (A domains), a unique region of 983 amino acids (B domain), and a carboxy-terminal duplicated region of 150 amino acids (C domain), that are arranged in the order AlFactor VIII is susceptible to proteolysis by thrombin, plasmin and other serine proteases [3, 41. Factor VIII purified from plasma fractions of intermediate purity, consists of variable active forms with molecular masses ranging over 290-170 kDa, though a single chain precursor of 330 kDa has been reported [5]. Continuous proteolysis of the 210-kDa proteins yields a series of polypeptides of 210-90 kDa [6, 71.…”

Human factor VIII from heparinized plasma

“…The other 30% of the mass of purified F.VII1 probably represents denatured or inactivated protein, which remains immunologically reactive, but does not contribute to procoagulant activity. By comparison, the specific activity of F.VII1 purified from commercial factor VIII cryoprecipitates and concentrates by other groups has been reported as 2294 U/mg [14,40], 4986 U/mg [15], 4500 U/mg [17], 6700 U/ mg [I91 and 4740 U/mg [20]. The degree of endogenous thrombin activation, if any, in these various F.VIII preparations is not known.…”

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

“…O concentrado de fator VIII (crioprecipitado) desprovido de plasma e tratado com Tris-HCl 0,02M pH 6.7 acrescido de heparina 3UI/ml e Al(OH) 3 foi aplicado sobre o gel. 22,24 …”

“…A partir daí iniciaram-se inúmeras pesquisas com o objetivo de introduzir novos produtos na terapêutica da hemofilia. [3][4][5][6][7][8][9][10][11][12] As novas técnicas de engenharia celular e biotecnologia avançaram muito nas últimas décadas e entre elas a produção de anticorpos monoclonais utilizados tanto para o aprimoramento diagnóstico em rotinas laboratoriais como na produção de medicamentos e purificação de proteínas utilizadas na terapêutica. [13][14][15][16][17] Os concentrados de FVIII purificados através de anticorpos monoclonais (denominados produtos ultrapuros) colocados no mercado no início dos anos 90 vieram solucionar complicações desastrosas no tratamento da hemofilia, pois se trata de terapêutica eficaz e segura apresentando alta especificidade, segurança viral e redução de efeitos colaterais.…”

“…A partir daí iniciaram-se inúmeras pesquisas com o objetivo de introduzir novos produtos na terapêutica da hemofilia. [3][4][5][6][7][8][9][10][11][12] …”