The significance of erythrocyte antigen site density

169

The human factor VIII procoagulant protein (VIUC) was purified from the VIUC-factor VIII-related antigen (VIURAg) complex in commercial factor VIII concentrate by immunoadsorbent chromatography with a monoclonal anti-VIURAg antibody bound to Sepharose. In this complex, VHIC is noncovalently bound to factor VIJII-related antigen. The VIHC eluted from the complex was free of VIURAg as determined by immunoassay and had a specific activity of 2,294 VIUC units/mg of protein, representing a 164,000-fold purification from plasma. Electrophoresis of this VIUC preparation in reduced sodium dodecyl sulfate containing 5% polyacrylamide slab gels and subsequent staining with Coomassie blue showed the VUIC to be a strongly staining doublet ofMrs 79

Section: Resultsmentioning

confidence: 53%

Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.

Fulcher¹,

Zimmerman²

1982

169

“…ated by using sucrose density gradients and gel filtration columns show that the elution profiles of factor VIII and factor VIII antigen do not always coincide with one another (19). The explanation for this separation of factor VIII from factor VIII antigen is not known, but it may be due to heterogeneity ofeither the antibody or the antigen or both; the latter is demonstrated clearly by the multiple elution peaks from QAE cellulose columns.…”

Section: Discussionmentioning

confidence: 95%

“…The ammonium sulfate precipitates were thawed at 40C and resuspended in 10-15 ml of 50 mM imidazole HCl, pH 7.0/150 mM NaCl (buffer A). This material was dialyzed against the same buffer for [18][19][20][21][22][23][24] hr (three changes). The protein sample was made 250 mM in CaCl2 by adding 1/9th vol of a 2.5 M solution prior to chromatography on Sepharose CL-4B (2 X 90 cm) equilibrated in buffer A containing 250 mM CaCl2.…”

mentioning

confidence: 99%

Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain.

Fay¹,

Chavin²,

Schroeder³

et al. 1982

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.-1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VI.The most common inherited bleeding disorders are associated with deficiencies or defects in factor VIII or von Willebrand protein. Much of the physiological role and structural features of the von Willebrand protein have been characterized (1); much less is known about factor VIII. Two major factors have hindered the study offactor VIII-the low concentration of factor VIII in plasma, and the difficulty of obtaining a purified, homogeneous preparation. Recent reports have described a high degree ofpurification and some characterization of bovine factor VIII (2), porcine factor VIII (3), and human factor VIII (4). However, the final preparations contained several polypeptide chains when analyzed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis under reducing conditions. We report here a procedure for purification of human factor VIII from plasma which utilizes calcium dissociation and differential size and charge chromatography. The (8). The source ofvon Willebrand protein used for the enzyme-linked immunosorbant assay was the void volume fraction eluted from Sepharose CL-4B obtained during the purification of factor VIII. Hemophiliac plasma was obtained from a patient with severe factor VIII deficiency as shown by a glass clotting time >60 min (9). The factor VIII inhibitor plasma used in this study had a titer of 6,400 Bethesda units. BioGel A-15m, Sepharose CL-4B, and QAE cellulose (fine mesh) were purchased from Bio-Rad Laboratories, Pharmacia, and Sigma, respectively. The protein silver staining kit was purchased from Bio-Rad.Assays. Protein concentration was determined by absorbance at 280 nm corrected for light scattering; an E" corrected of 10.0 for factor VIII was assumed. Factor VIII procoagulant activity was quantitated by a one-stage clotting assay (10). In the presence of0.25 M CaCl2, factor VIII procoagulant activity was measured by using a modified one-stage assay (11). Factor VIII antigen was measured according to the procedure of Reisner et al. (12); 1 unit of factor VIII antigen r...

“…As more studies are reported dealing with the structure of factor VIII, comparisons with structural data on factor V further reinforce the similarities between these two proteins. The products of thrombin activation, factor Va and factor VIIIa, are similar in size (5,11,12), and (from consideration of Stokes radii, mass, and sedimentation coefficient) both cofactors are apparently highly asymmetrical (4,5). In our studies of porcine factor VIII and bovine factor V, we isolated proteolytic fragments of these molecules and subjected the peptides to NH2-terminal amino acid sequence analysis.…”

mentioning

confidence: 99%

Internal duplication and sequence homology in factors V and VIII.

Fass¹,

Hewick²,

Knutson³

et al. 1985

(Fig. 1) was isolated as a single 330-kDa chain by procedures developed in these laboratories (14). ucts of 94 and 74 kDa remain linked together through divalent cation binding (9,10). The bovine factor V thrombingenerated peptides were isolated by ion-exchange procedures carried out in 0.005 M EDTA (15).The porcine factor VIII was isolated as three chains from an immobilized anti-porcine factor VIII mouse monoclonal antibody column (11). The constituent chains consisted of 166-, 130-and 76-kDa polypeptides as determined by NaDodSO4/polyacrylamide gel electrophoresis (Fig. 1). When the anti-factor VIII agarose gel was loaded with porcine factor VIII, the 166-and 130-kDa chains were released by treatment of the immobilized immune complex with EDTA under conditions previously described (11). The 76-kDa chain was retained and was obtained by subsequent elution with 50% ethylene glycol (11). The treatment of porcine factor VIII with thrombin results in the loss of the 166-, 130-, and 76-kDa peptides with the appearance of new chains at 82 and 69 kDa (11,16). With longer incubation in the presence of thrombin, there appear, in the digestion mixture, new polypeptide chains at 44 and 35 kDa concurrent with the loss of the 82-kDa chain (16). The peptides were isolated from radioactive bands cut from NaDodSO4/polyacrylamide gel slab electrophoresis gels that were used to analyze mixtures containing 99% unlabeled and 1% 125I-iodinated samples (17) of either porcine factor VIII or ttrombin-activated porcine factor VIII. The acrylamide slices containing 1251-iodinated protein were subjected to electroelution and concentration (18), thereby providing samples suitable (0.1-1 nmol) for gas-phase amino acid sequence analysis.Sufficient quantities of the factor V thrombin-generated 1688The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.