Molecular Weights of Factor VIII (AHF) and Factor IX (PTC) by Electron Irradiation

1962 ◽  
Vol 08 (02) ◽  
pp. 270-275 ◽  
Author(s):  
David L Aronson ◽  
John W Preiss ◽  
Michael W Mosesson
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Electron Irradiation ◽  
Factor Ix ◽  
Molecular Weights

SummaryThe molecular weights of AHF (factor VIII) and of PTC (factor IX) have been estimated by their sensitivity to inactivation by 7 kilovolt electrons. The molecular weight of AHF was found to be 180 000 by this method and that of PTC was found to be 110 000.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94 ◽  
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Abstract Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

scholarly journals Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1114-1117 ◽  
Author(s):  
MJ Weinstein ◽  
CA Fulcher ◽  
LE Chute ◽  
TS Zimmerman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Discontinuous System ◽  
Major Band ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
Electrophoretic Techniques

Abstract We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4–7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4–4% polyacrylamide/0.5% agarose gels (NaDodSO4–4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4–4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4–4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.

Conditions Affecting the Molecular Weight of Factor VIII.

1979 ◽  
Author(s):  
G. Rock ◽  
E. Tackaberry ◽  
D. Palmer
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Calcium Concentration ◽  
Low Molecular Weight ◽  
Biochemical Purification ◽  
Molecular Weights ◽  
Will Self ◽  
And Storage ◽  
Molecular Weight Form

By purifying Factor VIII while maintaining physiological concentrations of calcium we have recently demonstrated that about 50% of the procoagulant activity is in a very low molecular weight (VLHW) form not associated with the carrier (VIII: RAG). The remainder is carrier associated and elutes at Vo as a high molecular weight (HMW) compound upon Sepharose 6B chromatography. Reduction of the calcium concentration by increasing the amount of citrate added to heparin results in decreasing the ratio of VLMW:HMW from 1:1 in pure heparin to 1:5 in pure citrate. If citrate is replaced with the more strongly chelating EDTA no VLMW is detectable in the plasma. It has also been found that most of the biochemical purification techniques which have been previously used to prepare Factor VIII for study actually result in the aggregation of this VLMW with the carrier to produce the high molecular weight form. This includes: cryoprecipitation, precipitation by polyethylene glycol and storage -80°C. As well, the VLMW material will self-associate upon freezing to produce an aggregate with a molecular weight of 106. However, this material does not cross-react with rabbit antibody directed against VIII: RAG. The data indicate that many of the previously reported biochemical characteristics, including molecular weights, actually describe species which are artifacts of the isolation process rather than those of the physiologically occuring Factor VIII.

Determination Of Molecular Size Of VIII:C In Whole Plasma By Electron Irradiation

1981 ◽  
Author(s):  
J Harmon ◽  
G A Jamieson ◽  
G Rock
Keyword(s):  
Molecular Weight ◽  
Electron Irradiation ◽  
Gel Filtration ◽  
Molecular Size ◽  
Target Size ◽  
Platelet Poor Plasma ◽  
Molecular Weights ◽  
Normal Plasma ◽  
Biological Mixtures

Loss of activity during electron irradiation provides a means of determining the molecular size of specific molecules in complex biological mixtures. This technique has established a molecular weight of 200,000 for Factor VIII:C in concentrates prepared from citrated plasma in which calcium is sequestered by chelation (Aronson, et al. Thromb. Diath. Haemorrh. 8:270, 1962): this value is similar to that obtained by more conventional techniques. However recent data suggest that in heparinized plasma, where physiological levels of calcium are maintained, about half of the VIII:C activity has a molecular weight of 50,000 as determined by gel filtration and ultracentrifugation (Rock, et al. Thromb. Res. 13:85, 1978). When heparinized plasma was subjected to electron irradiation in the frozen state there was a perceptible loss of VIII:C activity at 1 megarad and 80% loss with 60 megarads of irradiation. Analysis of the course of inactivation showed a biphasic curve with 73% of the VIII:C activity having a target size of 40,000 daltons while 28% had a molecular weight in excess of one million. Similar results were obtained when blood was collected in citrate and rapidly processed (∼5 min) to platelet-poor plasma. Following electron irradiation, 62% of VIII:C activity showed a target size of 35,000 daltons while the remaining 38% gave a target size of 275,000. These results provide further evidence that circulating VIII:C activity in normal plasma has a molecular weight of about 40,000 and suggest that reports of higher molecular weights are an artifact of the chelation of calcium as evidenced by the biphasic decay of VIII:C activity in citrated plasma.

scholarly journals Low Molecular Weight Factor VIII: A Comparison of the Electrophoretic Maps of the Normal and Hemophiliac Molecule

1977 ◽  
Author(s):  
G. A. Rock ◽  
W. H. Cruickshank
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Paper Electrophoresis ◽  
Low Molecular Weight ◽  
Molecular Weights ◽  
Elution Pattern ◽  
Acidic Peptide ◽  
Relative Structure ◽  
Two Populations ◽  
Insight Into

Considerable debate exists as to the relative structure of normal and hemophiliac Factor VIII. In order to obtain some insight into the differences which exist at the molecular level we examined the various subunits found after dissociation of Factor VIII from these two populations.Factor VIII was obtained by column chromatography of either cryoprecipitate or concentrated plasma on Sepharose CL6B. The protein eluting at the void volume was rechromatographed in buffer containing 0.25 M CaCl2. Under these conditions, both normal and hemophiliac Factor VIII underwent dissociation with an identical elution pattern. The low molecular weight faction from normal Factor VIII displayed procoagulant activity: that from the hemophiliac did not.As determined by their elution volume and ultrafiltration, the molecular weights were identical. When digested with pepsin (protein: pepsin 50:1) for 16 hours at 37°C and “mapped” by pH 6.5/2.1 paper electrophoresis the two compounds were significantly different in the neutral and acidic peptide regions and identical in the basic region.It therefore appears that the inactivity of hemophiliac Factor VIII is a consequence of an amino acid substitution or deletion in the low molecular weight portion of the molecule rather than a complete absence of the procoagulant molecule.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

INACTIVATION OF FACTOR VIII BY ACTIVATED PROTEIN C AND PROTEIN S

1987 ◽  
Author(s):  
P J Fay ◽  
S I Chavin ◽  
F J Walker
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Ix ◽  
Heavy Chains ◽  
Factor Va

Human factor VIII has been isolated from factor VIII concentrates. The isolated protein is composed of a heavy chain and light chain. The heavy chain was heterogenous with respect to molecular weight ranging from 110-170 kDa. The light chain appeared as a 81/84 kDa dimer, 'when factor VIII was treated with activated protein C in the presence of calcium and phospholipids factor VIII procoagulant activity was rapidly lost. Analysis of the activated protein C catalyzed cleavage products of factor VIII indicated that loss of activity was correlated with cleavage of the heavy chains. The heavy chains appeared to be converted into 93 kDa and 53 kDa peptides. A separate factor VIII preparation has been prepared that contained only a 93 kDa heavy chain as well as the 81/83 kDa light chain. When this preparation was inactivated with activated protein C, a pathway in which the 93 kDa peptide was degraded into a 68 kDa peptide which was subsequently degraded into 48 and 23 kDa polypeptides. This result suggested that the 53 kDa polypeptide was not derived from the 93 kDa domain of the heavy chain, but must have been derived from the variable molecular weight portion of the heavy chain. These results suggest that activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. Protein S has been observed to be a protein cofactor both the anticoagulant and proteolytic action of activated protein C with factor Va. It is thought that protein S forms a lipid bound complex with activated protein C which then can rapidly inactivate factor Va. When factor VIII was inactivated in the presence of both activated protein C and protein S the rate of activity loss was enhanced. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products including the 93, 68, and 53 kDa polypeptides. In an analogous reaction, the addition of factor Xa has been observed to inhibit the inactivation of factor Va by activated protein C. The addition of factor IX to the factor Vlll-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Finally, as both factor Va and factor VIII have structural similarities and are substrates for activated protein C the possibility that they might compete as substrates was tested. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.

scholarly journals Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1114-1117
Author(s):  
MJ Weinstein ◽  
CA Fulcher ◽  
LE Chute ◽  
TS Zimmerman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Discontinuous System ◽  
Major Band ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
Electrophoretic Techniques

We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4–7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4–4% polyacrylamide/0.5% agarose gels (NaDodSO4–4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4–4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4–4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.

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