scholarly journals Immunologic evidence that the properties of human antihemophilic factor (factor VIII) are attributes of a single molecular species

Blood ◽  
1976 ◽  
Vol 47 (4) ◽  
pp. 657-667
Author(s):  
OD Ratnoff ◽  
CC Slover ◽  
MC Poon
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Molecular Species ◽  
Gel Filtration ◽  
Antigenic Sites ◽  
Heterologous Antiserum ◽  
Aggregation Of Platelets ◽  
Antihemophilic Factor

Preparations of human plasma rich in antihemophilic factor (AHF, factor VIII) correct the coagulative defect of classic hemophilic plasma, form precipitates with specific heterologous antiserum, and support aggregation of platelets by ristacetin and retention of platelets by columns of glass beads. Whether these various properties can all be attributed to a single molecular species is disputed. Antiserums were prepared in rabbits to partially purified AHF and to high molecular weight (MW) and low MW fragments separated by gel filtration through columns of agarose in the presence of 0.25 M calcium chloride. Antiserums to AHF and to its high or low MW fragments all inactivated procoagulant AHF in plasma or in preparations of AHF. In contrast, antiserums to AHF and its low MW fragment inactivated procoagulant AHF in the low MW fragment, while that against the high MW fragment lacked this property. Thus, the low MW fragment appeared to have some antigenic sites not present or accessible to the antiserum against the high MW fragment. In agreement with this, the low MW fragment did not block antiserum against the high MW fragment as tested by the capacity of this antiserum to inactivate functional AHF in plasma. These immunologic studies support the view that the various properties of preparations of human AHF are attributes of a single molecular species.

Immunologic evidence that the properties of human antihemophilic factor (factor VIII) are attributes of a single molecular species

Blood ◽  
1976 ◽  
Vol 47 (4) ◽  
pp. 657-667 ◽  
Author(s):  
OD Ratnoff ◽  
CC Slover ◽  
MC Poon
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Molecular Species ◽  
Gel Filtration ◽  
Antigenic Sites ◽  
Heterologous Antiserum ◽  
Aggregation Of Platelets ◽  
Antihemophilic Factor

Abstract Preparations of human plasma rich in antihemophilic factor (AHF, factor VIII) correct the coagulative defect of classic hemophilic plasma, form precipitates with specific heterologous antiserum, and support aggregation of platelets by ristacetin and retention of platelets by columns of glass beads. Whether these various properties can all be attributed to a single molecular species is disputed. Antiserums were prepared in rabbits to partially purified AHF and to high molecular weight (MW) and low MW fragments separated by gel filtration through columns of agarose in the presence of 0.25 M calcium chloride. Antiserums to AHF and to its high or low MW fragments all inactivated procoagulant AHF in plasma or in preparations of AHF. In contrast, antiserums to AHF and its low MW fragment inactivated procoagulant AHF in the low MW fragment, while that against the high MW fragment lacked this property. Thus, the low MW fragment appeared to have some antigenic sites not present or accessible to the antiserum against the high MW fragment. In agreement with this, the low MW fragment did not block antiserum against the high MW fragment as tested by the capacity of this antiserum to inactivate functional AHF in plasma. These immunologic studies support the view that the various properties of preparations of human AHF are attributes of a single molecular species.

scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

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.

Characterization Of Soluble DES-A And DES-AB Fibrin In Human Plasma At 37°C By Agarose-Gel Filtration

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J-C Bernhard ◽  
I Mahn
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Agarose Gel ◽  
High Molecular Weight Material ◽  
Different Temperatures ◽  
Lower Temperature

In two consecutive steps, thrombin cleaves the fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin. Labeled des-A fibrin was prepared by batroxobin and labeled des-AB fibrin by clotting of 125I-fibrinogen with thrombin. Fibrin solubilized in buffered urea was mixed with plasma containing 131I-fibrinogen (fibrin:fibrinogen ratio = 1:20). These fibrinfibrinogen mixtures were applied to sepharose CL- 6B columns eq ui librated with buffered plasma (0.0025 M EDTA, 0.1 M NaCl, 0.05 M tris, 0.005 M EACA, 2 AT U hirudin/ml, 500 KIU a protinin/ml, 0.003 M NaN3, pH 7.4). Plasma was used as an equilibration and elution medium to prevent precipitation of fibrin in the columns. At 20°C, labeled des-A fibrin as well as des-AB fibrin were eluted in the void volume as high-molecular weight aggregates (peak A) and separated from m onomeric labeled fibrinogen (peak B). At 37°C, however, des- A fibrin was eluted at the same position as monomeric fibrinogen (peak B), whereas des-AB fibrin was eluted in the void volume as at 20°C. Rechromatography of isolated fractions of peak A and peak B at different temperatures showed that monomeric fibrin isolated at 37°C formed high molecular weight material at 20°C, and high-molecular weight fibrin isolated at 20°C dissociated at 37 ° C. The results suggest that des-A fibrin solubilized in plasma in the absence of calcium ismonomeric at 37°C but forms high-molecular weight aggregates at lower temperature, whereas des-AB fibrin is oligomeric at 20°C as well as at 37°C.

scholarly journals Immunologic Studies of Antihemophilic Factor (AHF, Factor VIII). V. Immunologic Properties of AHF Subunits Produced by Salt Dissociation

Blood ◽  
1973 ◽  
Vol 42 (5) ◽  
pp. 737-747 ◽  
Author(s):  
Margaret E. Rick ◽  
Leon W. Hoyer
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Plasma Protein ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Antibody Neutralization ◽  
Antihemophilic Factor ◽  
Immunologic Properties

Abstract Human antihemophilic factor (AHF, factor VIII), a large plasma protein with a molecular weight of approximately two million, is dissociated by changes in ionic strength. The immunologic properties of subunits obtained by sucrose density ultracentrifugation in 1 M NaCl and by agarose gel filtration in 0.24 M CaCl2 have been determined using human and rabbit anti-AHF. Asymmetric dissociation of AHF has been identified with formation of two subunits in these separations: a nonfunctional highmolecular-weight (HMW) subunit similar in size to plasma AHF which is identified by immunoprecipitation and radioimmunoassay for AHF antigen, and an active lowmolecular-weight (LMW) subunit which is not detected by these antigen assays. The LMW subunit retains AHF antigens, however, for it is inactivated by both human and rabbit anti-AHF. Antibody neutralization studies verify the presence of AHF antigens on the HMW subunit. These immunologic studies provide constraints which must be incorporated into models of AHF structure.

scholarly journals Autosomal Factor VIII Deficiency in Rabbits: Size Variations of Rabbit Factor VIII.

1977 ◽  
Author(s):  
R.E. Benson ◽  
W.J. Dodds
Keyword(s):  
Molecular Weight ◽  
New Zealand ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Antigenic Relationship ◽  
Genetic Studies ◽  
Coagulant Activity ◽  
Autosomal Inheritance

Many rabbits from our Flemish Giant-Chinchilla colony have moderate to severely reduced levels of factor VIII coagulant activity (FVIII-C). Some have shown prolonged bleeding after venipunctures and gastrointestinal and intramuscular hemorrhages. Genetic studies indicate autosomal inheritance. Gel filtration of plasma from these rabbits by the method of Rick et al. (Blood, 49, 209, 1977) at 25°C, pH 6.8 revealed two distinct peaks of FVIII-C; the majority of activity eluting as high molecular weight (HMW) material at the void volume (V°) followed by a much smaller low molecular weight (LMW) peak eluting close to that of fibrinogen. By contrast, filtration of plasma from New Zealand (NZ) rabbits produced threefold greater protein at the V° and equal amounts of HMW and LMW FVIII-C. Increasing the pH to 7.4 had little effect on FVIII-C recovery, although filtration at 4°C virtually abolished the HMW FVIII-C peak of NZ plasma. Rat antiserum (AS) to rabbit HMW FVIII-C, absorbed with precipitate low in FVIII-C, detected precipitating antigen in both HMW and LMW fractions. After absorption with rabbit fibrinogen, the AS no longer detected HMW V° material. The antigenic relationship between HMW and LMW FVIII-C and fibrinogen thus remains unclear. The differences in amount of HMW protein and the ratio of HMW to LMW FVIII-C suggest that in comparison to NZ rabbits our animals have a variant factor VIII molecule as well as low FVIII-C.

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.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

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