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.

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.

Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

1978 ◽  
Vol 40 (02) ◽  
pp. 316-325 ◽  
Author(s):  
Ira I Sussman ◽  
Harvey J Weiss
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Direct Evidence ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Bean Trypsin Inhibitor ◽  
Benzamidine Hydrochloride

SummaryWhen gel filtration of factor VIII is performed with buffers of high ionic strength (1.0 M NaCl or 0.25 M CaCl2), the procoagulant activity elutes with proteins of relatively low molecular weight. It has been suggested that in the presence of proteolytic inhibitors, the procoagulant activity would appear at the void volume. To test this hypothesis, chromatography with buffers of high ionic strength was performed in the presence of benzamidine hydrochloride, soy bean trypsin inhibitor, heparin, DFP, and aprotinin. Under all of these conditions, the procoagulant activity continued to elute with proteins of low molecular weight. Similar findings were obtained after chromatographing cryoprecipitate prepared from the plasma of a normal subject who had received heparin. Thus, at present there is no direct evidence to suggest that proteolysis is involved in the dissociation of factor VIII by buffers of high ionic strength.

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.

Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

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.

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.

Gel Filtration Patterns of Factor VIII Coagulant Antigen and Factor VIII Related Antigen in Normal and von Willebrand’s Disease

1983 ◽  
Vol 50 (02) ◽  
pp. 509-512
Author(s):  
Juan Chediak ◽  
Ian Peake ◽  
Arthur Bloom
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Type I ◽  
Type Ii ◽  
Von Willebrand's Disease ◽  
Factor Viii Related Antigen ◽  
Von Willebrand’S Disease ◽  
Coagulant Activity

SummaryGel filtration (sepharose 2B CL) patterns of factor VIII coagulant antigen (VIIIC :Ag) and factor VIII related antigen (VIIIR: Ag) were obtained using normal plasma and plasma from patients with von Willebrand’s disease. The latter group consisted of five individuals with normal mobility of factor VIIIR :Ag on cross-immunoelectrophoresis (Type I) and five others with abnormal (increased) mobility (Type II). Results showed that the elution of VIIIC: Ag was delayed in those subjects whose ratio of VIIIR :Ag to VIIIC :Ag was reduced. It has previously been reported that VIIIR :Ag exerts a stabilizing influence on the coagulant activity of factor VIII (VIII: C); our data suggests that when VIIIR :Ag is deficient, abnormal (low molecular weight) forms of VIIIC: Ag circulate.

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.

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