Characterization of a Form of Bovine Factor VIII Which Does Not Aggregate Human Platelets

1981 ◽  
Vol 46 (02) ◽  
pp. 479-484 ◽  
Author(s):  
Edward P Kirby
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Rabbit Antibody ◽  
Factor Viii Related Antigen ◽  
Agarose Electrophoresis ◽  
Bovine Factor

SummaryFractionation of partially purified bovine factor VIII on tricalcium citrate columns produces a material which contains high levels of factor VIII procoagulant activity and factor VIII- related antigen, but does not aggregate human platelets. The procoagulant activity can be blocked by human inhibitors of factor VIII: C and by rabbit antibody to bovine factor VIII. Its activity can be increased by thrombin modification. The apparent molecular weight of this form of factor VIII is significantly lower than that of the forms of factor VIII which contain platelet aggregating activity, and it exhibits a higher mobility on agarose electrophoresis.

Characterization Of The ADP-Induced Fibrinogen Btnd1Ng Sites On Human Platelets Using Photoaffinity Techniques: Some Preliminary Findings

1981 ◽  
Author(s):  
Ellinor I Peerschke ◽  
Mariorie B Zucker ◽  
Avner Rotman
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Fragment ◽  
Fibrinogen Receptor ◽  
Congenital Deficiency ◽  
Sds Page ◽  
Platelet Membrane Receptor

The interaction of fibrinogen with its, platelet membrane receptor was investigated using 125-labeled fibrinogen which was photoaffinity labeled with a light-sensitive azide. This photoreactive material (125I-NPA-fibr) was indistinguishable from unlabeled fibrinogen as well as from iodinated fibrinogen on SDS-PAGE. It bound specifically to platelets stimulated with ADP and was crosslinked to the platelet membrane after exposure to light ( λ >300 nm) for 4 min. No crosslinking was observed in the presence of EDTA or with platelets that failed to aggregate with ADP either due to the congenital deficiency thrombasthenia or following incubation with EDTA for 8 min at 37° , pH 7.8 and recalcification. SDS-PAGE of platelets bearing crosslinked 125I-NPA-fibr revealed a radiolabeled band of about 450,000 daltons in addition to the 340,000 dalton radioactive band of fibrinogen, suggesting that fibrinogen had been covalently bound to a platelet membrane component with an intact apparent molecular weight of approximately 110,000 daltons. Following reduction, an extra radioactive band was noted at 80,000 daltons. As the A∝-chains of fibrinogen were too weakly labeled to be detected by autoradiography, this indicated that either the Bβ or γchain of fibrinogen was attached to a 25,000-35,000 molecular weight platelet membrane fragment. We conclude that the additional radioactive bands observed after electrophoresis of platelets bearing specifically bound-photoaffinity labeled 125I-fibrinogen most likely represent the binding of the B β or γ chains of fibrinogen to the platelet fibrinogen receptor which may be GPIIb.

Absence of the 145,000 Molecular Weight, Soluble Platelet Membrane Glycoprotein - Lack of Platelet Agglutination

1979 ◽  
Vol 42 (05) ◽  
pp. 1626-1629 ◽  
Author(s):  
Nils Olav Solum ◽  
Inger Hagen ◽  
Miroslav Peterka ◽  
Torbjørn Gjemdal
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Related Protein ◽  
Giant Platelets ◽  
Platelet Membrane Glycoprotein ◽  
Human Factor Viii ◽  
Bovine Factor

SummaryOne step in the function of platelets in hemostasis is their adhesion to subendothelial tissue. The human factor VIII related protein (von Willebrand factor) is considered to be involved in the adhesion phenomenon (Baumgartner et al. 1977). One manifestation of the protein-cell interaction can be observed as a platelet agglutination after addition to the human platelets of a combination of the human protein and the glycopeptide ristocetin, or after addition of the bovine protein alone. The bovine factor VIII related protein as such directly binds to the platelet membrane (Kirby and Sha May Tang 1977) and thus represents a simpler system than ristocetin plus the human cofactor which may have to interact with each other before excerting their effect on the platelet membrane. The present paper concerns the se.One of the characteristics of the agglutination of human platelets brought about by the bovine factor VIII related protein (as well as by ristocetin plus the human cofactor) is that it is independent of the energy metabolism and the internal organization of the platelet. One would therefore expect that modified platelets and platelet “ghosts” would agglutinate as long as certain structures on the outer cell surface are chemically and sterically intact. Because of the hydrophilic character of the carbohydrate side chains, the membrane glycoproteins are considered of special importance for cell contact phenomena. Thus it has already been known for some years that giant platelets of the Bernard-Soulier type which do not agglutinate with the bovine protein (Bithell et al. 1972), contain a reduced amount of sialic acid related to protein content and surface area (Grottum and Solum 1969), and show a reduced glycoprotein stain in the GP I region on SDS polyacrylamide gel electrophoresis (Nurden and Caen 1975).This paper presents five observations which support a working hypothesis stating that the presence on the platelet membrane of the 145,000 molecular weight, soluble platelet membrane glycoprotein called GPS or glycocalicin is a prerequisite to the agglutination of human platelets by bovine factor VIII related protein.

Purification of Ristocetin Willebrand Factor (RWF)

1975 ◽  
Author(s):  
J. D. Olson ◽  
D. N. Fass ◽  
W. J. Brockway ◽  
E. J. W. Bowie ◽  
K. G. Mann
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Amino Sugar ◽  
Procoagulant Activity ◽  
Factor Viii Inhibitor ◽  
Induced Aggregation

A component required for the ristocetin-induced aggregation of platelets was isolated in yields of 20-30% from pooled human and porcine plasma by cryoethanol concentration of the RWF, after removal of the vitamin K dependent coagulation factors on barium citrate. The concentrate was further purified using gel filtration (4% agarose) and ion exchange (DEAE-cellulose) chromatography. Sodium dodecyl sulfate polyacrylamide gels of the isolated factor indicated an apparent molecular weight of greater than 500,000. After reduction of RWF with mercaptoethanol, a single band is resolved with an apparent molecular weight of 230,000. The purified component had no factor VIII procoagulant activity and did not compete for the activity of naturally occurring factor VIII inhibitor (human). Antisera raised in rabbits directed against the purified component inhibited the RWF activity but not the factor VIII procoagulant activity of plasma. Amino acid analysis indicated the presence of all normal amino acids and failed to detect any amino sugar. Analysis of lipid revealed a significant amount of lipid composed of mono, di and triglycerides, cholesterol, cholesterol esters and free fatty acid, with small portions of phospholipids.

Immunologic characterization of canine factor VIII

Blood ◽  
1976 ◽  
Vol 48 (4) ◽  
pp. 521-529
Author(s):  
RE Benson ◽  
WJ Dodds
Keyword(s):  
Factor Viii ◽  
Antigenic Site ◽  
Procoagulant Activity ◽  
Gel Chromatography ◽  
Rabbit Antibody ◽  
Similar Extent ◽  
Crossed Immunoelectrophoresis ◽  
Heat Treated ◽  
Canine Plasma

Canine factor VIII (FVIII) preparations isolated from cryoprecipitates by gel chromatography were pooled to provide one batch of antigen for simultaneous immunization of two rabbits and a goat. The goat and rabbit antisera had similar FVIII-neutralizing titers, but the latter had seven to ten times more precipitating titer for FVIII-related antigen (FVIII-RA). Absorption with material low in FVIII had little effect on the precipitating titer of the rabbit antibody, but it abolished the precipitating capacity of the goat antibody and caused a 20% reduction in the neutralizing titer of both antisera. Results obtained in the Laurell assay with the two different antisera were similar. This finding was true whether the FVIII-RA levels were reduced, normal, or elevated, as well as for heat-treated and frozen- thawed plasmas. Both antisera were neutralized by the same canine plasma to a similar extent. Analysis of FVIII concentrates by crossed immunoelectrophoresis suggested that canine FVIII-RA was heterogeneous, with slow-and fast-migrating components. The presence of more than one antigenic site on the FVIII complex was also supported by the disparity between the FVIII-neutralizing and -precipitating titers of goat antiserum and by the demonstration that FVIII-RA, FVIII-neutralizing antigen, and procoagulant activity varied independently.

scholarly journals The Binding of Bovine Factor VIII of Human Platelets

1977 ◽  
Author(s):  
Edward P. Kirby ◽  
Sha May Tang
Keyword(s):  
Factor Viii ◽  
Evans Blue ◽  
Sodium Iodide ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Human Fibrinogen ◽  
Total Binding ◽  
Antihemophilic Factor ◽  
Bovine Factor

Highly purified bovine Factor VIII (FVIII) was iodinated by the lactoperoxidase method. Subsequent chromatography on agarose and extensive dialysis against sodium iodide solutions was required to remove non-covalently bound 1-125 which adhered tightly to FVIII. Iodination destroyed the procoagulant activity (Antihemophilic Factor-AHF) of FVIII, but did not affect its Platelet Aggregating Factor (PAF) activity. Binding to human platelets was determined by incubating radio!abelled FVIII with formalin-treated platelets, centrifuging, and measuring both bound and free radioactivity. Results obtained by this method were much more precise than those obtained by measuring disappearance of unlabelled AHF, PAF, or FVIII-related antigen from the supernatant, although the estimates of total binding obtained were comparable. Binding of FVIII to formal in-treated platelets was approximately the same as to unfixed platelets, and the binding could be saturated by adding an excess of unlabelled FVIII. Maximal binding occurred within 1-2 minutes at 37° and binding could be blocked by dextran sulfate, Evans Blue or other inhibitors of FVIII-induced platelet agglutination. Treatment of platelets with trypsin inhibited binding of labelled F-VIII. Binding was not affected by the presence of plasma, or high levels of purified human fibrinogen or FVIII.

scholarly journals Bovine Factor VIII. Antigens and Biological Activities

1977 ◽  
Author(s):  
G. Casillas ◽  
C. Simonetti ◽  
A. Pavlovsky
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Biological Activities ◽  
Gel Filtration ◽  
Species Specificity ◽  
Procoagulant Activity ◽  
Rabbit Antibody ◽  
Von Willebrand ◽  
The Relationship ◽  
Bovine Factor

The procoagulant (PcgF) and the platelet aggregating (PAF) activities of bovine F. VIII were studied in order to establish their relation to the antigens A1 and A2, antigens similar to those synthetized by von Willebrand and hemophilia A patients respectively. Studies of stability to different agents (temperature. EDTA, thrombin, etc.) allowed us to establish that VIII (PcgF) and VIII (PAF) are not mutually dependenteAn homologous antibody of low species specificity against human F. VIII binds specifically with VIII (A,) antigenic moiety of bovine F. VIII. The complex so formed is purified by gel filtration and has VIII (PAF) activity but not VIII (PcgF) activity indicating that VIII(A1) and VIM (PcgF) activities associate,, Another complex, formed by bovine factor VIII and a rabbit antibody against the VIII (A2) moiety, was prepared,, This complex has VIII (PcgF) activity but not VIII (PAF) activity, indicating that VIII (A2) and VIII (PAF) associate,, The complex and its procoagulant activity sediment after centrifugation and may by re-covered by suspension of the precipitate. The following scheme showing the relationship between the antigenic moieties and the activities of bovine factor VIII is proposed: VIII(A1-PcgF) (A2-PAF).

scholarly journals Immunologic characterization of canine factor VIII

Blood ◽  
1976 ◽  
Vol 48 (4) ◽  
pp. 521-529 ◽  
Author(s):  
RE Benson ◽  
WJ Dodds
Keyword(s):  
Factor Viii ◽  
Antigenic Site ◽  
Procoagulant Activity ◽  
Gel Chromatography ◽  
Rabbit Antibody ◽  
Similar Extent ◽  
Crossed Immunoelectrophoresis ◽  
Heat Treated ◽  
Canine Plasma

Abstract Canine factor VIII (FVIII) preparations isolated from cryoprecipitates by gel chromatography were pooled to provide one batch of antigen for simultaneous immunization of two rabbits and a goat. The goat and rabbit antisera had similar FVIII-neutralizing titers, but the latter had seven to ten times more precipitating titer for FVIII-related antigen (FVIII-RA). Absorption with material low in FVIII had little effect on the precipitating titer of the rabbit antibody, but it abolished the precipitating capacity of the goat antibody and caused a 20% reduction in the neutralizing titer of both antisera. Results obtained in the Laurell assay with the two different antisera were similar. This finding was true whether the FVIII-RA levels were reduced, normal, or elevated, as well as for heat-treated and frozen- thawed plasmas. Both antisera were neutralized by the same canine plasma to a similar extent. Analysis of FVIII concentrates by crossed immunoelectrophoresis suggested that canine FVIII-RA was heterogeneous, with slow-and fast-migrating components. The presence of more than one antigenic site on the FVIII complex was also supported by the disparity between the FVIII-neutralizing and -precipitating titers of goat antiserum and by the demonstration that FVIII-RA, FVIII-neutralizing antigen, and procoagulant activity varied independently.

Studies on the Characterization of Factor VIII and a Co-factor VIII

1974 ◽  
Vol 31 (02) ◽  
pp. 328-338
Author(s):  
M. M. P Paulssen ◽  
H. L. M. A Vandenbussche-Scheffers ◽  
P. B Spaan ◽  
T de Jong ◽  
M. C Planje
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
The Body ◽  
Haemophilia A ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Polysaccharide Content ◽  
Plasma Factor ◽  
Two Factors

SummaryFactor VIII occurs in the body in two different forms. In lymph factor VIII is bound to chylomicra. In plasma, factor VIII is bound to a protein.After delipidation of chylomicra we obtained a glycoprotein with a high polysaccharide content and a molecular weight of approx. 160,000.In plasma, factor VIII is attached to a protein which is present in normal concentrations in plasma of patients with haemophilia A and in serum (co-factor VIII).This factor is deficient in both the plasma and the serum of patients with von Willebrand’s disease.The binding between factor VIII and co-factor VIII is reversible.Some properties of these two factors are described.

Partial characterization of cheese-ripening proteinases produced by the yeastKluyveromyces lactis

1983 ◽  
Vol 50 (4) ◽  
pp. 469-480 ◽  
Author(s):  
Paul A. Grieve ◽  
Barry J. Kitchen ◽  
John R. Dulley ◽  
John Bartley
Keyword(s):  
Molecular Weight ◽  
Kluyveromyces Lactis ◽  
Apparent Molecular Weight ◽  
Aminopeptidase Activity ◽  
Carboxypeptidase Y ◽  
Casein Hydrolysis ◽  
Cheese Ripening ◽  
Caseinolytic Activity ◽  
Proteinase A

SUMMARYAn extract ofKluyveromyces lactis416 and a β-galactosidase preparation (Maxilact 40000) contaminated with proteinase, showed similar pH profiles of caseinolytic activity. Similar modes of casein hydrolysis (κ-, > αs-, ≥ β-) were observed at pH 5·0 (the pH of Cheddar cheese), without detection of bitterness. The contaminated Maxilact preparation contained similar proteinase types to those detected in an autolysate ofK. lactis. Both the autolysate and the Maxilact preparation contained acid endopeptidase (proteinase A), serine endopeptidase (proteinase B) and serine exopeptidase (carboxypeptidase Y) activities. Some aminopeptidase activity was also detected in both preparations. There were some differences in apparent molecular weight and charge properties between proteinase A and B and carboxypeptidase Y from the 2 proteinase sources.

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain

1988 ◽  
Vol 8 (2) ◽  
pp. 564-570
Author(s):  
P A Maher ◽  
S J Singer
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Protein Complex ◽  
Apparent Molecular Weight ◽  
Integral Membrane Protein ◽  
Beta Chain ◽  
Attachment Sites ◽  
Wide Range ◽  
Membrane Attachment

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

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