scholarly journals Factor VIII/von Willebrand factor protein: sensitivity of periodic acid Schiff stain to carbohydrate deficiency

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1310-1316 ◽  
Author(s):  
HR Gralnick ◽  
GM Jackson ◽  
SB Williams ◽  
MC Cregger
Keyword(s):  
Sialic Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Periodic Acid ◽  
Periodic Acid Schiff ◽  
Human Factor Viii ◽  
Acid Deficiency ◽  
Or Organization ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have investigated the periodic acid Schiff (PAS) Coomassie staining ratio of the human factor VIII/von Willebrand factor (fVIII/vWf) protein. The PAS-Coomassie staining ratio is consistent over 8 days. The PAS-Coomassie ratio of fVIII/vWf protein purified from different starting materials does not appear to be significantly different. The PAS stain can detect as little as 300 ng of carbohydrate in the fVIII/vWf protein. Desialation did not affect the PAS-Coomassie ratio, while removal of penultimate galactose resulted in a marked reduction in the PAS-Coomassie ratio. This reduction was further accentuated with the removal of N-acetylglucosamine. The smaller multimers of the fVIII/vWf protein have a reduced sialic acid and PAS-Coomassie staining ratio. This difference does not appear to be related to the sialic acid deficiency but may be related to the distribution or organization of the carbohydrate moieties on the smaller fVIII/vWf multimers.

scholarly journals Factor VIII/von Willebrand factor protein: sensitivity of periodic acid Schiff stain to carbohydrate deficiency

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1310-1316
Author(s):  
HR Gralnick ◽  
GM Jackson ◽  
SB Williams ◽  
MC Cregger
Keyword(s):  
Sialic Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Periodic Acid ◽  
Periodic Acid Schiff ◽  
Human Factor Viii ◽  
Acid Deficiency ◽  
Or Organization ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the periodic acid Schiff (PAS) Coomassie staining ratio of the human factor VIII/von Willebrand factor (fVIII/vWf) protein. The PAS-Coomassie staining ratio is consistent over 8 days. The PAS-Coomassie ratio of fVIII/vWf protein purified from different starting materials does not appear to be significantly different. The PAS stain can detect as little as 300 ng of carbohydrate in the fVIII/vWf protein. Desialation did not affect the PAS-Coomassie ratio, while removal of penultimate galactose resulted in a marked reduction in the PAS-Coomassie ratio. This reduction was further accentuated with the removal of N-acetylglucosamine. The smaller multimers of the fVIII/vWf protein have a reduced sialic acid and PAS-Coomassie staining ratio. This difference does not appear to be related to the sialic acid deficiency but may be related to the distribution or organization of the carbohydrate moieties on the smaller fVIII/vWf multimers.

Factor VIII/Von Willebrand Factor Protein Sensitivity Of Periodic Acid Schiff Stain To Carbohydrate Deficiency

1981 ◽  
Author(s):  
H Gralnick ◽  
G Jackson ◽  
S Williams ◽  
E Cregger
Keyword(s):  
Sialic Acid ◽  
Von Willebrand Factor ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Reaction ◽  
Periodic Acid Schiff ◽  
Sialic Acid Content ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Carbohydrate (C) deficiency of the factor VUI/von Willebrand factor (f. VIII/vWf) protein has been implicated as one of the molecular defects of von Willebrand’s disease. The periodic acid Schiff (PAS) reaction in polyacrylamide gel electrophoresis (PAGE) has been used as a screening test for C deficiency. To ascertain the sensitivity of this reaction to the C content of the f. VIII/vWf protein we have purified the normal protein and compared the Coomassie to PAS staining reaction of the subunit of the intact, asialo (neuraminidase-treated), asialo-agalacto (neuraminidase and β-galactosidase-treated) derivatives and a fourth derivative which was treated with a mixed glycosidase which included neuraminidase, β-galactosidase and N-acetyl-glucosaminidase. Gels were run simultaneously, one stained with Coomassie blue and the other with PAS and at various time intervals after staining the gels were scanned.Studies of the intact f. VIII/vWf protein revealed that the PAS and Coomassie stains could be compared from 24 hours up to 7 days with no significant changes. The desialated f. VIII/vWf protein had insignificant differences in the PAS stain compared to normal (range 81-108% of intact). However, the asialo-agalacto derivative (36% of intact) and the f. VIII/vWf treated with the mixed glycosidase (24% of intact) had significantly reduced PAS staining in relation to normal. C analysis of the different f. VIII/vWf proteins revealed that removal of the sialic acid reduced the C 30% while in the asialo-agalacto the C was reduced 47% and in the mixed glycosidase-treated material the C was reduced 78%. The PAS stain is not very sensitive to the sialic acid content of the f. VIII/vWf protein and reduction of 30% of the C does not affect the PAS stain. Since the PAS stain is relatively insensitive to significant carbohydrate deficiencies of the f. VIII/vWf protein, the use of the PAS stain in PAGE may not be an adequate test to ascertain C content of the f. VIII/vWf protein.

scholarly journals Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 823-831 ◽  
Author(s):  
VT Turitto ◽  
HJ Weiss ◽  
TS Zimmerman ◽  
II Sussman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Human Factor ◽  
Rabbit Aorta ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Von Willebrand ◽  
Willebrand Factor

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

scholarly journals Characterization of the defect of the factor VIII/von Willebrand factor protein in von Willebrand's disease

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 542-548 ◽  
Author(s):  
HR Gralnick ◽  
MC Cregger ◽  
SB Williams
Keyword(s):  
Sialic Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Molecular Forms ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Platelet Receptor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N- acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.

scholarly journals von Willebrand Factor Activity in Plasmin Digests of Factor VIII

1977 ◽  
Author(s):  
J. A. Guisasola ◽  
C. Cockburn ◽  
R. M. Hardisty
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Factor Activity ◽  
Group Iii ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Group Ii ◽  
Von Willebrand ◽  
Willebrand Factor

Purified human factor VIII was incubated for up to 24 hours with plasmin, and the activity of the breakdown products studied at intervals. Factor VIII coagulant activity was lost within the first hour, but von Willebrand factor activity (FVIIIR:WF) was retained for two hours, and then declined slowly during the subsequent incubation. Analysis of the 24-hour breakdown products by immuno-electrophoresis, sepharose 4B chromatography and SDS Polyacrylamide electrophoresis revealed three main groups of fragments recognised by rabbit anti-human factor VIII anti-serum, and having molecular weights in the following ranges: Group 1 300,000=500,000; Group II, 150–200,000; Group III, 100,000. FVIIIR:WF activity, which was found only in Group II, appeared to be associated with glycopeptide(s) of up to 155,000 daltons.

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