Effects of Ultrasonication on Factor VIII

G, Casillas; Simonetti, C; Lazzari, M.A.; Kempfer, Ana C.

doi:10.1055/s-0038-1650016

Cited by 4 publications

(2 citation statements)

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“…Casillas et al (20) have noted that a decrease in platelet aggegating activity of bovine factor VIII occurred after sonication while VIII C activity remained unchanged. We have shown conclusively that sonication (15 p amplitude for 30 secs) reduced the larger multimeric forms (greater than 3.76 x 10u) in MED, plasma and CRYO.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

Chan

1982

Thromb Haemost

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SummaryCrossed immunoelectrophoresis showed that factor VIII related protein (VIII R) synthesized in the human endothelial cell (EC-VIII R) had faster electrophoretic mobility than that secreted into the culture medium (MED-VIII R). Sodium dodecyl sulphate agarose gel electrophoresis followed by radioimmunofixation showed that EC-VIII R consisted of molecules varying from 0.26 × 106 to 3.76 × 106 daltons while MED-VIII R had molecules ranging from 0.93 × 106 to greater than 10 × 106 daltons, similar to that present in plasma. The smallest VIII R molecule present in normal plasma or spent culture medium (0.93 × 106 daltons) corresponded to a tetramer of subunits of 0.22-0.24 × 106 daltons. Only molecular forms greater than 3.76 × 106 daltons possessed ristocetin cofactor activity. Sonication (15 μ. amplitued for 30 secs) effectively broke the non-covalent bonds of the VIII R multimers resulting in smaller molecules. Thus endothelial cells in culture synthesized VIII R subunits and assembled them into the higher multimeric forms on secretion. Different types of von Willebrand disease could result from defects of either of the two processes.

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Section: Discussionmentioning

confidence: 99%

Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

Chan

1982

Thromb Haemost

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“…The interchain disulfide bonds that form a dimer are quite stable, although mild reduction of a small number of sensitive disulfide bonds (Ohmori et al, 1982) causes stepwise disassembly of the vWF multimers (Counts et al, 1978). Similar changes in vWF multimers have been observed after modification or removal of penultimate galactose (Gralnick et al, 1983) or after sonication (Casillas et al, 1980;Chan & Chan, 1982). Biological activity of vWF has been shown to be related to the size of the multimers.…”

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confidence: 84%

Human von Willebrand factor: a multivalent protein composed of identical subunits

Chopek¹,

Girma²,

Fujikawa³

et al. 1986

Biochemistry

118

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A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.

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Purification of the factor VIII complex

Thorell

Blombäck

1984

Thrombosis Research

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Effects of Ultrasonication on Factor VIII

Cited by 4 publications

References 0 publications

Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

Characterization of Factor VIII Related Protein Synthesized by Human Endothelial Cell: A Study of Structure and Function

Human von Willebrand factor: a multivalent protein composed of identical subunits

Purification of the factor VIII complex

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