von Willebrand factor in lysates of washed canine platelets

Michael T. Parker From the Department of Veterinary Pathology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Mark A. Turrentine From the Department of Veterinary Pathology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Gary S. Johnson From the Department of Veterinary Pathology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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SUMMARY

Canine and human platelets (washed 4 times in a solution containing edta, prostaglandin E1, and theophylline to prevent release of agranule constituents) were lysed by being frozen and thawed in the presence of detergent. Radioelectroimmunoassay for von Willebrand factor (vWf) in 5 human platelet lysates produced precipitin rockets, shaped like those produced from vWf in plasma from healthy human beings, and indicated that the mean von Willebrand factor antigen (vWf:Ag) content in platelets from healthy human beings was 526 ± 87 human U/ 1012 platelets. Radioelectroimmunoassay for vWf in platelet lysates from 17 healthy dogs with normal plasma vWf:Ag concentration produced precipitin rockets that looked different from those produced from canine plasma and indicated vWfAg content of 59 ± 35 canine U/1012 platelets. Inclusion of protease inhibitors in the lysing solution did not normalize the appearance of the precipitin rockets or substantially alter the measured platelet content of vWf:Ag. The array of vWf multimers revealed by sodium dodecyl sulfate-agarose gel electrophoresis of canine platelet lysates had a distinct appearance that differed from that of vWf in canine or human plasma and platelets; the intensity of the canine platelet vWf multimer bands was skewed, with relatively greater density in the lower molecular weight region and faint or undetectable multimer bands in the higher molecular weight region. Electrophoretograms with visible multimers in the high molecular weight region had vWf components that had higher molecular weight than did any vWf components in canine plasma. Radioelectroimmunoassay for fibronectin in these same canine platelet lysates indicated that the fibronectin content in platelets was 2.89 ± 1.10 mg/1012 platelets. An Airedale Terrier with type-I von Willebrand disease (vWd), but lacking clinical signs of vWd, had normal platelet content of vWf:Ag (28 ± 12 canine U/1012 platelets), whereas a German Shorthaired Pointer with moderately severe type-II vWd and a mildly affected Doberman Pinscher with type-I vWd had only a trace or undetectable amounts of vWf:Ag in their platelets. The concentration of vWf: Ag in platelet lysates from the Doberman Pinscher with vWd remained undetectable when the platelets were isolated from the Doberman Pinscher's blood mixed with citrated plasma from dogs with normal plasma vWf:Ag concentration. In all 3 dogs with vWd, platelet fibronectin content was within the normal range.

SUMMARY

Canine and human platelets (washed 4 times in a solution containing edta, prostaglandin E1, and theophylline to prevent release of agranule constituents) were lysed by being frozen and thawed in the presence of detergent. Radioelectroimmunoassay for von Willebrand factor (vWf) in 5 human platelet lysates produced precipitin rockets, shaped like those produced from vWf in plasma from healthy human beings, and indicated that the mean von Willebrand factor antigen (vWf:Ag) content in platelets from healthy human beings was 526 ± 87 human U/ 1012 platelets. Radioelectroimmunoassay for vWf in platelet lysates from 17 healthy dogs with normal plasma vWf:Ag concentration produced precipitin rockets that looked different from those produced from canine plasma and indicated vWfAg content of 59 ± 35 canine U/1012 platelets. Inclusion of protease inhibitors in the lysing solution did not normalize the appearance of the precipitin rockets or substantially alter the measured platelet content of vWf:Ag. The array of vWf multimers revealed by sodium dodecyl sulfate-agarose gel electrophoresis of canine platelet lysates had a distinct appearance that differed from that of vWf in canine or human plasma and platelets; the intensity of the canine platelet vWf multimer bands was skewed, with relatively greater density in the lower molecular weight region and faint or undetectable multimer bands in the higher molecular weight region. Electrophoretograms with visible multimers in the high molecular weight region had vWf components that had higher molecular weight than did any vWf components in canine plasma. Radioelectroimmunoassay for fibronectin in these same canine platelet lysates indicated that the fibronectin content in platelets was 2.89 ± 1.10 mg/1012 platelets. An Airedale Terrier with type-I von Willebrand disease (vWd), but lacking clinical signs of vWd, had normal platelet content of vWf:Ag (28 ± 12 canine U/1012 platelets), whereas a German Shorthaired Pointer with moderately severe type-II vWd and a mildly affected Doberman Pinscher with type-I vWd had only a trace or undetectable amounts of vWf:Ag in their platelets. The concentration of vWf: Ag in platelet lysates from the Doberman Pinscher with vWd remained undetectable when the platelets were isolated from the Doberman Pinscher's blood mixed with citrated plasma from dogs with normal plasma vWf:Ag concentration. In all 3 dogs with vWd, platelet fibronectin content was within the normal range.

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