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Evaluation of glycoprotein Ib expression on feline platelets

Fern TablinDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Jocelyn D. JohnsrudeDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.
Present address is Idexx Veterinary Services, 2825 KOVR Dr, PO Box V, West Sacramento, CA 95691.

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Naomi J. WalkerDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Abstract

Objective—To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb).

Sample Population—Platelets obtained from 11 specific-pathogen-free cats.

Procedure—Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation.

Results—Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 µg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton.

Conclusions and Clinical Relevance—Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis. (Am J Vet Res 2001;62:195–201)

Abstract

Objective—To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb).

Sample Population—Platelets obtained from 11 specific-pathogen-free cats.

Procedure—Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation.

Results—Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 µg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton.

Conclusions and Clinical Relevance—Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis. (Am J Vet Res 2001;62:195–201)