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Evaluation of glycoprotein Ib expression on feline platelets
F Tablin1, J D Johnsrude, N J Walker
1Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, USA.
Insights
Feline platelets express a 140-kd membrane protein recognized by antibodies against glycoprotein Ib (GPIb). This finding may help in understanding feline platelet function and health.
Area of Science:
- Veterinary Hematology
- Platelet Biology
- Immunochemistry
Background:
- Glycoprotein Ib (GPIb) is a critical platelet surface receptor involved in hemostasis.
- The expression of GPIb on feline platelets has not been previously characterized.
Purpose of the Study:
- To investigate the presence and characteristics of glycoprotein Ib (GPIb) on platelets from domestic cats.
- To determine if feline platelets express a protein analogous to human GPIb.
Main Methods:
- Platelets were isolated from 11 specific-pathogen-free cats.
- Immunofluorescence microscopy and flow cytometry were used to detect the protein on the platelet surface.
- Biochemical analyses including SDS-PAGE, western immunoblotting, and immunoprecipitation were performed.
Main Results:
- A 140-kd membrane glycoprotein, recognized by anti-GPIb monoclonal antibodies, was detected on the surface of feline platelets.
- Feline GPIb demonstrated sensitivity to proteolysis, with fragments associating with the actin cytoskeleton.
- The proteolysis was influenced by calpain inhibitor concentrations.
Conclusions:
- Feline platelets express a 140-kd membrane protein homologous to GPIb.
- Quantifying glycocalicin, a GPIb fragment, using ELISA could offer insights into microvesicle production, platelet turnover, and proteolysis in cats.
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 microg/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.