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Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane
P W Modderman1, L G Admiraal, A Sonnenberg
1Department of Immunohematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Abstract:
Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.
Insights
Platelet glycoprotein V (GPV) and the GPIb-IX complex form a noncovalent bond in platelet membranes. This finding suggests GPV may influence platelet interaction with von Willebrand factor.
Area of Science:
- Biochemistry
- Hematology
- Cell Biology
Background:
- Platelet glycoprotein (GP) V is a plasma membrane protein with an unknown function.
- Platelet GPIb-IX complex is crucial for platelet adhesion via von Willebrand factor.
- Previous studies suggested an association between GPV and GPIb-IX due to their co-deficiency in Bernard-Soulier syndrome.
Purpose of the Study:
- To investigate the potential association between platelet GPV and the GPIb-IX complex.
- To elucidate the functional relationship between GPV and GPIb-IX in platelet membranes.
Main Methods:
- Coprecipitation assays using monoclonal antibodies against GPV, GPIb, or GPIX.
- Solubilization of platelets with digitonin and Nonidet P-40 detergents.
- Elastase treatment to remove the amino-terminal part of GPIb alpha.
- Quantification of GPV and GPIb-IX molecules per platelet using 125I-labeled monoclonal antibodies.
Main Results:
- GPV and GPIb-IX were coprecipitated, indicating they form a complex.
- The complex dissociated upon treatment with Nonidet P-40, suggesting noncovalent interaction.
- Removal of the amino-terminal region of GPIb alpha did not disrupt the GPV-GPIb-IX association.
- Quantitative analysis revealed approximately 24,370 GPIb-IX complexes and 11,170 GPV molecules per platelet.
Conclusions:
- Platelet glycoproteins GPV and GPIb-IX form a noncovalent complex within the platelet membrane.
- GPV's role in platelet adhesion, particularly in interaction with von Willebrand factor, is suggested.
- This finding provides new insights into platelet surface protein interactions and function.