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Structural and functional characterization of major platelet membrane components derived by limited proteolysis of
S Niewiarowski1, K J Norton, A Eckardt
1Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140.
Abstract:
The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.
Insights
Researchers identified a 66 kDa platelet membrane component resulting from glycoprotein IIIa (GPIIIa) proteolysis. This degraded GPIIIa fragment, formed by chymotrypsin, exhibits altered binding properties compared to intact GPIIIa.
Area of Science:
- Biochemistry
- Molecular Biology
- Platelet Physiology
Background:
- Glycoprotein IIIa (GPIIIa) is a key component of the GPIIb/IIIa complex on human platelets.
- Proteolytic degradation of platelet membrane proteins can alter their function.
- Understanding GPIIIa processing is crucial for platelet biology.
Purpose of the Study:
- To characterize the 66 kDa component, a product of GPIIIa proteolytic degradation.
- To investigate the relationship between GPIIIa degradation and fibrinogen receptor exposure.
- To identify intermediate products in GPIIIa proteolysis.
Main Methods:
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced and non-reduced conditions.
- N-terminal sequencing of protein subunits.
- Affinity chromatography using GRGDSPK-agarose, fibrinogen-agarose, and monoclonal antibodies.
- Platelet incubation with chymotrypsin, porcine pancreatic elastase, and human granulocytic elastase.
Main Results:
- A 66 kDa component, a proteolytic product of GPIIIa, was isolated and characterized.
- This component yielded two subunits (14-17 kDa and 65 kDa) upon reduction, linked by S-S bridges.
- The 66 kDa component showed reduced binding to fibrinogen and antibodies recognizing the GPIIb/IIIa complex.
- An intermediate 120 kDa (non-reduced) / 80 kDa (reduced) product was identified as a precursor.
- Similar fragments were generated by elastase treatment.
Conclusions:
- The 66 kDa component represents a significantly degraded form of GPIIIa with altered functional properties.
- Fibrinogen receptor exposure precedes the formation of this specific GPIIIa degradation product.
- GPIIIa undergoes complex proteolytic processing, yielding intermediate and final fragments with distinct characteristics.