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Membrane expression of platelet calpain
A H Schmaier1, H N Bradford, D Lundberg
1Hematology/Oncology Section, Temple University School of Medicine, Philadelphia, PA 19140.
Blood
|March 15, 1990
Summary
Platelet calpain II, a major platelet protein, is associated with platelet membranes. Upon activation by thrombin or platelet activating factor, calpain II externalizes to the platelet surface.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Platelet calpain is known to interact with platelet membrane proteins.
- The localization and membrane association of platelet calpain II in different platelet activation states require further investigation.
Purpose of the Study:
- To investigate the association of platelet calpain II with platelet membranes in both unstimulated and activated platelets.
- To determine if platelet calpain II externalizes upon platelet activation.
Main Methods:
- Competitive enzyme-linked immunosorbent assay (ELISA) to quantify calpain antigen.
- Enzyme activity assay using a fluorogenic substrate.
- Immunoblotting to detect calpain II heavy chain in cytoskeletons.
- Subcellular fractionation to determine calpain II distribution.
- Immunofluorescence and 125I-anti-calpain II Fab' binding assays to assess surface exposure.
Main Results:
- Platelet calpain II is a major platelet protein (2% of total protein) and is substantially membrane-associated.
- Calpain II is found in both cytosol and membrane fractions of platelets.
- While not present on the surface of unstimulated platelets, calpain II externalizes to the surface of thrombin-activated platelets.
- Dibucaine and platelet activating factor also induce calpain II externalization, but collagen and ionophore A23187 do not.
Conclusions:
- Platelet calpain II is a significant membrane-associated protein within platelets.
- Platelet activation, particularly by thrombin and platelet activating factor, leads to the externalization of calpain II antigen onto the platelet surface.