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Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation
Published on: April 6, 2017
Solubilization and characterization of a platelet membrane ADP-binding protein
The Journal of Biological Chemistry
|May 25, 1979
Summary
Researchers solubilized a platelet ADP-binding protein, crucial for ADP-induced platelet aggregation. This protein, distinct from nucleoside diphosphokinase activity, has a high affinity for ADP and may serve as the primary receptor.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Platelet membranes exhibit ADP-binding and nucleoside diphosphokinase activity.
- These activities are involved in transforming adenosine diphosphate (ADP) to adenosine triphosphate (ATP).
Purpose of the Study:
- To characterize the ADP-binding and nucleoside diphosphokinase activities of purified platelet membranes.
- To solubilize and purify the ADP-binding protein from platelet membranes.
Main Methods:
- Platelet membranes were solubilized using freeze-thaw and saline extraction.
- A Millipore filter binding assay with radiolabeled [3H]ADP was developed.
- Scatchard analysis, ultracentrifugation, and gel filtration were used to characterize the binding protein.
Main Results:
- Solubilization yielded a 4-fold purification of ADP-binding activity.
- The binding protein demonstrated rapid, reversible, saturable binding with a Kd of 3.8 x 10(-7) M.
- ADP-binding activity was separable from nucleoside diphosphokinase activity, suggesting distinct molecules.
- Hydrodynamic parameters indicated a molecular weight of 61,000 for the binding protein.
Conclusions:
- A soluble ADP-binding protein was purified from platelet membranes.
- This protein exhibits characteristics consistent with a receptor involved in ADP-induced platelet aggregation and release.
- The ADP-binding activity is distinct from the membrane-associated nucleoside diphosphokinase activity.
