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Affinity modulation in platelet alpha 2 beta 1 following ligand binding
N Gofer-Dadosh1, A Klepfish, H Schmilowitz
1Hemostasis Research Laboratory, Rabin Medical Center, Petah Tiqva, Israel.
Biochemical and Biophysical Research Communications
|March 27, 1997
Summary
Platelet alpha-2 beta-1 receptors bind collagen with higher affinity after ligand binding. This interaction involves a new, intrareceptor disulfide bond, enhancing receptor-ligand interactions.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Platelet adhesion to collagen is crucial for hemostasis.
- The alpha-2 beta-1 integrin is a key receptor mediating platelet-collagen interactions.
- The precise mechanism of affinity modulation for alpha-2 beta-1 remains incompletely understood.
Purpose of the Study:
- To investigate ligand-induced changes in the affinity of platelet alpha-2 beta-1 for collagen.
- To identify the molecular mechanisms underlying enhanced alpha-2 beta-1 receptor-ligand binding.
Main Methods:
- Labeled viable platelets were passed through fibrillar collagen columns.
- Stringent lysis conditions were used to isolate high-affinity receptor fractions.
- Elution with DTT and SDS, followed by Western blotting and antibody-based functional assays.
- Investigated elution with EDTA, chaotropic agents, low pH, DTT, and acetic acid.
Main Results:
- A high-affinity fraction of alpha-2 beta-1 was eluted using DTT and SDS.
- Antibodies against this fraction inhibited collagen-specific platelet adhesion and aggregation.
- EDTA, chaotropic agents, or low pH did not elute the high-affinity fraction.
- DTT followed by acetic acid successfully eluted the high-affinity fraction.
Conclusions:
- Ligand binding induces the formation of a new, intrareceptor disulfide bond in a fraction of alpha-2 beta-1.
- This disulfide bond formation increases the receptor's affinity for its ligand (collagen).
- This mechanism contributes to the functional regulation of platelet-collagen interactions.