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Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets
T H Mondoro1, B C Shafer, J G Vostal
1Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, FDA, Bethesda, MD, USA.
Free Radical Biology & Medicine
|January 1, 1997
Summary
Peroxynitrite causes protein tyrosine nitration and alters phosphorylation in platelets. This impairs platelet activation and reduces sensitivity to thrombin, particularly at lower concentrations.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Peroxynitrite (ONOO-) is known to modify proteins by nitrating tyrosine residues.
- This modification can interfere with cellular signaling pathways, including tyrosine phosphorylation.
- Platelets play a crucial role in hemostasis and thrombosis, and their activation is tightly regulated by signaling events.
Purpose of the Study:
- To investigate the impact of peroxynitrite on protein tyrosine nitration and phosphorylation in human platelets.
- To determine how peroxynitrite affects thrombin-induced platelet activation responses.
Main Methods:
- Gel-filtered human platelets were treated with peroxynitrite (ONOO-).
- Protein tyrosine nitration and phosphorylation levels were assessed using gel electrophoresis and Western blotting.
- Platelet activation markers, including P-selectin expression, serotonin secretion, and aggregation, were measured.
Main Results:
- Peroxynitrite induced significant tyrosine nitration of multiple platelet proteins, which was sustained over time.
- Peroxynitrite initially increased platelet tyrosine phosphorylation, but this effect was transient and decreased within 5 minutes.
- Pre-treatment with peroxynitrite reduced thrombin-induced tyrosine phosphorylation and key platelet activation responses (P-selectin expression, serotonin secretion, aggregation) at lower thrombin concentrations.
- These effects suggest that peroxynitrite exposure and subsequent tyrosine nitration decrease platelet sensitivity to thrombin.
Conclusions:
- Peroxynitrite modifies platelet proteins through tyrosine nitration and impacts tyrosine phosphorylation dynamics.
- Peroxynitrite impairs platelet activation responses and reduces platelet sensitivity to thrombin, suggesting a role in modulating platelet function.