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PAR-3 is a low-affinity substrate, high affinity effector of thrombin.
1Department of Biochemistry and Molecular Biology, Mayo Foundation for Medical Education and Research, Rochester, MN 55905-0002, USA. wgo@mayo.edu
Summary
Protease-activated receptor 3 (PAR-3) binds thrombin in a unique way, affecting its activity. Low concentrations of PAR-3 allosterically modulate thrombin, impacting substrate hydrolysis and fibrinogen clotting.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Thrombin, a key enzyme in coagulation, interacts with various substrates.
- Protease-activated receptors (PARs) are G protein-coupled receptors activated by proteolysis.
- The interaction between thrombin and PAR-3 has been previously characterized as slow hydrolysis.
Purpose of the Study:
- To investigate the detailed mechanism of thrombin's interaction with PAR-3.
- To elucidate the allosteric effects of PAR-3 on thrombin's enzymatic activities.
- To compare the effects of PAR-3 with PAR-4 on thrombin function.
Main Methods:
- Enzyme kinetics assays measuring substrate hydrolysis rates.
- Thrombin activity assays including fibrinogen clotting.
- Characterization of thrombin-PAR-3 binding interactions.
Main Results:
- Thrombin exhibits biphasic binding to PAR-3, with slow hydrolysis of one bound molecule.
- PAR-3 enhances nitroanilide substrate hydrolysis in Na+-free conditions (1.6-fold, K(1/2)=2.5 microM).
- PAR-3 completely inhibits thrombin's fibrinogen clotting activity (K(I)=3 microM) by blocking the anion-binding exosite.
Conclusions:
- PAR-3 acts as an allosteric modulator of thrombin, distinct from simple substrate hydrolysis.
- Low concentrations of PAR-3 induce a conformational change in thrombin, affecting substrate access and activity.
- The allosteric modulation by PAR-3 resembles the effect of Na+ on thrombin's activity.