A naturally homozygous deletion mutation of LYS9 or LYS10 in the A-chain of alpha-thrombin reduces the catalytic efficiency of the enzyme
Abstract number: P1211a
Akhavan* S., De Cristofaro R., Peyvandi* F., Mannucci* P. M.
*Angelo Bianchi Bonomi, University of Milan, Italy; Catholic University School of Medicine, Italy
We have recently identified a homozygous deletion mutation of one of the two contiguous Lys9/Lys10 residues in the A-chain of a -thrombin (FII-Del) in two apparently unrelated patients with a severe prothrombin deficiency and hemorrhagic diathesis. The level of prothrombin antigen measured in plasma was 15%, while the coagulant activity ranged from < 1% to 2.5%. To clarify the role of the deletion of Lys 9 or 10 in the thrombin activity, we used in vitro expression analysis. The wild-type prothrombin (FII-WT) and mutant cDNAs were transiently expressed in COS-7 cells. The FII-Del antigen level in the cell conditioned media reflected the patients, plasma pattern. Stable transfected CHO cell lines containing either FII-WT or FII-Del cDNAs permitted us to obtain a single clone stably transfected with each construct and expressing high levels of prothrombin for further functional experiments. Activation of FII-Del could be accomplished by Taipan snake venom only, as ecarin venom failed to activate the zymogen. Steady state kinetic experiments showed that the association rate constant of FIIa-Del with the synthetic substrate Phe-Pip-Arg-pNA decreased from 1.3 × 108 M1 sec1-2.7 × 106 M-1 s-1. The kcat value of the substrate hydrolysis at 25 °C decreased from 90 s-1 in the FIIa-WT to 4 s-1 in the FIIa-Del, whereas the Km values were comparable (1.9 and 1.6 µM, respectively). The kcat/Km value of fibrinopeptide A release by FIIa-Del was about 70 fold lower than in FIIaWT. Interaction with ATIII was also reduced, being the association rate value about 60-fold lower than in the FII-WT. The sensitivity to sodium ion of the mutated thrombin form was found significantly attenuated compared to the FIIa-WT form, suggesting a more stiff conformational state in FIIa-Del. Finally, the mutated thrombin has a very weak platelet activating capacity, attributed to a defective PAR-1 interaction, as the specificity of the PAR-1 3860 peptide hydrolysis was about 100 fold lower than in FIIa-WT. These experiments show that the deletion of Lys 9/10 in the A-chain of thrombin significantly impairs its catalytic machinery, likely through long-range conformational effects on the catalytic site residues.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2003; 1 Supplement 1 July: abstract number
|Subject:||Thrombin, thrombin receptors|
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