Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib

The activation of human platelets by alpha-thrombin is mediated in part by cleavage of the protease-activated receptor (PAR) 1 and 4 and by the glycoprotein (Ib)alpha, (Gp(Ib)alpha), which binds with high affinity to alpha-thrombin. Recent studies have shown that the thrombin domain referred to as heparin binding site (HBS) is involved in the interaction with the platelet Gp(Ib)alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to Gp(Ib)alpha, we have performed alanine scanning mutagenesis of the basic HBS R93, R97, R101, R233, K236, K240, R233/K236/Q239, as well as of the neutral Q239 residues, located in different regions of the domain. For comparison, mutation at R67 within the fibrinogen recognition site (FRS) of thrombin was performed as well. Solid-phase binding experiments showed that the Kd of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutant R233A/K236A/Q239A, while the Q239A and R67A forms did not show any significant affinity change. The platelet activating capacity of these mutants was evaluated as well. Using gel-filtered platelets, the EC50 value of thrombin-induced aggregation was from 5- to 13-fold higher in the HBS mutants than in the WT form, and was linearly and positively correlated with the corresponding Kd values pertaining to thrombin binding to GpIb. Measurements of PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R233A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the apparent kcat/Km value. These results are a consequence of a defective binding to GpIb, which is known to optimize the interaction with PAR-1 in situ. A confirm of this hypothesis came from the demonstration that the kcat/Km value pertaining to the hydrolysis by the HBS-mutated thrombins of the synthetic PAR-1 38-60 peptide in solution was similar to that one obtained with the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding to the platelet Gp(Ib)alpha and in the cell activation.