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 glyco protein Ib alpha, (GpIb alpha), which binds with high affinity to alpha -th rombin. Recent studies have shown that the thrombin domain referred to as h eparin binding site (HBS) is involved in the interaction with the platelet GpIb alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to GpIb alpha, we have performed ala nine 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 diffe rent regions of the domain. For comparison, mutation at R67 within the fibr inogen recognition site (FRS) of thrombin was performed as well. Solid-phas e binding experiments showed that the K-d of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R 233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutan t R233A/K236A/Q239A, while the Q239A and R67A forms did not show any signif icant affinity change. The platelet activating capacity of these mutants wa s evaluated as well. Using gel-filtered platelets, the EC50 value of thromb in-induced aggregation was front 5- to 13-fold higher in the HBS mutants th an in the WT form, and was linearly and positively correlated with the corr esponding K-d values pertaining to thrombin binding to GpIb. Measurements o f PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R23 3A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the app arent k(cat)/K-m value. These results are a consequence of a defective bind ing 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 k(cat)/K -m value pertaining to the hydrolysis by the HBS-mutated thrombins of the s ynthetic PAR-1 38-60 peptide in solution was similar to that one obtained w ith the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding t o the platelet GpIb alpha and in the cell activation.