Inhibition of Platelets Aggregation by [D‐Phe‐Pro‐D‐Arg‐P1′‐CONH2] Peptides Inhibitors of Thrombin

A structure‐activity relationship (SAR) for reversible inhibitory activity toward thrombin of tetrapetides from series D‐Phe‐Pro‐D‐Arg‐P1′‐CONH2 is reported. The P1′ position was varied with D and L amino acids. There are significant differences between the inhibitory constants (Kis) of tetrapeptides from the series D‐Phe‐Pro‐D‐Arg‐P1′‐CONH2 suggesting that the interaction between the amino acid at P1′ position and the S1′ subpocket in thrombin is very specific. A 2 to 500 fold difference between the inhibitory constants of tetrapeptide inhibitors was determined using an in vitro assay for thrombin inhibition combined with isothermal titration calorimetry (ITC). The SAR studies suggests that P1′ position requires small hydrophobic and polar amino‐acids. In addition, a switch from L‐Thr into D‐Thr in P1′ was correlated with a 13 fold increase in the peptides inhibitory activity. Functional assays, such as thrombin‐activated platelets aggregation showed that the peptides were effective in inhibiting the platelets aggregation completely at concentration 5–15 fold their Ki determined from in vitro inhibition assays. These results strongly support our original structure‐based design of peptides as reversible inhibitors for thrombin and propose these tetrapeptides as very potent anticoagulants.