Characterization of proexosite I on prothrombin

Activation of prothrombin by factor Xa is accompanied by expression of regu latory exosites I and II on the blood coagulation proteinase, thrombin, Qua ntitative affinity chromatography and equilibrium binding studies with a fl uorescein-labeled derivative of the exosite I-specific peptide ligand, hiru din(54-65) ([5F]Hir(54-65) (SO3-), were employed to identify and characteri ze this site on human and bovine prothrombin and its expression on thrombin . [5F]Hir(54-65)(SO3-) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human p rothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 mu M and 25 +/- 2 nM, respectively, demonstrating a 130-fold increase in affinity f or the active proteinase. The bovine proteins similarly showed a 150-fold h igher affinity of [5F]Hir(54-65)(SO3-) for thrombin compared with prothromb in, despite a 2-5-fold lower affinity of the peptides for the bovine protei ns. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound comp etitively with [5F]Hir(54-65)(SO3-) to human and bovine prothrombin and thr ombin, exhibiting similar, 40-70-fold higher affinities for the proteinases , although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovi ne prothrombin that is present in a conformationally distinct, low affinity state and is activated with a similar to 100-fold increase in affinity whe n thrombin is formed.