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Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin 54 -65 (SO 3 ؊ ) to human and bovine prothrombin and thrombin, 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 bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a ϳ100-fold increase in affinity when thrombin is formed.Thrombin is generated in the penultimate step of the blood clotting cascade through activation of the zymogen, prothrombin, by the proteinase factor Xa in reactions regulated by phospholipid membrane surfaces, calcium, and the protein cofactor, factor Va (1). Cleavage of two peptide bonds in prothrombin by factor Xa activates the serine proteinase catalytic domain (prethrombin 2) and releases it as thrombin along with prothrombin activation fragments 1 and 2 (1). Activation of prothrombin is accompanied by the expression of regulatory exosites I and II on thrombin (2-4). Exosite I has been well characterized structurally and functionally on thrombin as an electropositive site that binds fibrinogen (5), thrombomodulin (6), the platelet thrombin receptor (7), factor V and Va (8 -10), heparin cofactor II (11), and COOH-terminal hirudin peptides and their analogs specifically (12)(13)(14). This site plays a critical role in mediating the binding of these and other specific protein substrates, inhibitors, and macromolecular effectors to thrombin (2, 15, 16). The characteristics of this site on prothrombin and prothrombin activation intermediates, however, are not clearly established. Equilibrium binding studies with a fluorescein-labeled hirudin peptide, hirudin 53-64 1 (14), as an exosite I-specific probe showed that bovine prothrombin had no detectable affinity for the peptide (3). Expression of exosite I on thrombin was concluded to result from conformational changes that accompany either of the two factor Xa cleavages that give rise to the alternate prothrombin activation intermediates, prethrombin 2 and meizothrombin (3). Contrasting the results for hirugen, an anti-exosite I antibody and thrombomodulin showed no detectable affinity for the human prothrombin activation intermediates, whereas these ligands bind to exosite I of thrombin specifically (4). Binding to human prothrombin of a nonsulfated hirudin peptide analog, N-acetyl-Hir 55-65 , containing a Gly substitution at residue 65 was demonstrated by NMR, but the peptide bound to prothrombin with an estimated dissociation constant of ϳ500 M, indicating a very low affinity of uncertain significance (17). Although there is good agreement that the affin...

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Hole is a novel gene product isolated from a chick heart subtractive hybridization. Hole is a six-transmembrane protein (predicted size 311 and 317 amino acids in chick and mouse) expressed in the cardiac crescent and later in the myocardium of the developing chick heart, as well as in the fusing neural tube and ganglia. Mouse hole is not expressed in the developing heart, although it does share neural expression seen in the chick.

Inhibition of Trypanosoma cruzi Epimastigotes in vitro by Iron-Chelating Agents.-Twenty new iron-chelating agents are investigated for their trypanosoma cruzi Epimastigotes inhibiting activities. Imines of type ( VI) are generally inactive, whereas the hydroxypyridones (III) show inhibitory activity comparable to the clinically used benznidazole ( VII). -(JONES, M. M.; SINGH, P. K.; LANE, J. E.; RODRIGUES, R. R.; NESSET, A.; SUAREZ, C. C.; BOGITSH, B.