Binding of Exosite Ligands to Human Thrombin

Abstract

The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. The specificity of thrombin toward its procoagulant and anticoagulant physiological substrates is allosterically regulated by interactions of macromolecular substrates, inhibitors, and effectors with either of two electropositive sites, exosites I and II, in near-opposition on the enzyme surface (1, 2). Exosite I binds fibrinogen (Fbg) 1 (3), fibrin I and II (3, 4), the 12-residue carboxyl-terminal hirudin 54 -65 sequence (5, 6), thrombomodulin (7), the thrombin receptor (8, 9), and an acidic sequence on the serpin, heparin cofactor II (10, 11). Exosite II binds heparin and other glycosaminoglycans (2, 12, 13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15, 16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18). Factors V (19 -22), Va (21,22), and VIII (19), platelet glycoprotein Ib␣ (23-25), and the snake venom protein bothrojaracin (26) have been reported to interact with both exosites I and II.Binding of exosite ligands to thrombin is correlated with significant changes in the kinetics of hydrolysis of peptide ester and peptide p-nitroanilide substrates (3,7,9,18,(27)(28)(29)(30)(31) in addition to profound effects on specificity and reactivity toward its natural macromolecular substrates and inhibitors (10,11,15,(32)(33)(34)(35)(36). These studies indicate that exosite binding of allosteric effectors is coupled to conformational changes affecting the S1-S3 substrate specificity subsites in the thrombin catalytic site (37-39). Binding studies of F2, thrombomodulin, fibrin, and heparin with various active site-labeled thrombin derivatives in which the S1-S4 subsites were occupied (16,34,40), and studies of the effect of exosite ligand binding on the hydrolysis of tripeptide p-nitroanilide substrates suggest that structurally different ligands produce ligand-specific changes in the catalytic site. Extreme allosteric linkage between exosites I and II (30,31,41) has been reported to prevent simultaneous occupation of exosites I and II (30,41), whereas other studies provide contrasting evidence for binary and ternary complex formation with similar affinities among thrombin and exosite I and II ligands (18). In favor of inter-exosite linkage, the dissociation constant for fluorescently labeled, Tyr 63 -sulfated hirudin [53][54][55][56][57][58][59][60][61][62][63][64] and bovine thrombin was weakened 10-fold by F2 binding, although ternary complex formation was demonstrated (31). Extremely negative interexosite interactions were reported for Tyr 63 -sulfated hirudin 54 -65 (Hir 54 -65 (SO 3 Ϫ )) and human F2 or a synthetic peptide...

Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

“…Spectra were collected and analyzed as described under "Experimental Procedures." same as previously determined (37,45). Competitive titrations of F2 binding to labeled thrombin and unlabeled Pre 2 gave a dissociation constant of 3 Ϯ 1 M for F2 binding to Pre 2, which represented a small (ϳ2-fold) and probably insignificantly higher affinity for Pre 2 in comparison to thrombin (Fig.…”

“…The novel conformation of the autolysis loop in MzT(-F1) has also been suggested to inhibit the interaction with fibrinogen (51). In the case of fibrinogen, however, previous studies have found that the active site-blocked human thrombin⅐F2 complex and MzT(-F1) bind fibrinogen through exosite I with only 2-to 4-fold lower affinity than thrombin (45). Together, the above results indicate that access of a variety of macromolecules to exosite I on MzT(-F1) is only modestly restricted.…”

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

“…The effect of ␥Ј peptide on PAR4 cleavage would be negligible because this latter substrate does not bind to ABE-I. This working hypothesis is worthy of attention in the light of the proposed, but still debated, allosteric linkage existing between ABE-I and ABE-II (25,26). To test this hypothesis, we investigated the effect of ␥Ј on the binding of the fluorescein-conjugated C-terminal 54 -65 peptide of hirudin, [F]-hirudin 54 -65 (PO 3 H 2 ), a well known ligand for ABE-I (52).…”

“…54 -65 (PO 3 H 2 ), having the sequence GDFEEIPEEY(PO 3 H 2 )LQ, was synthesized as described previously (24). Binding of this peptide to ABE-I of thrombin was studied by monitoring the decrease of the peptide fluorescence occurring upon interaction with thrombin, as reported previously (25). Fluorescence spectra were recorded on a Jasco (Tokyo, Japan) spectrofluorometer, as detailed above.…”

Crystal Structure of Anticoagulant Thrombin Variant E217K Provides Insights into Thrombin Allostery

“…Thrombin interacts with many cofactors capable of inducing conformational change and altered protease activity; however, the physiological significance of thrombin allostery is unclear (7)(8)(9)(10). The most relevant alteration of thrombin activity is caused by its binding to TM, but similar changes can also be induced by other cofactors.…”