Na؉ binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na ؉ site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na ؉ binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na ؉ . Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G-protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90-fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies.

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Human fibrinogen 1 is homodimeric with respect to its ␥ chains ('␥ A -␥ A '), whereas fibrinogen 2 molecules each contain one ␥ A (␥ A 1-411V) and one ␥ chain, which differ by containing a unique C-terminal sequence from ␥408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the ␥ chains. Second, cross-linking of fibrin ␥ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to ␥ chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to ␥ chains. IntroductionFibrinogen is a multidomain disulfide-linked protein composed of symmetric halves, each consisting of 3 polypeptide chains termed A␣, B␤, and ␥. 1 Human fibrinogen can be separated by ion exchange chromatography into 2 major fractions, fibrinogen 1 (peak 1 fibrinogen) and fibrinogen 2 (peak 2 fibrinogen). 2,3 Plasma fibrinogen contains approximately 15% fibrinogen 2. Structurally, the 2 fibrinogens differ from each other with respect to the composition of their ␥ chains. Fibrinogen 1 contains 2 ␥ A chains, each composed of 411 amino acids, whereas heterodimeric fibrinogen 2 molecules each contain one ␥ A and one ␥Ј chain. 3,4 The variant ␥Ј chain is longer (427 residues) and has a more anionic, carboxyl terminal sequence than the ␥ A chain beyond position 408. 4 Alternative mRNA splicing at the exon 9-exon 10 boundaries gives rise to the variant ␥Ј chain. 5 Thrombin binds to fibrinogen at the substrate site through its exosite 1, 6-8 thereby mediating cleavage of fibrinopeptide A 9-12 and slower cleavage of fibrinopeptide B. 13,14 Fibrin assembly commences with the formation of double-stranded twisting fibrils in which fibrin molecules are arranged in a staggered, overlapping manner. 15 Subsequently, lateral fibril associations occur, resulting in thicker fibrils and fibers. Concomitant with converting fibrinogen to fibrin, thrombin activates factor XIII to factor XIIIa. [16][17][18][19][20][21] In the presence of factor XIIIa and Ca 2ϩ , fibrin undergoes intermolecular covalent cross-linking by the formation of ⑀-amino(␥-glutamyl) lysine isopeptide bonds. 22 Generally speaking, intermolecular cross-linking occurs rapidly between ␥ chains to form ␥-dimers and more slowly among ␣ chains to create oligomers and larger ␣ chain...

Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K ؉ , whereas a few of them show preferential activation by Na ؉ . The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na ؉ -activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K ؉ -specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K ؉ -activated enzyme.

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na ؉ . The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na ؉ -activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsinlike proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na ؉ to the smaller cation Li ؉ or to the larger cation Rb ؉ , respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na ؉ (Asp-189) to Li ؉ (Ser-189) or Rb ؉ (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na ؉ site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.

The anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/ Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity. IntroductionSystemic anticoagulants can completely interrupt thrombus formation, but their usefulness is limited because their antithrombotic and antihemostatic activities are mechanistically tied. Highly effective plasma concentrations of systemic anticoagulants, such as those used for temporary thrombo-prophylaxis in interventional cardiology, prevent thrombin generation in both the blood vessel and the wound and can paralyze hemostasis with potentially fatal consequences. Due to safety considerations, the vast majority of patients who receive antithrombotic treatment are not anticoagulated to full efficacy. Thrombotic blood vessel occlusions causing myocardial infarction and ischemic stroke thus continue to contribute to mortality statistics. 1 Until more thrombosis-specific intravascular anticoagulants become available, balancing the potential benefits and risks of systemic anticoagulation remains a critical hurdle for the clinician.Occlusive thrombus formation is naturally down-regulated without bleeding complications most of the time following thrombogenic stimuli, such as blood vessel injuries or infections. It thus seems possible that selective pharmacological enhancement of the natural antithrombotic systems could achieve thrombosis specificity. Enhancement of the endogenous protein C pathway in the vicinity of thrombus formation might offer this wider therapeutic window. Thrombin is the natural protein C activator enzyme that activates protein C on such anatomic surfaces as the endothelial lining of blood vessels. ...