Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.     

Binding of hirudin to meizothrombin

Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.     

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.     

Identification and characterization of the thrombin binding sites on fibrin

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing gamma' chains, which are fibrinogen gamma chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 microM respectively). The presence of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on gamma' chains is a constitutive feature of fibrin as well as fibrinogen.     

Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist

A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing "tethered-ligand" then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn- Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by alpha-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of gamma-thrombin, and the alpha- and zeta-thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of alpha-thrombin involved in human platelet activation.     

Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases

In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, factor Va, and to release the activation fragments E and C1. For the identification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobilizing thrombin and other serine proteinases specifically (>/=92%) through their active sites and used in affinity chromatography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation pathway from the 93% +/- 12% inhibition of the rate of activation correlated with specific binding of hirudin54-65 to this exosite. Chromatography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically and with apparently higher affinity, in an interaction that was prevented by hirudin54-65. The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and fragment E had no detectable affinity. These results demonstrate a previously undescribed, exosite I-dependent interaction of thrombin with factor Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombin to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteolytic activation of the protein cofactor.     

Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.     

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.     

Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting

BACKGROUND: Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis more effectively. METHODS AND RESULTS: We first studied antithrombotic potency in vitro, comparing fibrin-targeted hirudin (recombinant hirudin covalently linked to the Fab' fragment of the anti-fibrin monoclonal antibody 59D8) to recombinant hirudin in baboon plasma. Fibrin-targeted hirudin was nine times more effective than recombinant hirudin in inhibiting fibrin deposition on experimental clot surfaces in baboon plasma (P < .01). The potency of fibrin-targeted hirudin was then compared with that of recombinant hirudin in a baboon model of thrombus formation. 111In-labeled platelet deposition was measured in a synthetic graft segment of an extracorporeal arteriovenous shunt in control animals and in animals receiving either fibrin-targeted hirudin or hirudin. In these experiments, fibrin-targeted hirudin was 10-fold more potent than hirudin in inhibiting platelet deposition and thrombus formation (P < .05). CONCLUSIONS: These data indicate that targeting a thrombin inhibitors such as hirudin to an epitope present in thrombi results in increased antithrombotic potency.     

CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3

To elucidate the differential roles of N- and C-terminal halves of hirudins in thrombin inhibition, we produced novel recombinant hirudin analogs, CX-397 and CX-397R, having a hybrid amino acid sequence of hirudin variants-1 (HV-1) and -3 (HV-3). CX-397 is composed of the N-terminal half of HV-1 (HV-11-36) combined with the C-terminal half of HV-3 (HV-337-66). CX-397R is the opposite combination. Their anti-thrombin activity was determined by a fluorogenic enzyme assay and compared with that of recombinant HV-1 (rHV-1) and rHV-3. The order of the magnitude of dissociation constants (Ki) of these four hirudin analogs in thrombin inhibition was as follows: CX-397R (0.294 pM) > rHV-1 (0.148 pM) > rHV-3 (0.0593 pM) > CX-397 (0.0433 pM), indicating that CX-397 is the strongest inhibitor among them.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.     

Protein engineering thrombin for optimal specificity and potency of anticoagulant activity in vivo

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.     

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.     

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.     

Thrombin induces S-D-lactoylglutathione accumulation by enhancing platelet glycolytic pathway

1. Thrombin addition to human platelets stimulates L(+)lactate formation and S-D-lactoylglutathione (SDL) accumulation. 2. Monoiodoacetamide decreases lactate formation and potentiates SDL accumulation through a significant increase of dihydroxyacetone phosphate and fructose1,6bisphosphate intracellular levels both in resting and in activated platelets. 3. A similar effect is produced by exogenous methylglyoxal on L(+)lactate formation and SDL accumulation. 4. Resting platelets completely transform (1 hr at 37 degrees C) the ketoaldehyde into D(-)lactate: 5. When platelets are incubated in the presence of thrombin only 60% of the ketoaldehyde is found as D(-)lactate and the accumulated S-D-lactoylglutathione represents about the 0.7% of the initial substrate. 6. During platelet stimulation with thrombin the hemithioacetal adduct, formed as a by-product of glycolytic pathway, can be rapidly removed for important steps of cellular activation.     

Experimental kallikrein gene therapy in hypertension, cardiovascular and renal diseases

Argatroban, a direct thrombin inhibitor, is used clinically because of its safe and effective antithrombotic action. This drug of low molecular weight shows reversible inhibition of thrombin irrespective of whether thrombin is fibrin-bound or soluble. Optimal anticoagulant effects can easily be attained by monitoring with the activated partial thromboplastin time or whole-blood activated clotting time when a therapeutic range of argatroban equivalent to that of heparin is used. The antithrombotic action is simply detected with a chromogenic substrate assay. The clinical use of the drug in Japan was approved for the treatment of chronic peripheral arterial obstructive disease and acute ischemic stroke. For coronary artery disease in patients with deficiency of antithrombin activities attributable to either antithrombin III or heparin cofactor II deficiency, argatroban is effective as an anticoagulant. Acute coronary occlusion during and after percutaneous transluminal coronary angioplasty can be treated by argatroban as an alternative to heparin. The presence of platelets activated by a trace amount of thrombin is evidenced by modified methods of platelet aggregometry in acute ischemic stroke. Therefore, argatroban can render the platelets insensitive against the platelet hyperaggregation enhanced by thrombin.     

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.     

The clinical course of asymptomatic mesenteric arterial stenosis

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied. Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50) of 14 +/- 3 nM compared to an IC50) of 12 +/- 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 +/- 0.4 nM compared to 1.6 +/- 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50) = 69 +/- 9 mU/ml) than against thrombin in solution (IC50 = 15 +/- 5 mU/ml). This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.