Pulsed cavitational therapy using high-frequency ultrasound for the treatment of deep vein thrombosis in an in vitro model of human blood clot.

Post-thrombotic syndrome, a frequent complication of deep venous thrombosis, can be reduced with early vein recanalization. Pulsed cavitational therapy (PCT) using ultrasound is a recent non-invasive approach. We propose to test the efficacy and safety of high-frequency focused PCT for drug-free thrombolysis (thrombotripsy) in a realistic in vitro model of venous thrombosis. To reproduce venous thrombosis conditions, human whole blood was allowed to clot by stasis in silicone tubes (6 mm internal diameter) at a 30 cm H2O pressure, maintained during the whole experiment. We engineered an ultrasound device composed of dual 2.25 MHz transducers centered around a 6 MHz imaging probe. A therapeutic focus was generated at a 3.2 cm depth from the probe. Thrombotripsy was performed by longitudinally scanning the thrombus at three different speeds: 1 mm s-1 (n = 6); 2 mm s-1 (n = 6); 3 mm s-1 (n = 12). Restored outflow was measured every three passages. Filters were placed to evaluate the debris size. Twenty-four occlusive thrombi, of 2.5 cm mean length and 4.4 kPa mean stiffness, were studied. Flow restoration was systematically obtained by nine subsequent passages (4.5 min maximum). By varying the device's speed, we found an optimal speed of 1 mm s-1 to be efficient for effective recanalization with 90 s (three passages). Within 90 s, flow restoration was of 80, 62 and 74% at respectively 1, 2 and 3 mm s-1. For all groups, cavitation cloud drilled a 1.7 mm mean diameter channel throughout the clot. Debris analysis showed 92% of debris <10 µm, with no fragment > 200 µm.

Multimodal assessment of non-specific hemostatic agents for apixaban reversal.

BACKGROUND: Non-specific hemostatic agents, namely activated prothrombin complex concentrate (aPCC), PCC and recombinant activated factor (F) VII (rFVIIa), can be used, off-label, to reverse the effects of FXa inhibitors in the rare cases of severe hemorrhages, as no approved specific antidote is available. We have evaluated the ability of aPCC, PCC and rFVIIa to reverse apixaban. METHODS: Healthy volunteer whole blood was spiked with therapeutic or supra-therapeutic apixaban concentrations and two doses of aPCC, PCC or rFVIIa. Tests performed included a turbidimetry assay for fibrin polymerization kinetics analysis, scanning electron microscopy for fibrin network structure observation, thrombin generation assay (TGA), thromboelastometry, prothrombin time and activated partial thromboplastin time. RESULTS: aPCC generated a dense clot constituting thin and branched fibers similar to those of a control without apixaban, increased fibrin polymerization velocity and improved quantitative (endogenous thrombin potential and peak height) as well as latency (clotting and lag times) parameters. Adding PCC also improved the fibrin and increased quantitative parameters, but fibrin polymerization kinetics and latency parameters were not corrected. Finally, rFVIIa improved latency parameters but failed to restore the fibrin network structure, fibrin polymerization velocity and quantitative parameters. CONCLUSION: aPCC was more effective than PCC or rFVIIa in reversing in vitro the effects of apixaban. aPCC rapidly triggered the development of an apparently normal fibrin network and corrected latency and quantitative parameters, whereas PCC or rFVIIa had only a partial effect.

Recombinant activated factor VII and prothrombin complex concentrates have different effects on bleeding and arterial thrombosis in the haemodiluted rabbit.

BACKGROUND: Recombinant activated factor VII (rFVIIa) is indicated in bleeding patients when a life-threatening haemorrhage occurs. Prothrombin complex concentrates (PCCs) are also used for this indication in several countries, without any evidence-based rationale. Our objective was to compare the efficacy and safety of PCC and rFVIIa in a model of bleeding and thrombosis in haemodiluted rabbits. METHODS: Forty-eight rabbits were randomly allocated into four groups: a control group and three treatment groups, in which animals were haemodiluted with hydroxyethyl starch 130/0.4 then administered either placebo, 160 µg kg(-1) rFVIIa, or 25 IU FIX kg(-1) PCC. The primary endpoint was hepatosplenic (HS) blood loss. Secondary endpoints were: (i) ear immersion bleeding time (IBT); (ii) thrombosis risk assessed by cyclic flow reductions (CFRs) of the carotid artery; and (iii) activated partial thromboplastin time (aPTT), and progress of thrombin activity. RESULTS: Haemodilution increased HS blood loss by 80% from 8 g (5-16) (control group) to 14 g (8-45) (placebo group) (P<0.01). HS blood loss was not different in animals receiving either rFVIIa [10 g (7-22)] or PCC [15 g (4-33)] (P<0.05) compared with the placebo group. Ear IBT was reduced with both rFVIIa and PCC. CFRs disappeared after haemodilution and were not restored with any treatment. Although PCC nearly doubled the total amount of thrombin generated, no significant change in the total amount of thrombin was seen in animals treated with rFVIIa. CONCLUSIONS: Neither rVIIa nor PCC reduced HS blood loss, whereas they both controlled the bleeding time, without increasing the thrombosis risk.

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules.

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.

Dose-effect relationship for several coagulation markers during administration of the direct thrombin inhibitor S 18326 in healthy subjects.

AIMS: We conducted a phase I placebo-controlled trial with two i.v. doses (0.5 mg h-1 and 3 mg h-1) of S 18326, a selective thrombin inhibitor that interacts with the catalytic site of thrombin, with the aim to study the relationships between increasing plasma levels of S 18326 and changes in coagulation tests and thrombin generation markers. METHODS: Thirty-six healthy male volunteers were divided into three groups. In each group, 10 volunteers were randomly assigned to receive S 18326 and two to receive a placebo. Following a bolus of 4.5 mg, doses were 0.5 mg h-1 in the first group and 3 mg h-1 in the two other groups, administered as an i.v. infusion for 24 h. Blood was drawn repeatedly up to 36 h after the bolus, and tested for the activated clotting time (ACT) and activated partial thromboplastin time (APTT). The APTT reagent was chosen among five commercial reagents to yield a linear increase in the clotting time among possible therapeutic S 18326 concentrations in vitro. To accurately measure the thrombin-inhibiting effects of low doses of S 18326 (< 0.5 microm), we developed a specific chromogenic assay. We also measured F1 + 2 prothrombin fragment levels to assess the effect of S 18326 on thrombin generation in vivo. RESULTS: A two-compartment pharmacokinetic model was fitted to the S 18326 plasma concentration vs time data by using population pharmacokinetic methods. Results of the pharmacodynamic-pharmacokinetic relationships showed that both the ACT and APTT methods yielded a linear increase according to the S 18326 concentration measured using a highly sensitive analytical method. At the end of infusion, ACT was prolonged 1.20 and 1.95-fold in the 0.5 mg h-1 and the 3 mg h-1 groups, respectively, and APTT was prolonged 1.27 and 2.75-fold. Thrombin inhibition plateaued above 0.5 microm of S 18326 according to an Emax model, confirming that the test was highly sensitive. F1 + 2 levels fell significantly after the 24 h S 18326 infusion (0.83 nm to 0.6 nm and 0.80 nm to 0.44 nm in the 0.5 mg h-1 and the 3 mg h-1 groups, respectively), but remained stable after the placebo infusion. CONCLUSIONS: Our results support specific monitoring of the thrombin inhibitor S 18326 with ACT and APTT to establish the safety range of the drug in further studies. Moreover, the fall in F1 + 2 prothrombin fragments suggests that S 18326 effectively reduces the retroactivation of factors V and VIII by thrombin.

Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the gamma-carboxyglutamic acid-rich domain.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy.

The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombin(S195A)) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys(232), Cys(P3'), and Cys(313)) were covalently modified with the fluorescence probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change upon forming a complex with either active or inactive proteinase. Steady-state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombin(S195A) is also able to induce a conformational change in the serpin.

The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1.

The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.

Cleaved antitrypsin polymers at atomic resolution.

Alpha1-antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of alpha1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that alpha1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the beta-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a beta-strand linked polymer form of alpha1-antitrypsin: the crystal structure of a cleaved alpha1-antitrypsin polymer.

The role of Glu(192) in the allosteric control of the S(2)' and S(3)' subsites of thrombin.

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir(52-65)) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S(2)' and S(3)' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P(2)' and P(3)'. Regardless of the amino acids, Hir(52-65) decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu(192) participates in this modulation. We have examined the role of Glu(192) by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P(2)' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the "least favorable" P(2)' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P(3)' residues and its strong aversion for negatively charged P(3)' residues. Furthermore, both Hir(52-65) and heparin increased the k(cat)/K(m) value of substrate hydrolysis. We conclude that Glu(192) is critical for the P(2)' and P(3)' specificities of thrombin and for the allostery mediated through exosite 1.

Topology of the stable serpin-protease complexes revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin.

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P'1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.

Thrombomodulin modulates the mitogenic response to thrombin of human umbilical vein endothelial cells.

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 microM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin.

In southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.

Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I.

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.

Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease.

The role of the protease active-site serine residue in the formation of protease-serpin complexes has been investigated by using a mutant of thrombin in which Ser195 was mutated to alanine (S195A). The structural integrity of S195A was established by examining the kinetics of its interaction with the inhibitor hirudin, which does not have substantial interactions with Ser195. The affinity of S195A for hirudin was only tenfold less than that of thrombin and the kinetic constants for the formation of the S195A-hirudin complex were very similar to those observed with thrombin. In contrast to hirudin, the dissociation constants (Ki) for S195A with serpins (antithrombin, protease nexin-1 and alpha1-antitrypsin with a P1 arginine) were 2 x 10(3) to 2 x 10(5)-fold higher than those observed with thrombin. These results indicate a critical role for interactions with Ser195 in stabilizing the thrombin-serpin complexes. The cofactor heparin compensated partially for the loss of interactions with Ser195; it increased the affinity of S195A for protease nexin-1 and antithrombin by 140-fold and 1000-fold, respectively. In the case of heparin/antithrombin, the increase in affinity could be attributed mainly to interactions outside the active site of S195A. Kinetic studies with antithrombin and protease nexin-1 in the presence of heparin indicated that Ser195 was not involved in any rate-limiting process in the formation of protease-serpin complexes. Interactions with Ser195 increased the stability of the complex by markedly reducing its rate of breakdown rather than by increasing its rate of formation. Overall, the results of the kinetic studies were consistent with a mechanism in which the binding of the protease induces a rate-limiting conformational change in the serpin and interactions with the protease's active-site serine residue, occurring in a subsequent faster step, greatly stabilize the complex.

Allosteric modulation of the activity of thrombin.

Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu192-->Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192-->Gln mutant. A peptide based on the sequence of hirudin (rhir52 65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192-->Gln mutant; a 5-fold decrease in the K(m) value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52-65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both alpha-thrombin and the Glu192-->Gln mutant. However, the increases in the ki values were greater with the Glu192-->Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable D-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 site of thrombin.

Role of the P2 residue in determining the specificity of serpins.

The importance of the P2 residue in determining serpin specificity was examined by making a series of substitutions in the P2 position of recombinant alpha 1-antichymotrypsin that contained an arginine P1 residue. The importance of the P2 residue in governing the association rate constant (Kon) of the serpin varied with the protease examined. For trypsin, the P2 residue played a relatively minor role, whereas the nature of this residue markedly influenced the rates of inhibition of thrombin, factor Xa, and APC. A 1000-fold difference in Kon values was observed between the fastest (P2 proline) and the slowest (P2 threonine) inhibitors of thrombin. Similar differences were observed with factor Xa; the best inhibitor (P2 glycine) displayed a 200-fold higher Kon value than the poorest (P2 threonine). The nature of the P2 residue also affected whether the interaction of the serpin with the protease resulted in inhibition of the protease or cleavage of the serpin; a P2 proline residue increased the rate of cleavage of alpha 1-antichymotrypsin by trypsin. By using mutants of thrombin, it was possible to show that the B-insertion loop, which partially occludes the active site, is important in determining the P2 specificity of this enzyme. Deletion of three amino acids from this loop yielded a protease (des-PPW) that became more like trypsin in its specificity. In addition, it was shown that Glu192 dramatically restricts thrombin's ability to accommodate a threonine in the P2 position. Taken together, the results demonstrated the importance of complementary interactions between the P2 residue of the serpin and the S2 binding site of the protease in regulating the specific interaction between serpin and protease.

Characterization of the P2' and P3' specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis.

The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.