Kininogenase activity of alpha and beta/gamma forms of bovine thrombin

The kininogenase activity of alpha- and beta/gamma-forms of bovine thrombin with respect to the high molecular weight (HMW) and low molecular weight (LMW) human kininogens was studied. It was shown that both forms of the enzyme split of bradykinin from these kininogens. The kininogenase activity of alpha-thrombin is completely blocked by the highly specific thrombin inhibitor Nalpha-dansyl-L-arginine-p-ethylpiperidineamide, but not by the soya bean trypsin inhibitor. The alpha- and beta/gamma-forms of thrombin hydrolyze HMW (Km(app) = 4.5 and 3.3 microM, respectively) and LMW (Km(app) = 10.1 and 4.7 microM, respectively). The specific constants (kcat/Km(app) ) for thrombin with respect to the substrates differ about 7-fold, predominantly due to the high catalytic rates of HMW as compared to LMW; the kcat values are 0.18 and 0.06 min-1, respectively. alpha-Thrombin upon a long-term (over 1 hour) exposure to HMW, besides bradykinin, splits off the product inhibiting the kininogenase activity of thrombin. No differences in the specificity of the beta/gamma-form of thrombin with resect to HMW and LMW were detected.     

Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation

The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.     

Kinetic characterization of the substrate reaction between a complex of antithrombin with a synthetic reactive-bond loop tetradecapeptide and four target proteinases of the inhibitor.

A tetradecapeptide corresponding to the P1 to P14 region of the reactive-bond loop of antithrombin (AT) binds to the inhibitor, presumably as a middle strand of the A beta-sheet, thereby converting AT from an inhibitor to a substrate of thrombin (Bj?rk, I., Ylinenj?rvi, K., Olson, S.T., and Bock, P. E. (1992) J. Biol. Chem. 267, 1976-1982). The kinetics of cleavage of the AT reactive bond in the AT-peptide complex by four target proteinases were quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. The kcat/Km values for thrombin and factor IXa were indistinguishable from the second-order rate constants for AT inhibition of these enzymes, whereas the values for factor Xa and plasmin were 10-17-fold higher than the inhibition rate constants. Heparin with high affinity for AT accelerated the substrate reaction with thrombin to an extent consistent with the reduced heparin affinity of the AT-peptide complex. These data show that blocking by the peptide of the putative intramolecular association of the P1 to P14 region of the AT reactive-bond loop with the A beta-sheet leads to AT functioning as a substrate of its target enzymes with an efficiency that equals or exceeds the action of uncomplexed AT as an inhibitor and with the expected heparin activation. The results thus suggest that a substrate-like attack of the proteinase on the inhibitor reactive bond in an exposed loop initiates the inhibition reaction. This attack presumably induces the subsequent trapping of the enzyme by the insertion of the reactive-bond loop into the A beta-sheet.     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

DNA-enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium

We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni²⁺ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. I. Effects on thrombin generation

The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K(i) values. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors.     

A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin.Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.     

Conformationally restricted analogs of the direct thrombin inhibitor FM 19

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme’s active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure–activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH₂). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC₅₀ values of 0.51 and 0.45 μM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.     

YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells

Yersinia spp. inject effector proteins ( Y ersinia o uter p roteins, Yop s ) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro . To investigate the intracellular, ' in vivo ' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia .     

Aprotinin can inhibit the proteolytic activity of thrombin. A fluorescence and an enzymatic study.

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human alpha-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x 10(-6) mol.l-1 was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride++ + (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 microM and a Km of 1.72 microM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 +/- 0.12 and an apparent dissociation constant of 57.0 +/- 13.1 microM (mean +/- SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

BSTI, a trypsin inhibitor from skin secretions of Bombina bombina related to protease inhibitors of nematodes.

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.     

Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions

Activated protein C (APC) reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models. APC anticoagulant activity involves inactivation of factors Va and VIIIa, whereas APC cytoprotective activities involve the endothelial protein C receptor and protease-activated receptor-1 (PAR-1). The relative importance of the anticoagulant activity of APC versus the direct cytoprotective effects of APC on cells for the in vivo benefits is unclear. To distinguish cytoprotective from the anticoagulant activities of APC, a protease domain mutant, 5A-APC (RR229/230AA and KKK191-193AAA), was made and compared with recombinant wild-type (rwt)-APC. This mutant had minimal anticoagulant activity but normal cytoprotective activities that were dependent on endothelial protein C receptor and protease-activated receptor-1. Whereas anticoagulantly active rwt-APC inhibited secondary-extended thrombin generation and concomitant thrombin-dependent activation of thrombin activable fibrinolysis inhibitor (TAFI) in plasma, secondary-extended thrombin generation and the activation of TAFI were essentially unopposed by 5A-APC due to its low anticoagulant activity. Compared with rwt-APC, 5A-APC had minimal profibrinolytic activity and preserved TAFI-mediated anti-inflammatory carboxypeptidase activities toward bradykinin and presumably toward the anaphlatoxins, C3a and C5a, which are well known pathological mediators in sepsis. Thus, genetic engineering can selectively alter the multiple activities of APC and provide APC mutants that retain the beneficial cytoprotective effects of APC while diminishing bleeding risk due to reduction in APC's anticoagulant and APC-dependent profibrinolytic activities.     

Involvement of nitric oxide and potassium channels in the bradykinin-induced vasodilatation in the rat kidney perfused ex situ

The role of nitric oxide (NO), K(+) channels, and arachidonic acid metabolism, via cytochrome P450 and cyclooxygenase pathways, in the renal vasodilatory effect of bradykinin was examined in the isolated rat kidney perfused ex situ with a blood-free solution. Bradykinin (BK, 0.25-1.0 microM) induced a dose-dependent reduction of 10-35% in the relative renal vascular resistance (rRVR) of isolated kidneys preconstricted with phenylephrine (PHE, 0.17-0.35 microM). The vasodilating effect of 0.5 microM bradykinin was significantly inhibited by the nitric oxide synthase inhibitors, N(G)-nitro-L-arginine (95% inhibition) and N(G)-nitro-L-arginine methyl ester (45-75% inhibition). Clotrimazole, an inhibitor of cytochrome P450 pathway but not indomethacin, a cyclooxygenase inhibitor, reduced the renal vasodilator response to bradykinin by 84%. The nonspecific K(+) channel inhibitor, tetraethylammonium ion (TEA) and the selective inhibitor of Ca(2+)-activated K(+) channels, charybdotoxin (ChTX) greatly attenuated the vasodilator response to bradykinin by approximately 84% and 79%, respectively. These two K(+) channel inhibitors showed similar effects on vasodilatation induced by S-nitroso-acetyl-D,L-penicillamine (1 microM), a nitric oxide donor. The results suggest that bradykinin releases nitric oxide which, by opening potassium channels specifically the Ca(+)-dependent type, mediates the renal vasodilator response to bradykinin in the isolated kidney perfused ex situ.     

The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.     

Cell survival signalling in heart derived myofibroblasts induced by preconditioning and bradykinin: The role of p38 MAP kinase

Fibroblasts possess receptors for compounds released during ischemia, including bradykinin. The aims of the present study were to investigate tyrosine kinase and p38 MAP kinase signalling in heart derived myofibroblasts in response to bradykinin and preconditioning ischemia. Fibroblasts from neonatal rat hearts were subjected to pharmacological agents and/or simulated ischemia. Cell viability was measured by the conversion of a tetrazolium salt to its formazan derivative. Preconditioning with 30 min of simulated ischemia followed by 30 min recovery resulted in an 85.4% +/- 7.8% increase in cell survival above that of cells treated with prolonged ischemia alone. Cells treated with bradykinin showed a 35% +/- 7.9 increase in cell survival after lethal ischemia. The B2 receptor antagonist Hoe 140 blocked the protective effect of bradykinin, but did not block preconditioning. The K(ATP) channel blocker glibenclamide and the mitochondria specific K(ATP) blocker 5, hydroxydecanoate, abolished the cytoprotection induced by both preconditioning and bradykinin. The non specific tyrosine kinase inhibitor genistein also abolished the cytoprotection. Effective blockade of cytoprotection was obtained with K(ATP) channel blockers and the tyrosine kinase inhibitor when these compounds were given prior to the preconditioning stimulus and not during the lethal insult. The stress activated protein kinase p38 MAP kinase was investigated by Western blotting and by the use of a specific inhibitor (SB203580). Preconditioning reduced phospho-p38 MAP kinase; in contrast, bradykinin administration markedly increased phosphorylation of p38 MAP kinase. SB203580 protected cells from lethal simulated ischemia. In conclusion, cell survival-signalling pathways activated by bradykinin or simulated ischemia in heart fibroblasts protect via the opening of K(ATP) channels and are independent of the stress-activated p38 MAP kinase and/or related to inhibition of this kinase.     

A Thermodynamic Characterization of the Binding of Thrombin Inhibitors to Human Thrombin, Combining Biosensor Technology, Stopped-Flow Spectrophotometry, and Microcalorimetry

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.     

Characterization of endothelium-derived hyperpolarizing factor in the human forearm microcirculation

The identity of endothelium-dependent hyperpolarizing factor (EDHF) in the human circulation remains controversial. We investigated whether EDHF contributes to endothelium-dependent vasomotion in the forearm microvasculature by studying the effect of K+ and miconazole, an inhibitor of cytochrome P-450, on the response to bradykinin in healthy human subjects. Study drugs were infused intra-arterially, and forearm blood flow was measured using strain-gauge plethysmography. Infusion of KCl (0.33 mmol/min) into the brachial artery caused baseline vasodilation and inhibited the vasodilator response to bradykinin, but not to sodium nitroprusside. Thus the incremental vasodilation induced by bradykinin was reduced from 14.3 +/- 2 to 7.1 +/- 2 ml x min(-1) x 100 g(-1) (P < 0.001) after KCl infusion. A similar inhibition of the bradykinin (P = 0.014), but not the sodium nitroprusside (not significant), response was observed with KCl after the study was repeated during preconstriction with phenylephrine to restore resting blood flow to basal values after KCl. Miconazole (0.125 mg/min) did not inhibit endothelium-dependent or -independent responses to ACh and sodium nitroprusside, respectively. However, after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and NG-monomethyl-L-arginine, the forearm blood flow response to bradykinin (P = 0.003), but not to sodium nitroprusside (not significant), was significantly suppressed by miconazole. Thus nitric oxide- and prostaglandin-independent, bradykinin-mediated forearm vasodilation is suppressed by high intravascular K+ concentrations, indicating a contribution of EDHF. In the human forearm microvasculature, EDHF appears to be a cytochrome P-450 derivative, possibly an epoxyeicosatrienoic acid.     

Biosensor analysis of drug-target interactions: direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specificity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 microM concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel.