Human protease nexin-I. Further characterization using a highly specific polyclonal antibody

We have used purified protease nexin-I (PN-I) from human fibroblasts to develop a polyclonal antibody that specifically blocks the PN-I-mediated cellular binding of thrombin and urokinase. Anti-PN-I IgG did not inhibit the binding of 125I-epidermal growth factor-binding protein to fibroblasts, which is mediated by protease nexin-II, another cell-secreted, serine protease inhibitor that is distinct from PN-I. This furthers the belief that the protease nexins are distinct from one another. In addition, while anti-PN-I IgG immunoprecipitated PN-I X thrombin complexes, it did not do so with antithrombin-III X thrombin. Metabolically labeled PN-I was also immunoprecipitated by IgG, indicating that the protein can be labeled in vivo. The antibody also recognized primarily one band on Western transfers of conditioned medium from fibroblast cultures. These results suggest that anti-PN-I will be useful in probing the physiological role of PN-I as well as its biosynthesis.     

Biosynthesis of protease nexin-I

Protease nexin-I (PN-I) is representative of a newly described class of serine protease inhibitors secreted by human fibroblasts, the protease nexins. Protease nexins form covalent complexes with their target proteases, subsequently binding to cells via specific receptors. PN-I preferentially binds thrombin, urokinase, trypsin, and plasmin, and its binding to thrombin is accelerated by heparin. We have previously described the production of a polyclonal antibody against PN-I which is able to block the binding of PN-I X proteinase complexes to cells and will immunoprecipitate metabolically labeled PN-I. Anti-PN-I was used to investigate the biosynthesis and regulation of PN-I in human fibroblasts. Unlabeled PN-I could compete for the binding of metabolically labeled PN-I to anti-PN-I, as shown by the elimination of the 43-kDa band representing PN-I on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs. Excision of this 43-kDa band from gels, followed by amino-terminal sequencing, showed a homogeneous protein that is homologous with that described by Scott et al. (Scott, R. W., Bergman, B. L., Bajpai, A., Hersh, R. T., Rodriguez, H., Jones, B. N., Barreda, C., Watts, S., and Baker, J. B. (1985) J. Biol. Chem. 260, 7029-7034). An analysis of the biosynthesis of the PN-I revealed that a lower Mr precursor exists intracellularly. This apparent rough endoplasmic reticulum form appears as a doublet on sodium dodecyl sulfate gels, as does mature PN-I. The PN-I precursor was also sensitive to endoglycosidase H, suggesting that it contains N-linked carbohydrates of the high mannose form. Mature PN-I is not sensitive to endoglycosidase H, but does contain 3 kDa of N-linked carbohydrate. PN-I appears to be constitutively secreted by fibroblasts. PN-I levels in conditioned media reach a steady state within 48 h, although PN-I synthesis maintains a constant rate. This steady state is due to the continuous uptake of PN-I from medium, presumably through a specific receptor.     

The glioma cell-derived neurite promoting activity protein is functionally and immunologically related to human protease nexin-I

Protease nexin-I (PN-I, Mr approximately 43,000) is representative of a newly described class of cell-secreted protease inhibitors. PN-I has been purified to apparent homogeneity, partially sequenced, and monospecific antibodies have been raised against it. PN-I is a potent inhibitor of urokinase, thrombin, plasmin, and trypsin. In addition, cells have specific receptors that mediate the uptake of covalently linked complexes formed between PN-I and its protease substrates. In the present studies, we have investigated the relationship between human PN-I and a protease inhibitor derived from C6 glioma cells in culture that has neurite-promoting activity. On the basis of co-purification on heparin-Sepharose, identical molecular weight, antibody cross-reactivity, and receptor cross-reactivity, we conclude that PN-I and the glioma-cell-derived inhibitor are equivalent molecules.     

Binding of protease nexin-1 to the fibroblast surface alters its target proteinase specificity

Protease nexin-1 is a protein proteinase inhibitor that is secreted by a variety of cultured cells and rapidly forms complexes with thrombin, urokinase, and plasmin; the complexes then bind back to the cells and are internalized and degraded. In fibroblast cultures, protease nexin-1 is localized to the extracellular matrix. Here we report that protease nexin-1, which is bound to the surface of fibroblasts, forms complexes with thrombin, but not urokinase or plasmin. Experiments were conducted to determine directly if protease nexin-1 binding to the fibroblast surface alters its proteinase specificity. To do this, cell surface protease nexin-1 was inhibited using anti-protease nexin-1 monoclonal antibodies that stoichiometrically block its ability to form complexes with target proteinases. Then, purified protease nexin-1 was added to these cells; the cell-bound molecule formed complexes with thrombin, but not urokinase or plasmin. Similar experiments showed that protease nexin-1 bound to preparations of fibroblast extracellular matrix also formed complexes with thrombin, but not urokinase or plasmin. Components of the extracellular matrix other than heparin-like glycosaminoglycans are required for this regulation since heparin did not block the formation of complexes between protease nexin-1 and urokinase or plasmin. These results suggest that protease nexin-1 is primarily a thrombin inhibitor in interstitial fluids where much of it would be bound to cell surfaces.     

Interactions of serine proteases with cultured fibroblasts

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.     

Binding uptake and degradation of antithrombin III X protease complexes by cultured corneal endothelial cells

Interaction of 125I-labeled human antithrombin III (125I-AT III) X protease complexes with bovine corneal endothelial cells has been studied in tissue culture. 125I-AT III does not bind to endothelial cells, but its complexes with either thrombin or trypsin bind specifically to the cultures. The binding of 125I-AT III X protease complexes is not via the moiety of the free antithrombin III (AT III) or the free protease, since neither AT III nor thrombin compete on the binding of 125I-AT III X thrombin complexes. Only unlabeled AT III X thrombin complexes compete on the binding of the iodinated ligand. 125I-AT III X trypsin complexes bind with a KD of 1.4 X 10(-7) M to high affinity-binding sites present on the cell surface of corneal endothelial cells. Saturation of binding to the cell surface is observed at a concentration of 2.5 X 10(-7) M 125I-AT III X trypsin complexes and the number of binding sites per cell is about 4 X 10(4). The cell surface binding reaches a maximum by 15 min and then decreases with time. The cells, when incubated at 37 degrees C, appear to internalize the bound complexes by adsorptive endocytosis which proceeds at a rate of 0.5-0.8 pmole/1 X 10(6) cells/h. The internalization process of 125I-AT III X protease complexes is saturated at a concentration of 2.5 X 10(-7) M. Since the cells release 125I-labeled material into the extracellular media which cannot be precipitated by trichloroacetic acid (TCA), it probably represents degradation of 125I-AT III X protease complexes into small fragments at a linear rate of about 0.5 pmole/1 X 10(6) cells/h. The described process of AT III X protease complexes binding, internalization and subsequent degradation by corneal endothelial cells may represent a clearing mechanism for extracellular AT III X protease complexes formed under pathological conditions.     

Specific interaction of vitronectin with the cell-secreted protease inhibitor glia-derived nexin and its thrombin complex.

Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex.     

Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.     

A fibroblast-derived urokinase-inhibitor differing from protease nexin

UK-I, a 60-kDa urokinase-inhibitor derived from human fibroblasts, inhibited 54-kDa urokinase (EC 3.4.21.31) activity dose-dependently on ordinary fibrin-agar autograms. This UK-I formed an SDS-stable approximately 75-kDa complex with radioiodinated urokinase (33 kDa) on an autoradiogram following SDS/polyacrylamide gel electrophoresis. Benzamidine hydrochloride inhibited its formation, indicating UK-I to bind at the active site of urokinase and form an inactive complex. UK-I did not form a complex with [125I]thrombin (EC 3.4.21.5). It is thus evident that UK-I is one type of urokinase-inhibitor derived from human fibroblasts with properties differing from protease nexin, another urokinase-inhibitor derived from the same source.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog.Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.     

Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization

We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.     

Covalent binding of human thrombin to a human endothelial cell-associated protein

Binding of 125I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. This binding is specific and requires the catalytically active site of the enzyme. Formation of the complex could be detected as early as 3 min after addition of thrombin or with a thrombin concentration as low as 0.5 nM. This irreversible binding exhibits thrombin dose-dependence and reaches maximum levels at a concentration of 50 nM (10 fmol/10(5) cells). Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (i) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (ii) heparin prevents the appearance of the complex on the cell surface, (iii) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblast or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.     

Functional and structural similarities between protease nexin I and C1 inhibitor

Protease nexin I is a proteinase inhibitor that is secreted by human fibroblasts and forms stable complexes with certain serine proteinases; the complexes then bind to the fibroblasts and are rapidly internalized and degraded. In this report, we show that this inhibitor, which is present in very low concentrations in plasma, has functional and structural similarities to C1 inhibitor, an abundant proteinase inhibitor in plasma. Both inhibitors complex and inactivate certain proteinases that previously were known to rapidly react only with C1 inhibitor. Kinetic inhibition studies show that protease nexin I inhibits Factor XIIa and plasma kallikrein with second-order rate constants of 2.3 x 10(3) and 2.5 x 10(5) M-1 s-1, respectively, which are similar to the rate constants for inhibition of these proteinases by C1 inhibitor. Protease nexin I inhibits C1s about one-tenth as rapidly as does C1 inhibitor. Alignment of the amino acid sequences of protease nexin I and C1 inhibitor shows that these proteins have similarity at their reactive centers (from sites P7 to P1). The remaining regions of the two proteins share much less similarity. In contrast to protease nexin I, C1 inhibitor is not secreted by human fibroblasts. Although 125I-C1s-protease nexin I complexes readily bind to human fibroblasts, binding of 125I-C1s-C1 inhibitor complexes or other 125I-proteinase-C1-inhibitor complexes to these cells is not detectable. Thus, protease nexin I and C1 inhibitor may control some common regulatory proteinases in the extravascular and vascular compartments, respectively.     

Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding.

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.     

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e–m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a–d, 3a–d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.     

Crystallographic determination of the structures of human alpha-thrombin complexed with BMS-186282 and BMS-189090.

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.