Conformationally restricted thrombin inhibitors resistant to proteolytic digestion.

A new type of thrombin exo-site inhibitor has been designed with enhanced inhibitory potency and increased metabolic stability. With the aid of the model of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio, J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5, 77-85], cyclic analogs of the hirudin fragment (hirudin55-65) were designed and synthesized. In these analogs, the side chains of appropriately substituted residues, 58 and 61, were joined in order to restrict the conformation of the inhibitor. An analog with an 18-membered lactam ring showed higher antithrombin activity (IC50 = 0.57 microM) than the corresponding analogs with 17- or 16-membered rings and was 2-fold more potent than its linear counterpart. Even 4-fold greater enhancement was obtained when a shorter fragment, hirudin 55-62, was cyclized. This cyclization not only improved the potency but, more importantly, dramatically increased the resistance to proteolytic digestion. Remarkable enhancement of stability to proteolysis was observed for peptide bonds located in the exocyclic linear peptide segments. These results are discussed using molecular modeling.     

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.     

Sequence specificities of human fibroblast and neutrophil collagenases.

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.     

The proteolytic activity of human prostate-specific antigen

Human prostate-specific antigen has been found to exhibit a mild activity of protease at neutral pH. This finding is based on two observations: a proteolytic activity was always associated with the antigen fractions during purification, and the proteolytic activity and the antigen were precipitated with specific antibody to the antigen. In comparison with physico-chemical and catalytic properties of known proteases, human prostate-specific antigen is a distinct neutral protease.     

The extended cleavage specificity of human thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.     

Multiple proteolytic action of rat liver cathepsin B: specificities and pH-dependences of the endo- and exopeptidase activities.

Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.     

Binding of human thrombin to cultured human endothelial cells.

Binding of thrombin to monolayer cultures of human umbilical vein endothelium is studied. Binding is measured as inhibition by unlabeled ligand of the binding of 125I-thrombin to the cells. Radioactivity bound to cultures at equilibrium is measured after draining but not washing the cells. To correct for unremoved supernatant, 131I-albumin is included as a second label in the medium. Equilibrium between bound and free thrombin is attained within 1 min, and Scatchard analysis indicates a population of approximately 3 x 10(3) sites/cell with a dissociation constant of 10(-10) M, and a larger population with a dissociation constant greater than 10(-8) M. The two populations of sites are also indicated by a biphasic dissociation of bound label. Thrombin inactivated with diisopropyl fluorophosphate binds to the same receptor, with an affinity similar to that of active thrombin. Binding is unaffected by albumin (an acidic protein) and cytochrome c (a basic protein). Cultures of umbilical cord smooth muscle and fibroblasts bind thrombin at least 100 times more weakly than endothelium, and no binding to erythrocytes or a monolayer culture of mouse neuroblastoma is detected.     

Platelet plasma membrane glycoproteins Identification of a proteolytic substrate for thrombin

The surface glycoproteins of the platelet plasma membrane were labeled by oxidation with galactose oxidase followed by reduction with (³H)-sodium borohydride. Of the glycoproteins labeled, only glycoprotein V (apparent molecular weight of 89,000) was decreased as a result of thrombin action. The affected glycoprotein appeared to be completely removed at a concentration of 1 U thrombin per 10⁹ platelets. A soluble glycopeptide hydrolytic product with an apparent molecular weight of 70,000 was released into solution.     

Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.

We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization.     

Receptor specificities of human respiroviruses

Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galbeta1-4GlcNAc) by the alpha2-3 linkage (NeuAcalpha2-3Galbeta1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galbeta1-4GlcNAc through an alpha2-6 linkage (NeuAcalpha2-6Galbeta1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galbeta1-4GlcNAc (NeuGcalpha2-3Galbeta1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcalpha2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcalpha2-3Gal as receptors and that hPIV-3 also recognizes NeuAcalpha2-6Gal- or NeuGcalpha2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.     

Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.     

Cyclic and linear oligocarbamate ligands for human thrombin.

Several classes of compounds have been tested as potential inhibitors of the serine protease thrombin, an important regulator of blood coagulation cascades. We describe here the discovery of a new class of thrombin inhibitors based on an unnatural carbamate biopolymer. Oligocarbamate thrombin inhibitors were identified through the screening of diverse cyclic trimer, cyclic tetramer, and linear tetramer libraries using the one bead, one peptide method. Whereas the cyclic trimer oligocarbamate ligands bound thrombin with modest affinity, a cyclic tetramer oligocarbamate inhibited thrombin with an apparent Ki of 31 nM. Linear oligocarbamate tetramers bound thrombin with inhibition constants in the 100-nM range. These nonpeptidic, oligomeric molecules may provide the basis for further drug development and studies of thrombin ligand interactions.     

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.