Inactivation of thrombin by murine peritoneal macrophages.

We recently showed that murine peritoneal macrophages cultured in vitro express potent prothrombinase activity (Lindahl, U., Pejler, G., B?gwald, J., and Seljelid, R. (1989) Arch. Biochem. Biophys. 273, 180-188). In the present report, we demonstrate that the macrophages also express anticoagulant activity by inactivating the thrombin that is formed due to the action of the prothrombinase. Addition of exogenous purified thrombin to the macrophage cultures resulted in inactivation of the enzyme at a maximum rate of approximately 5 micrograms/h/10(6) cells. The inactivation appeared to be specific for thrombin, since neither Factor Xa, chymotrypsin, nor trypsin, three serine proteases exhibiting homology with thrombin, were inactivated by the macrophages. Thrombin-inactivating activity was not secreted into the culture medium. Inhibitors of endocytosis did not decrease the rate by which thrombin was inactivated, suggesting that internalization of the coagulation factor was not required. In contrast, the thrombin-inactivating activity was strongly inhibited by the polycation Polybrene. Anion-exchange chromatography of extracts obtained after Triton X-100-solubilization of the macrophages demonstrated that the thrombin-inactivating activity exhibited a high negative charge. Incubation of the thrombin-inactivating activity recovered after anion-exchange chromatography with unlabeled thrombin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, showed that thrombin was proteolytically cleaved into defined fragments. Similar proteolytic fragments were obtained when 125I-labeled thrombin was added to macrophage cultures. Degradation of thrombin was blocked by phenylmethanesulfonic fluoride, an inhibitor of serine proteases, but not by inhibitors of other classes of proteases. Thrombin that had been chemically modified at its active site was degraded at the same rate by the macrophages as active thrombin. Taken together, these findings indicate that the murine macrophages express surface-bound serine protease activity that specifically inactivates thrombin by proteolytic cleavage. The significance of thrombin-inactivating activity in relation to the involvement of macrophage procoagulant activity in the immune response is discussed.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Engineering of a macromolecular scaffold to develop specific protease inhibitors

The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications. Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface. Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface. The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal). Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (K(i)*) of 11 pM, whereas K(i)* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher. The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa.     

Design and synthesis of macrocyclic indoles targeting blood coagulation cascade Factor XIa

The synthesis of a series of novel macrocyclic compounds designed to target blood coagulation Factor XIa is described. The compounds were evaluated for their inhibition of a small set of serine proteases. Several compounds displayed modest activity and good selectivity for Factor XIa. Within the series, a promising lead structure for developing novel macrocyclic inhibitors of thrombin was identified.     

Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans

Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field.     

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.     

Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

Thrombin: a paradigm for enzymes allosterically activated by monovalent cations

Enzymes activated by monovalent cations are abundantly represented in plants and in the animal world. The mechanism, of activation involves formation of a ternary intermediate with the enzyme-substrate complex, or binding of the cation to an allosteric site in the protein. Thrombin is a Na+-activated enzyme with procoagulant, anticoagulant and signaling roles. The binding of Na+ influences allosterically thrombin function and offers a paradigm for regulatory control of protease activity and specificity. Here we review the molecular basis of thrombin allostery as recently emerged from mutagenesis and structural studies. The role of Na+ in blood coagulation and the evolution of serine proteases are also discussed.     

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses

Background

Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.

Methodology/Principal Findings

Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K_D = 0.4 nM) and to the active site of FXa (K_D = 3.07 nM). We propose the name “Simukunin” for this novel protein.

Conclusions

We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.     

Coagulation factor V

The coagulation cascade involves sequential enzymatic activations of serine protease zymogens that converge on the generation of thrombin. Factor V (FV) takes part in this process as a component of the prothrombinase complex. Besides its role as procoagulant factor, it is also involved in the physiologic anticoagulant pathway, by participating in the inactivation of activated factor VIII (FVIIIa). Given the dual role of FV, genetic defects in FV gene may result in opposite hemorrhagic or thrombotic phenotypes. This review focuses on the structure, function (procoagulant and anticoagulant), regulation (activation and inactivation) of FV as well as on the genetic defects associated with mutations in the FV gene.     

Selective boron-containing thrombin inhibitors--X-ray analysis reveals surprising binding mode

Based on the structural comparison of the S1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesized as inhibitors of thrombin. Among the novel compounds, a number of derivatives were synthesized which appeared to have side-chain variants too big to fit into the S1 pocket. Nevertheless, these compounds inhibited thrombin in the nM range. The X-ray structure of one of these inhibitors bound to the active side of thrombin reveals that a new binding mode is responsible for these surprising results.     

Sphingolipids as bioactive regulators of thrombin generation

Sphingolipids contribute to modulation of two opposing cell processes, cell growth and apoptotic cell death; ceramide and sphingosine promote the latter and sphingosine-1-phosphate triggers the former. Thrombin, a pro-inflammatory protease that is regulated by the blood coagulation cascade, exerts similar effects depending on cell type. Here we report a new mechanism for cross-talk between sphingolipid metabolism and thrombin generation. Sphingosine and sphinganine, but not ceramide or sphingosine-1-phosphate, down-regulated thrombin generation on platelet surfaces (IC(50) = 2.4 and 1.4 microm for sphingosine and sphinganine, respectively) as well as in whole plasma clotting assays. Thrombin generation was also inhibited by glucosylsphingosine, lysosphingomyelin, phytosphingosine, and primary alkylamines with >10 carbons. Acylation of the amino group ablated anticoagulant activities. Factor Va was required for the anticoagulant property of sphingosine because prothrombin activation was inhibited by sphingosine, sphinganine, and stearylamine in the presence but not in the absence of factor Va. Sphingosine did not inhibit thrombin generation when Gla-domainless factor Xa was used in prothrombinase assays, whereas sphingosine inhibited activation of Gla-domainless prothrombin by factor Xa/factor Va in the absence of phospholipids (IC(50) = 0.49 microm). Fluorescence spectroscopy studies showed that sphingosine binds to fluorescein-labeled factor Xa and that this interaction required the Gla domain. These results imply that sphingosine disrupts interactions between factor Va and the Gla domain of factor Xa in the prothrombinase complex. Thus, certain sphingolipids may be bioactive lipid mediators of thrombin generation such that certain sphingolipid metabolites may modulate proteases that affect cell growth and death, blood coagulation, and inflammation.     

Serine proteases of the complement system

The complement system in blood plasma is a major mediator of innate immune defence. The function of complement is to recognize, then opsonize or lyse, particulate materials, including bacteria, yeasts and other microrganisms, host cell debris and altered host cells. Recognition occurs by binding of complement proteins to charge or saccharide arrays. After recognition, a series of serine proteases is activated, culminating in the assembly of complex unstable proteases called C3/C5 convertases. These activate the complement protein C3, which acts as an opsonin. The complement serine proteases include the closely related C1r, C1s, MASPs 1-3 (80-90 kDa), C2 and Factor B (100 kDa), Factor D (25 kDa) and Factor I (85 kDa). Each of these has unusually restricted specificity and low enzymic activity. The C1r, C1s and MASP group occur as proenzymes. When activated, they are regulated, like many plasma serine proteases, by a serpin, C1-inhibitor. C2 and Factor B, however, have complex multiple regulation by a group of complement proteins called the Regulation of Complement Activation (or RCA) proteins, whereas Factors I and D appear to have no natural inhibitors. Advances in structure determination and protein-protein interaction properties are leading to a more detailed understanding of the complement-system proteases, and are indicating possible new routes for potential therapeutic control of complement.     

A new concept of action of hemostatic proteases on inflammation,neurotoxicity, and tissue regeneration

Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

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.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.