Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor D (C3 proactivator convertase) of human serum has been shown to be absolutely necessary for alternative pathway function, for activation of the C3/C5 convertase of that pathway and not to be a subunit of this enzyme. Factor D was found to be present in human plasma in active form only, at a concentration of 2 microgram/ml, and not to be controlled by plasma protease inhibitors or by spontaneous decay. Unlike trypsin, factor D cleaves and activates factor B only when it is in Mg++-dependent complex with C3b, has no esterolytic activity, and is unable to cleave the B chain of insulin. The alleged functional and antigenic relationship of factor D to alpha-thrombin could not be verified. The results of this study led to the description of the mechanism of action of factor D in terms of the cryptic site hypothesis.
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PMID:Mechanism of action of factor D of the alternative complement pathway. 8 4

Thrombin-mediated platelet membrane-specific uptake of C3 and C5 was demonstrated by radiolabeled components and was visualized electron microscopically utilizing a ferritin marker conjugated to monospecific antibody to each component. The role of complement in thrombin-induced platelet function was determined. Though complement was not essential for thrombin-induced platelet aggregation and release of serotonin, these activities were significantly increased if complement was present. The release of serotonin was found to be a nonlytic process because under the conditions employed, no lactic dehydrogenase was released. The activation of complement was induced by a mechanism which has not been previously described. Thrombin associated with the platelet membrane presumably formed a C3 convertase that entered the known complement sequence at the C3 stage and proceeded to activate the terminal components through the known sequence to C9.
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PMID:The human complement system in thrombin-mediated platelet function. 68 79

The alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.
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PMID:Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H. 295 7

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.
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PMID:Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anti-coagulant and anti-complementary sites on the heparin molecule. 321 Nov 61

A small fragment of C3, called C3a, which has smooth muscle contracting activity, was isolated by three different methods. At pH 8.6, C3a behaved as cation, and using the Archibald method, its mol wt was determined to be 7000. A specific antiserum to C3a showed the fragment to be antigenically distinct from the rest of the C3 molecule, i.e., the C3b portion. The same antiserum and an anti-whole C3 were able to inhibit the biologic activity of C3a. In addition to anaphylatoxin activity, leukocyte chemotactic activity was shown to reside in C3a. Treatment with trypsin caused the cationic fragment to become anionic and abolished the anaphylatoxin but not the chemotactic activity. C3a fragments with identical biologic activity and comparable cationic properties, as determined by acid disc electrophoresis, were obtained by treatment of C3 with C3 convertase, C3 inactivator complex, trypsin, and plasmin. Thrombin produced a similar C3 fragment which was inactive. It was concluded that C3a corresponds to an unusually basic portion of C3 which may be liberated by attack of a variety of enzymes on a highly susceptible region of the native C3 molecule. C3b was cleaved by trypsin and less efficiently by thrombin or plasmin into two antigenically distinct pieces: the larger C3c fragment corresponding to beta(1A) and the smaller C3d fragment to alpha(2D) of aged serum. The c- and the d-fragments were separated and characterized. Isolated C3a rapidly lost its anaphylatoxin activity when treated with small amounts of a partially purified, thermolabile 10S alpha-pseudoglobulin of human serum. The conditions of inactivation suggested an enzymatic reaction. The anaphylatoxin inactivator also destroyed the activity of C5-derived anaphylatoxin and of lysyl bradykinin.
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PMID:Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. 577 86

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.
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PMID:Amino acid sequence of human D of the alternative complement pathway. 638 66

Hexapeptides mimicking the partial amino acid sequence of factor B surrounding the bond that is cleaved by factor D have been synthesized. These peptides have been assessed for their ability to inhibit factor D enzymatic activity and for their susceptibility to serine proteases. The synthetic peptides were cleaved by bovine trypsin and C1s but not by alpha-thrombin and factor D. The peptides inhibited factor B cleavage and fluid-phase or cell-bound alternative pathway C3 convertase activation by factor D. Altogether, these results suggest that peptides analogous to factor B specifically inhibit factor D enzymatic activity. Thus, they constitute an interesting tool for study of alternative pathway activation and can be of use when attempting to manipulate this pathway, since factor D is an essential component for alternative pathway initiation and amplification.
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PMID:Inhibition of alternative pathway factor D by factor B-related synthetic hexapeptides. 692 Feb 99

The mannan-binding lectin (MBL)-associated serine proteases (MASPs) circulate in serum complexed with mannan-binding lectin, a recognition molecule of the complement system. MASP-2 cleaves the complement components C4 and C2 to form the C3 convertase C4b2a. A definitive natural substrate for MASP-1 has not yet been described. We investigated the substrate specifities of MASP-1 and MASP-2 using cleavage of fluorescent amide substrates by recombinant and serum-derived MASPs. Recombinant MASP-1 cleaved Phe-Gly-Arg-aminomethylcoumarin (AMC) most rapidly at a rate of 16.8 nmol min(-1) microg(-1) rMASP-1. Recombinant MASP-2 barely cleaved any of 14 substrates used. This provides means of measuring MASP-1 activity in the absence of a known natural substrate. An assay for MBL-bound MASP-1 was established using the substrate Val-Pro-Arg-AMC. Assay of MBL-bound MASP-2 was done by cleavage of a natural protein substrate, C4. The condition of the serum used for the assays is important; simulated aging showed decreased detectable MASP-1 and MASP-2 activity. The inhibitors Z-D-Phe-Pro-methoxy-propylboroglycinepinanediol ester (boroMpg), anti-thrombin III in the presence and absence of heparin, hirudin and C1 inhibitor were tested against the MASPs. C1 inhibitor inhibits both enzymes, but the protease-serpin complex is unusually unstable at alkaline pH. The thrombin inhibitor boroMpg inhibited MASP-1 but not MASP-2 while hirudin did not inhibit either protease. Anti-thrombin III alone was not inhibitory, but in the presence of heparin inhibited both MASP-1 and MASP-2. The ancient origin of MASP-1 and its thrombin-like activity suggests its involvement in a coagulation-based defense mechanism in the early evolution of innate immunity.
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PMID:Differential substrate and inhibitor profiles for human MASP-1 and MASP-2. 1472 88

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.
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PMID:Generation of C5a in the absence of C3: a new complement activation pathway. 1671 88

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.
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PMID:Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot. 2000 87


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