The balance between inhibition and substrate-type reactions of recombinant C1 inhibitor P5/P3 variants.
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Amino Acid Sequence↗
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The effect of chemical modification of basic amino acid residues on the activation and amidolytic activity of Hageman factor (factor XII).
Modification of arginyl residues of Hageman factor by phenylglyoxal hydrate inhibits activation of this clotting factor in a plasma-free system, that is, in the absence of the other constituents of the contact activation system. Activation is also inhibited by alteration of the other two basic amino acid residues present, lysine and histidine. Chemical modification of histidine and arginine residues does not inhibit the amidolytic activity of activated Hageman factor. In contrast, modification of amino group(s) in N-terminal and lysine residues inhibits activated Hageman factor. Thus, basic amino acid residues essential to the activation or activity of Hageman factor appear to be variably accessible to chemical modification.
Amides↗
Generation of plasmin during acute attacks of hereditary angioedema.
Hereditary angioedema is caused by a genetic deficiency of C1-inhibitor, a serine protease inhibitor that regulates activation of complement, contact, and fibrinolytic systems. Symptoms (bouts of subcutaneous and mucous swelling) depend on the release of a vasoactive mediator, probably through activation of these three systems. We studied the interrelationship among complement, contact, and fibrinolytic activation in 23 patients with hereditary angiodema, 18 during remission and five during an attack, by measuring plasma levels of C1-C1 inhibitor, factor XIIa-C1 inhibitor, kallikrein-C1 inhibitor, and plasmin-alpha 2-antiplasmin complexes, tissue plasminogen activator, and urokinase plasminogen activator. In addition, cleavage of high-molecular weight kininogen was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and quantified by densitometry. During remission, plasma levels of C1-C1 inhibitor complexes were elevated (p = 0.0002), whereas the other parameters were within the normal range. During acute attacks, not only plasma levels of C1-C1 inhibitor complexes but also those of plasmin-alpha 2-antiplasmin complexes (P = 0.0009) and cleaved high-molecular weight kininogen were elevated. A positive correlation between plasmin-alpha 2-antiplasmin complexes and cleaved high-molecular weight kininogen was observed (r = 0.75, p < 0.001). This article presents the first in vivo evidence that supports the concept that release of vasoactive mediators in hereditary angiodema attacks is associated with the activation of the fibrinolytic system.
Acute Disease↗
Activation of the contact system in patients with sepsis and with septic shock.
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Biomarkers↗
Tridegin, a novel peptidic inhibitor of factor XIIIa from the leech, Haementeria ghilianii, enhances fibrinolysis in vitro.
Tridegin is a potent inhibitor of factor XIIIa from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet-free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.
Animals↗
Impact of n-3 fatty acid supplemented parenteral nutrition on haemostasis patterns after major abdominal surgery.
In various diseases n-3 fatty acids exert anti-inflammatory properties. These effects seem to be related to the uptake and incorporation of eicosapentaenoic acid (EPA) into the cellular substrate pool after dietary intake of EPA, which is contained in fish oils (FO). In the state of inflammation EPA is released to compete with arachidonic acid (AA) for metabolism at the cyclo-oxygenase and the 5-lipoxygenase level. The metabolites of EPA have less inflammatory and chemotactic potency than the substances derived from AA. In addition to positive effects, early studies pointed towards prolonged bleeding times after dietary intake of n-3 fatty acids. This study was undertaken to address the issue of potential coagulation disturbances associated with postoperative parenteral FO administration. This was a prospective, randomised, double blinded clinical trial, carried out in two operative intensive care units (13 and 16 beds) in a university hospital. Forty-four patients undergoing elective major abdominal surgery participated in the trial. Patients were randomly assigned to receive total parenteral nutrition (TPN) supplemented with either soybean oil (SO, Lipovenoess 10% PLR; 1.0 g/kgBW per day; n = 20) for five days or with a combination of FO and SO (FO, Omegaven; 0.2 g/kgBW per day plus SO, Lipovenoes 10% PLR; 0.8 g/kgBW per day, n = 24), respectively. Blood samples were taken preoperatively (day -1), prior to (day 1) during (days 2-5) and after TPN (day 6). The coagulation parameters thromboplastin time (Quick), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin III were measured. To differentially assess activation levels of extrinsic and intrinsic coagulation pathway, factors VIIa and XIIa were quantified. Moreover platelet function was determined by resonance thrombography. Baseline values of coagulation and platelet function were comparable in both groups, but coagulation activity dropped after surgery. Over the observation period of 6 days, however, physiological levels were regained. No clinically significant differences were observed between the SO- and SO + FO- group. These findings suggest that infusion of fish oil in doses up to 0.2 g/kgBW per day is safe regarding coagulation and platelet function.
Aged↗
Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1.
Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.
Alanine↗
Intraindividual comparison of the impact of two selective apheresis methods (DALI and HELP) on the coagulation system.
We performed an intraindividual comparison of the effect on the coagulation system of two selective apheresis procedures: Direct Adsorption of Lipoproteins (DALI) and Heparin-induced Lipoprotein Fibrinogen Precipitation (HELP). Six patients suffering from heterozygous familial hypercholesterolemia have been treated with 2 sessions of each procedure. Anticoagulation was carried out according to usual recommendations. Blood samples were taken before, immediately after and on the second day after the sessions. We assessed global coagulation tests (prothrombin time, activated partial thromboplastin time), fibrinogen, prothrombin fragment F 1 + 2 and a variety of factors (Factors II, V, VII, XIII, IX, X, XI, XII, XIIa; von Willebrand Factor; collagen-binding activity, prekallikrein, high-molecular weight kininogen) and antagonists (antithrombin III, protein S activity, free protein S). In fact, all parameters measured have been influenced by the apheresis treatment. Fibrinogen is lowered more by HELP which also has a more definite impact on factors belonging to the prothrombin complex (II, VII, X). In contrast, the major effects of the DALI system have been seen on the intrinsic pathway of the coagulation system (IX, XI, prekallikrein, high-molecular-weight kininogen). With both systems, no increases in activated Factor XII or in prothrombin fragment F 1 + 2 have been observed. These data provide a solid basis for individual adaptations of anticoagulant doses.
Adult↗
Activation of factor XII in acetone-treated human plasma: significance of the functional state of plasma kallikrein for the extent of activation.
The kaolin-induced activation of factor XII (XII) to XIIa was studied in plasminogen-free human citrated plasma treated with acetone in the presence of benzamidine 7.5 mM. XIIa was assayed as prekallikrein (PK) activator. The significance of the concentrations of XII, PK and high molecular weight kininogen (HMrK) was examined using mixtures of normal plasma and plasma genetically deficient in these factors. At the high plasma dilution used (1 + 23 v/v in the kaolin incubate) a joint estimation of the factors was obtained. A reduction in amount of XII, PK or HMrK resulted in a correspondingly reduced yield of XIIa. Plasma kallikrein present was assayed as S-2302 amidase. The concentration of PK in XII-deficient plasma was normal, in HMrK-deficient plasma about 30% of normal. The activation of XII was studied in fresh plasma as well as in plasma stored for 3-6 months at -70 degrees, and the activation with acetone was carried out in the presence and in the absence of benzamidine, EDTA or purified HMrK. In previous work benzamidine was found to protect the cofactor function of purified HMrK in the assay system used, and EDTA was found to inhibit purified human plasma kallikrein assayed as plasminogen activator. The present results support the previous observations, and indicate that acetone treatment of fresh human plasma (benzamidine present) results in the activation of plasma kallikrein in a functional state that requires kinin-free, but otherwise native HMrK as a cofactor for the activation of XII.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetone↗
Scaled interfacial activity of proteins at the liquid-vapor interface.
A principal conclusion drawn from observations of time- and concentration-dependent liquid-vapor (LV) interfacial tension gamma(lv) of a diverse selection of proteins ranging from albumin to ubiquitin spanning nearly three decades in molecular weight (MW) is that concentration scaling substantially alters perception of protein interfacial activity as measured by reduction in gamma(lv). Proteins appear more similar than dissimilar on a weight/volume basis, whereas molarity scaling reveals a "Traube-rule" ordering by MW, suggesting that adsorption is substantially driven by solution concentration rather than diversity in protein amphilicity. Scaling as a ratio-to-physiological-concentration demonstrates that certain proteins exhibit the full possible range of interfacial activity at and well-below physiological concentration, whereas others are only weakly surface active within this range, requiring substantially higher solution concentration to achieve reduction in gamma(lv). Important among this latter category of proteins are the blood factors XII and XIIa, assumed by the classical biochemical mechanism of plasma coagulation to adsorb to procoagulant surfaces, even in the presence of overwhelming concentrations of other blood constituents such as albumin and immunoglobulin that are shown by this work to be among the class of highly surface-active proteins at physiologic concentration. A comparison of pendant drop and Wilhelmy balance tensiometry as tools for assessing protein interfacial activity shows that measurement conditions employed in the typical Wilhelmy plate approach fails to achieve the steady-state adsorption condition that is accessible to pendant drop tensiometry.
Adsorption↗
Coumarin and isocoumarin as serine protease inhibitors.
Serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. Within the class of serine proteases, HLE is one of the most destructive enzymes in the body. It is implicated in the promotion or exacerbation of a number of diseases including pancreatitis, acute respiratory syndrome, rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and cystic fibrosis. Thrombin, a trypsin-like serine protease, plays a dual role in thrombogenesis, including fibrin formation and platelet activation. As a result, thrombin constitutes one of the most widely studied targets for antithrombotic strategy. Numerous inhibitors of serine proteases have been reported during the past three decades. Among them, coumarin-type molecules displayed a high inhibitory potency towards various serine proteases. At that time, halomethyl dihydrocoumarins have been shown to behave as the first general suicide inhibitors of serine protease. These molecules inhibit several proteases such as human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin. Isocoumarins are very effective as mechanism-based inhibitors of serine proteases. Pharmacomodulation on the 3-alkoxy-4-chloroisocoumarins and the 3-alkoxy-7-amino-4-chloroisocoumarins led to strong inhibitors of numerous serine proteases such as HLE, human factor XIa and XIIa, thrombin, urokinase and kallikrein. Recently, a series of coumarins characterised by an alkyl, aryl ester, amide, thioester or ketone in the position 3 and an electrophilic chloromethyl moiety in the position 6 have been developed. These compounds were found to be high inhibitors of alpha-chymotrypin, HLE and human thrombin.
Binding Sites↗
[A substrate inhibitor analysis of the urinary excretion of tissue and plasma kallikreins in patients with chronic glomerulonephritis].
The usage of substrate inhibitor analysis made it possible to estimate the levels of excretion of plasma proteinases, including plasma kallikrein in the urinary DValLeuArgpNA (S-2266)- and DProPheArgpNA (S-2302)-amidase activity in patients with latent and nephrotic types of chronic glomerulonephritis (CGN). The soya bean trypsin inhibitor, an inhibitor of plasma kallikrein and other plasma proteinases, such as that of the blood coagulative factors XIa and XIIa, and the high selective plasma kallikrein inhibitor DPhePheArgCH2Cl were used as those differentiating kallikreins of tissue and plasma origin. The S-2266 and S-2302-amidase activity of the urine from healthy subjects was shown to be determined by only tissue (renal) kallikrein. The urine from the patients with a latent CGN type displayed the activity of plasma proteinases, but plasma kallikrein made no significant contribution to the urine amidase activity in these patients. With a nephrotic CGN type, great quantities of trypsin-like proteinases were secreted from the plasma through the glomerular filter into the urine, the proportion of plasma kallikrein in the urinary S-2266 and S-2302-amidase activities being approximately 27%. The compensatory and pathogenetic role of plasma kallikrein is discussed if there is lower excretion of tissue (renal) kallikrein in CGN with the nephrotic syndrome.
Amino Acid Chloromethyl Ketones↗
Squash inhibitor family of serine proteinases.
Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor beta 2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: Xa and XIIa, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exibit at neutral pH a high kcat/K(m) index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant.
Amino Acid Sequence↗
Murine monoclonal antibodies to human factor XI.
Murine monoclonal antibodies to human factor XI (F.XI) are described. The monoclonal antibodies (2-1, 4-1, 7-1 and 10-1) consisted of IgG1. 4-1 inhibited the activation of F.XI completely in the presence of high molecular weight kininogen and kaolin and the others did so partially, whereas these antibodies had no effect on the activation of F.XI with activated factor XII (beta-XIIa). Four antibodies had no effect directly on the amidolytic activity of activated F.XI (F.XIa). 10-1 inhibited the activation of factor IX in coagulant assay for F.XIa by Mannhalter. And 4-1 and 7-1 did so partially, whereas 2-1 did not. In immunoblotting analysis, all antibodies bound to F.XI, its reduced form and F.XIa. All were directed against the heavy chain of F.XI. All antibodies recognized F.XIa-alpha 1 antitrypsin complex.
Antibodies, Monoclonal↗
Plant and marine derived (n-3) polyunsaturated fatty acids do not affect blood coagulation and fibrinolytic factors in moderately hyperlipidemic humans.
Dietary alpha-linolenic acid (ALA) can be converted to long-chain (n-3) PUFA in humans and may potentially reproduce the beneficial effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on risk factors for coronary heart disease (CHD). This study compared the effects of increased intakes of ALA with those of dietary EPA and DHA on blood coagulation and fibrinolytic factors in fasting subjects. A placebo-controlled, parallel study was conducted in 150 moderately hyperlipidemic subjects, age 25-72 y. Subjects were randomly assigned to one of five interventions and consumed a total intake of 0.8 or 1.7g/d EPA+DHA, 4.5 or 9.5g/d ALA or control (linoleic acid; LA) for 6 mo. Fatty acids were incorporated into 25 g of fat spread, which replaced the subject's normal spread and three capsules. Long-term supplementation with either dietary EPA+DHA or estimated biologically equivalent amounts of ALA did not affect factors VIIa, VIIc, VIIag, XIIa, XIIag, fibrinogen concentrations, plasminogen activator inhibitor-1 or tissue plasminogen activator activity compared with the control. (n-3) PUFA of plant or marine origin do not differ from one another or from LA in their effect on a range of blood coagulation and fibrinolytic factors.
Adult↗
Role of heparin and heparinlike molecules in thrombosis and atherosclerosis.
Antithrombin is a protease inhibitor that neutralizes the activity of the serine proteases of the coagulation cascade, such as factors IXa, Xa, XIa, XIIa, and thrombin by forming a 1:1 stoichiometric complex between enzyme and inhibitor via a reactive site (arginine)-active center (serine interaction). Heparin binds to lysyl residues on antithrombin and accelerates the rate of complex formation. Studies of the binding parameters and kinetic characteristics of the heparin-antithrombin-hemostatic enzyme interactions have revealed that binding of heparin to antithrombin is responsible for a approximately 1000-fold acceleration of the thrombin-antithrombin or factor IXa-antithrombin and factor Xa-antithrombin interactions (allosteric effect). The reactions between free thrombin or free factor IXa and heparin provide an additional 4- to 15-fold enhancement in the rate of these processes (approximation effect) and account for 1-2% of the total rate of enhancement. It has been shown that commercial heparin is composed of anticoagulantly active and anticoagulantly inactive species. The anticoagulantly active mucopolysaccharide contains a unique antithrombin-binding site. Anticoagulantly inactive heparin does not possess this structure and does not bind to the protease inhibitor. Anticoagulantly active heparin also contains a critical region required for the acceleration of the various enzyme-inhibitor interactions. The two different domains of the heparin molecule interact with separate areas of antithrombin and induce distinct conformational transitions within the protease inhibitor. Anticoagulantly active heparinlike molecules (most likely a heparan sulfate with an appropriate sequence for anticoagulant activity) are found on the luminal surface of the endothelium. This heparinlike substance appears to alter the conformation of antithrombin in a manner virtually identical to that of commercial heparin. Both anticoagulantly active heparin and inactive heparin are able to suppress smooth muscle cell proliferation in vitro and in vivo and can reverse the effects of mitogenic factors such as platelet-derived growth factor. Furthermore, it has been shown that bovine aortic endothelial cells produce heparinlike molecules with growth inhibitory potency.
Animals↗
[Pharmacology of heparin].
After introductory notes on the history of heparin research the chemistry of this mucopolysaccharide is described. It is shown that the chemistry of heparin cannot be exactly described by one chemical formula because heparin represents a family of compounds with different chain-length. Molecular features responsible for the high structural specificity of anticoagulant activity are described. The present status of knowledge on the mechanism of the anticoagulant action of heparin is described in detail. It is shown that basic mechanism is the acceleration of the interaction of antithrombin III with the clotting factors IXa, Xa, XIa, XIIa, and thrombin which are known to be serine proteases. The effects of heparin of platelet function, lipid composition of blood, and on fibrinolysis reflect its complex influence on hemostasis. After a description of side effects and toxicity clinically relevant data on its pharmacocinetics are given.
Animals↗