Synthetic Thrombin Inhibitor Research Articles

Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway.

Argatroban is a synthetic thrombin inhibitor approved by U.S. Food and Drug Administration for the treatment of thrombosis. However, whether it plays a role in the repair of spinal cord injury is unknown. In this study, we established a rat model of T10 moderate spinal cord injury using an NYU Impactor Moder III and performed intraperitoneal injection of argatroban for 3 consecutive days. Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord. RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway, which is involved in astrogliosis and glial scar formation. Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway. Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord. Taken together, these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway, thereby promoting the recovery of neurological function after spinal cord injury.

Heparin-Induced Thrombocytopenia in Infants after Heart Surgery.

Heparin-induced thrombocytopenia (HIT) in infants is a rare disorder, and the diagnosis and management of HIT still remains challenging. Argatroban is a synthetic direct thrombin inhibitor (DTI) that is widely used for treating HIT. However, little is known about the efficacy of the activated clotting time (ACT) test in monitoring DTI treatment as an alternative to the routinely used activated partial thromboplastin time (aPTT). Between July 2013 and January 2015, four infants were diagnosed with HIT after surgical correction of congenital anomalies. In all cases, heparin was used during cardiopulmonary bypass (CPB). Diagnosis of HIT was based on the "4 Ts" pretest clinical scoring system, and platelet factor 4 (PF4) antibody was detected using enzyme-linked immunosorbent assay. Argatroban was used in treating HIT. When argatroban was infused, anticoagulation tests (aPTT, prothrombin time [PT], thrombin time [TT], and fibrinogen) were performed every 4 to 12 hours. ACT was used in addition to monitor the anticoagulation effect of argatroban. The target ACT was 1.5 to 3.0 times the baseline. ACT was measured every 2 to 4 hours and remeasured 1 hour after each dosage adjustment. Thrombocytopenia (defined as a 50% decrease in platelet count) occurred during the 3rd to 6th day postoperatively. After the diagnosis of HIT, argatroban was started immediately, and platelet counts stabilized and gradually increased. Anticoagulation effect of argatroban was successful monitored by ACT and aPTT. Poor correlation between the ACT test and aPTT test (R = 0.270, p = 0.092) was noted in one patient. ACT values increased rapidly after 3 to 7 days on argatroban treatment. In most cases, low dosage of argatroban was given ranging from 0.04 to 5.00 μg/kg/min. Argatroban may be an effective medicine in treating HIT in infants, in a reduced dosage. The great fluctuation in argatroban dosage during the course of HIT treatment necessitates close monitoring. ACT test may be reliable and convenient for monitoring HIT treatment and may contribute to positive clinical outcomes in infants. The efficacy of argatroban and the use of ACT monitoring in the management of HIT infants needs further study.

Development of Orally Active Thrombin Inhibitors for the Treatment of Thrombotic Disorder Diseases

Thrombotic disorders represent the major share of the various cardiovascular diseases, and significant progress has been made in the development of synthetic thrombin inhibitors as new anticoagulants. In addition to the development of highly potent and selective inhibitors with improved safety and suitable half-life, several allosteric inhibitors have been designed and synthesized, that did not fully nullify the procoagulant signal and thus could result in reduced bleeding complications. Furthermore, natural products with thrombin inhibitory activity have been isolated, and some natural products have been modified in order to improve their inhibitory activity and metabolic stability. This review summarizes the development of orally active thrombin inhibitors for the treatment of thrombotic disorder diseases, which could serve as a reference for the interested researchers.

Heparin-Induced Thrombocytopenia and Hemodialysis

Hemodialysis-related-heparin-induced thrombocytopenia (HD-HIT) is a drug-induced, immunoglobulin- mediated disorder that it is suspected in dialysis patients with an unexpected fall in the platelet count, and/or unexplained thrombotic events, particularly visible clotting in the circuit under an adequate heparin dose, that begins between 5 and 10 days (nadir between 7 and 30 days, mostly by the third to fifth session) after heparin initiation. Although a positive result for anti-PF4/heparin complex antibodies (HIT antibodies) is presumably detected by sensitive ELISA, the diagnosis should be confirmed, whenever possible, using a functional assay. Immediately after the clinical suspicion of HIT, all sources of heparin should be discontinued including heparin used to flush or lock catheters. Alternative non-heparin anticoagulants, preferentially a direct thrombin inhibitor, should be restarted for dialysis. Early treatment is important as thrombus formation including a clotting circuit may complicate at a high rate within 30 days after the cessation of heparin. Argatroban, a synthetic direct thrombin inhibitor, as an alternative to heparin, must contribute to the rapid recovery of the platelet count and disappearance of visible circuit clotting. A steady decreasing of the ELISA titers can be expected after heparin discontinuation. A negative seroconversion of HIT antibodies is usually observed by ~30 to more than 100 days after discontinuation. Re-exposure to heparin can be selected at the same dose of heparin as used before the onset of HIT. A small peak of HIT antibodies may often appear after exposure, but a follow-up of the antibody titers shows that they not reach a threshold to induce the recurrence of HIT. When HD-HIT patients exhibit a high index of thrombotic formation or worsening thrombosis, the same alternative anticoagulant therapy may be needed on non-session days.

New Synthetic Thrombin Inhibitors: Molecular Design and Experimental Verification

BackgroundThe development of new anticoagulants is an important goal for the improvement of thromboses treatments.ObjectivesThe design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration.MethodsComputer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured.ResultsNew compounds that are both effective direct thrombin inhibitors (the best KI was <1 nM) and strong anticoagulants in plasma (an IC50 in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD50 values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C.ConclusionsThe high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

Use of Subcutaneous Lepirudin in an Obese Surgical Intensive Care Unit Patient with Heparin Resistance

To report the use of subcutaneous lepirudin in an obese patient with heparin resistance. A 34-year-old morbidly obese male (weight 145 kg) presented with hypoxia on postoperative day 1 following a sigmoid colectomy. A continuous unfractionated heparin infusion was started for a suspected pulmonary embolism. Doses were escalated without therapeutic activated partial thromboplastin time (aPTT) response and an antithrombin (AT) level was obtained. The AT level was reported as 78% (reference range 85-120%). Computed tomography angiography ruled out pulmonary embolism and lepirudin 50 mg administered subcutaneously twice daily was started (serum creatinine 1.3 mg/dL) for prevention of venous thromboembolism. The resulting aPTT values were therapeutic (63 and 60 sec, reference range 24-34, therapeutic heparin range 55-85). The dose was adjusted to 25 mg twice daily. aPTT values were 35 and 48 seconds. His serum creatinine increased to 1.6 mg/dL and minor bleeding was noted. The dose was decreased to 25 mg once daily, with resulting aPTT values of 31, 39, and 41 seconds. The patient was discharged to home without development of venous thromboembolism, as confirmed by duplex ultrasonography. Commonly administered anticoagulants, such as unfractionated heparin, low-molecular-weight heparins, and fondaparinux, exert their effect by complexing with AT and thrombin (Factor IIa), activating AT and preventing thrombin from exerting coagulation effect. Without AT present, these drugs have little effect on inhibiting the coagulation cascade. Lepirudin is a synthetic irreversible direct thrombin inhibitor and does not rely on AT to exert its anticoagulation action. It can be given subcutaneously and is eliminated primarily by the kidneys. Dosage adjustments for both renal function and obesity need to be considered and aPTT should be monitored. In obese patients or those with heparin resistance, subcutaneous lepirudin can be monitored and the regimen adjusted based on aPTT values. Further studies are warranted to maximize efficacy and define dosing.

New anticoagulants for secondary haemostasis

In contrast to heparins and oral anticoagulants, anti IIa inhibitors (thrombin inhibitors) are able to directly inhibit the protease activity of thrombin and can thereby precisely control the blood coagulation process. Direct thrombin inhibitors are either biosimilars (r-hirudin) or synthetically produced substances (bivalirudin, argatroban, dabigatran). In 1997 r-hirudin was introduced into clinical practice, however due to its narrow therapeutic range and the necessity of drug monitoring it has not gained widespread clinical use by now. Since 2004 and 2005 the synthetic thrombin inhibitors bivalirudin and argatroban, respectively, are available. With dabigatran the first oral synthetic thrombin inhibitor followed in 2008. These four drugs can inhibit even clot bound thrombin and show low plasma protein binding. They differ in respect to route and duration of application as well as elimination from the body, thereby offering a precise inhibition of blood coagulation adjusted to the individual case and without danger of HIT II. These advantages shall be used and advanced by the development of further direct thrombin inhibitors.

Antikoagulanzien

In contrast to heparins and oral anticoagulants, anti IIa inhibitors (thrombin inhibitors) are able to directly inhibit the protease activity of thrombin and can thereby precisely control the blood coagulation process. Direct thrombin inhibitors are either biosimilars (r-hirudin) or synthetically produced substances (bivalirudin, argatroban, dabigatran). In 1997 r-hirudin was introduced into clinical practice, however due to its narrow therapeutic range and the necessity of drug monitoring it has not gained widespread clinical use by now. Since 2004 and 2005 the synthetic thrombin inhibitors bivalirudin and argatroban, respectively, are available. With dabigatran the first oral synthetic thrombin inhibitor followed in 2008. These four drugs can inhibit even clot bound thrombin and show low plasma protein binding. They differ in respect to route and duration of application as well as elimination from the body, thereby offering a precise inhibition of blood coagulation adjusted to the individual case and without danger of HIT II. These advantages shall be used and advanced by the development of further direct thrombin inhibitors.

Hemodialysis and Argatroban

Hemodialysis is indicated in uremic patients with endstage renal failure. Unfractionated heparin (heparin) is routinely used as the anticoagulant for hemodialysis, but heparin is contraindicated for dialysis of patients with antithrombin deficiency or heparin-induced thrombocytopenia (HIT). With heparin, these patients often encounter clotted dialyzers and extracorporeal circuits that lead to discontinuation of dialysis. As one alternative to heparin, argatroban (a synthetic thrombin inhibitor mainly eliminated via the hepatobiliary tract) is indicated. In uremic patients with antithrombin deficiency, the alternative use of argatroban has made it feasible to carry out hemodialysis. In patients with HIT undergoing dialysis, the use of argatroban as an alternative to heparin improves thrombocytopenia and safely prevents clotting of the hemodialysis circuit. Because the elimination of argatroban does not depend on renal function and there is no need for strict dose monitoring, argatroban is recommended as a suitable anticoagulant for patients with HIT requiring hemodialysis.

Drug Monitoring of Argatroban Using the Ecarin Chromogenic Assay

The ecarin chromogenic assay (HaemoSys ECA; JenAffin GmbH, Jena, Germany) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle of measurement: the activation of prothrombin by ecarin, a snake venom from Echis carinatus. In the ECA, the prothrombin activation products meizothrombin and meizothrombin-des-F1 cleave a chromogenic substrate. The activity of meizothrombin/meizothrombin-des-F1 is inhibited in a concentration-dependent manner by direct thrombin inhibitors. The ECA-T is an assay for quantitative determination of active site directed synthetic thrombin inhibitors. For argatroban ECA-T, there is a very precise and sensitive method of quantitative drug monitoring. Only very low interindividual variations were found compared with activated partial thromboplastin time and even ECT. ECA-T is independent of variations in prothrombin levels of the plasma samples. Heparin does not influence the measuring result. ECA-T is performed on manual coagulation analyzers with an option for optical measurement but can also be applied to automated laboratory systems. By the use of ECA-T as a drug-monitoring method, the drug safety of argatroban, most of all in critically ill patients, can be increased.

Thrombin enhances the production of monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 in cultured rat glomerular epithelial cells

Glomerular crescents play an important role in progressive glomerular injury. The lesions consist of epithelial cells, macrophages and deposits of fibrin and extracellular matrix. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) are members of chemokine subfamilies. MCP-1 and MIP-2 are potent chemoattractant leukocyte cytokines, and they may be involved in crescent formation. Thrombin participates in fibrin formation. We hypothesized that thrombin stimulates the production of MCP-1 and MIP-2 by glomerular epithelial cells (GECs). Cultured rat GECs from the 19th to the 24th passage were used. We incubated GECs with or without thrombin to examine the effect of thrombin on the production of MCP-1 and MIP-2. The levels of MCP-1 and MIP-2 were measured in the cell supernatants by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of MCP-1 and MIP-2 were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). We also examined the inhibitory effect of argatroban, a synthetic thrombin inhibitor, and prednisolone in the production of MCP-1 and MIP-2 stimulated by thrombin. Thrombin stimulated the production of MCP-1 and MIP-2 in a dose- and time-dependent manner. Thrombin also enhanced the mRNA expressions of MCP-1 and MIP-2 in the GECs. The stimulating effect of thrombin on the production of MCP-1 and MIP-2 was inhibited by the addition of argatroban or prednisolone. We demonstrated a novel role of thrombin: it stimulates the production of MCP-1 and MIP-2 by GECs. It is clinically important that the inhibition of these chemokines leads to the improvement of crescentic glomerulonephritis. Anti-thrombin drugs and prednisolone may be useful in treating crescentic glomerulonephritis.

Heparin-induced thrombocytopenia in a pediatric patient receiving extracorporeal membrane oxygenation managed with argatroban???

Heparin-induced thrombocytopenia (HIT) is rare in the pediatric population, with a majority occurring in the pediatric intensive care unit setting. All cases reported to date have been associated with the use of unfractionated heparin. Because unfractionated heparin is the anticoagulant of choice for extracorporeal membrane oxygenation (ECMO) and other extracorporeal therapies, the development of HIT in these patients can be devastating, making management problematic. We report a case of HIT type II with evidence of small-vessel arterial thrombosis in a 17-month-old boy receiving ECMO and continuous renal replacement therapy successfully treated with argatroban. The patient was a 17-month-old boy with severe hypercapnic and hypoxemic respiratory failure secondary to asthma and mucus plugging that failed conventional and unconventional ventilation. Venovenous ECMO was initiated, and within 24 hrs, there was a precipitous decrease in the platelet count, with the development of cutaneous ischemia involving his lower limbs. Argatroban was started and the child maintained on ECMO and continuous renal replacement therapy, with resolution of the cutaneous ischemia and recovery of the platelet count. Heparin-associated antibodies were positive. HIT is rare in the pediatric population. Recognition of HIT is vital because withdrawal of heparin is the first and most important therapy. For patients receiving ECMO or continuous renal replacement therapy who develop HIT, synthetic thrombin inhibitors have been reported as an alternative. However, little information on their use in extracorporeal life support has been published, particularly in the pediatric population. This report documents a pediatric case of HIT type II successfully treated with argatroban, allowing continuation of the ECMO and continuous renal replacement therapy therapy, with resolution of the cutaneous ischemia and thrombocytopenia.

Heparin-induced thrombocytopenia in a pediatric patient receiving extracorporeal support and treated with argatroban

Heparin-induced thrombocytopenia (HIT) is rare in the pediatric population, with a majority occurring in the pediatric intensive care unit setting. All reported cases have been associated with the use of unfractionated heparin. Because unfractionated heparin is the anticoagulant of choice for extracorporeal life support, the development of HIT in these patients can be devastating. We report a case of HIT with evidence of small-vessel arterial thromboembolism in a 17-month-old child receiving extracorporeal membrane oxygenation and continuous renal replacement therapy successfully treated with argatroban. The patient was a 17-month-old boy with severe respiratory failure secondary to asthma and mucus plugging that failed conventional and unconventional ventilation. Venovenous extracorporeal membrane oxygenation was initiated, and within 24 hrs, there was a precipitous decrease in the platelet count, with the development of cutaneous ischemia involving his lower limbs. Heparin-associated antibodies were positive. Argatroban was started, and the child maintained on extracorporeal membrane oxygenation and continuous renal replacement therapy, with resolution of the cutaneous ischemia and rebound of the thrombocytopenia. HIT is rare in the pediatric population. Recognition of HIT is vital because withdrawal of heparin is the first and most important therapy. For patients on extracorporeal membrane oxygenation or continuous renal replacement therapy who develop HIT, synthetic thrombin inhibitors (hirulogs) have been reported as an alternative. However, little information on their use in extracorporeal life support has been published, particularly in the pediatric population. This report documents a pediatric case of HIT successfully treated with argatroban, allowing continuation of the venovenous extracorporeal membrane oxygenation and continuous renal replacement therapy, with resolution of the thromboembolic ischemia and thrombocytopenia.

Ecarin chromogenic assay: an innovative test for quantitative determination of direct thrombin inhibitors in plasma

The ecarin chromogenic assay (ECA) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle, the activation of prothrombin by ecarin a snake venom from Echis carinatus. In the ECA the prothrombin activation products meizothrombin and meizothrombin-desF1 cleave a chromogenic substrate, whereas in the clotting assay ECT plasma fibrinogen is converted to fibrin. The activity of meizothrombin/meizothrombin-desF1 is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. The ECA can be used as ECA-H for quantitative determination of hirudin and as ECA-T for determination of synthetic thrombin inhibitors. As shown for hirudin, argatroban and melagatran, the ECA turned out as a very precise and sensitive method, which combines the advantages of ECT with those of chromogenic assays. In ECA very low interindividual variations were found compared to aPTT and even ECT. The ECA is independent of the variations of the coagulation variables prothrombin and fibrinogen.

Thrombin inhibitors identified by computer-assisted multiparameter design.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.

A molecular dynamics study on liquid 1‐octanol. Part 3. Evaluating octanol/water partition coefficients of novel thrombin inhibitors via free‐energy perturbations

AbstractThrombin inhibition is an important strategy for the treatment of thrombotic and embolic disorders. Despite high in vitro affinity for the enzyme, synthetic thrombin inhibitors have limited oral bioavailability due to low intestinal permeability. In a previous work, we obtained the following order for thrombin binding by using molecular dynamics (MD) simulations and the finite difference thermodynamic integration (FDTI) method: p‐(2‐oxo‐1‐propyl)benzamidine (POPBz) &gt; p‐ethylbenzamidine (PEBz) &gt; p‐(1‐propyl)benzamidine (PPBz) &gt; p‐methylbenzamidine (PMBz) &gt; benzamidine (Bz). As the octanol/water partition coefficient (log Po/w) is a thermodynamic property that may be related to drug transfer across biological membranes, we now turn our attention to the calculation by the FDTI method of relative log Po/w for the benzamidine derivatives. To examine the reliability of the method, log Po/w values for Bz and PMBz were determined experimentally. Experimental log Po/w of −1.02 and −0.89 were obtained for Bz and PMBz, and theoretical log Po/w of −0.12, −0.01, and −1.00 were obtained for PEBz, PPBz, and POPBz. Thus, we improved the hydrophobicity of Bz by adding the methyl, ethyl, propyl, and 2‐oxo‐propyl groups, but further substitutions should be conducted to raise log Po/w above zero and into a region where reasonable oral absorption might be expected. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005

Argatroban Treatment for Patients with Intoleranceto Heparin and Hirudin

Argatroban is a synthetic thrombin inhibitor that acts independently of any cofactor. It inhibits free as well as fibrin- and clot-bound thrombin. The clinical history of two patients is presented, both with previous allergic reactions toward heparin and hirudin. In both patients, antibodies against hirudin were documented. Argatroban was successfully used in both patients without any side effects. We conclude argatroban to be the drug of choice in patients with intolerance to heparin and hirudin. Moreover, genetic variations affecting the hepatic catabolism of argatroban might exist, because very different dosages of argatroban were needed in the two patients.

Progress in the development of synthetic thrombin inhibitors as new orally active anticoagulants.

The trypsin-like serine protease thrombin is a multifunctional key enzyme at the final step of the coagulation cascade and is involved in the regulation of hemostasis and thrombosis. An increased activation of coagulation can result in severe thromboembolic disorders, one of the major reasons responsible for mortality and morbidity in western world. Therefore, an effective, safe, and orally available thrombin inhibitor could be a useful anticoagulant drug for the daily prophylaxis of venous and arterial thrombosis and prevention of myocardial infarction for high-risk patients. Synthetic thrombin inhibitors have a long history; initial compounds were derived from electrophilic ketone- and aldehyde-analogs of arginine. First potent leads of non-covalent inhibitors were developed in the early eighties, which were further optimised in the nineties, after the X-ray structure of thrombin became available. In the meantime a huge number of highly active and selective inhibitors has been published, however, only a few of them have an appropriate pharmacokinetic and pharmacodynamic overall profile, which could justify their further development. Very recently, with Ximelagatran a first orally available thrombin inhibitor has been approved in France for the prevention of venous thromboembolic events in major orthopaedic surgery after successful clinical phase III. However, it still has to be awaited, whether the extensive clinical use of Ximelagatran can demonstrate for the first time that direct thrombin inhibitors offer a real benefit in terms of efficacy and safety over established antithrombotic therapies. This review summarizes the current status of synthetic thrombin inhibitors with a focus on more recently published and promising new compounds.

Human complement factor I does not require cofactors for cleavage of synthetic substrates.

Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino-4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH(3)). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzenesulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, only Cr(2+) and Fe(3+) were found to inhibit both the proteolytic and amidolytic activity of fI.

Production and characterization of bioactive peptides from soy hydrolysate and soy-fermented food

Soy hydrolysate and the soy-fermented foods, natto and tempeh, were dephosphorylated, deglycosylated and digested with a variety of endoproteases (pronase, trypsin, Glu C protease, plasma proteases and kidney membrane proteases) to generate oligopeptides. The peptides were purified and characterized. They demonstrated a range of biological activities – angiotensin converting enzyme (ACE) inhibitory, anti-thrombotic, surface tension and antioxidant properties. The biologically active peptides were mostly derived from glycinin, a highly expressed soy protein, and were found mainly in the pronase, kidney membrane proteases and plasma proteases digests of the fermented foods. The proteases of lower specificity produced more oligopeptides and a higher percentage of bioactive peptides than the proteases of higher specificity, namely trypsin and Glu C. A peptide with ACE inhibitory activity was found in the pronase digest of natto, which also produced a peptide with surface active properties. The kidney membrane hydrolysate of natto contained an ACE inhibitor, in addition to a peptide with anti-thrombotic activity bearing homology to hirutonin, a previously described synthetic thrombin inhibitor [DiMaio, Gibbs, Lefebvre, & Munn, 1992, J. Med. Chem. 35, 3331]. Synthetic analogs were also evaluated as substrates for some inhibitors.