Low Molecular Weight Protease Inhibitor Research Articles

Identification and biochemical characterization of a Kunitz‐type serine protease inhibitor from an insect, Manduca sexta (772.1)

Hemolymph (blood) of insects contains serine proteases and serine protease inhibitors that function in innate immune responses. The objective for this research was to investigate low molecular weight protease inhibitors from hemolymph of a lepidopteran insect, Manduca sexta. We identified serine protease inhibitor activity against trypsin, chymotrypsin, and elastase in plasma from naïve larvae and in plasma from larvae that were previously injected with Micrococcus luteus. We separated hemolymph plasma proteins using size exclusion chromatography, and the fractions with protease inhibitor activity were analyzed by SDS‐PAGE and liquid chromatography‐electrospray ionization tandem mass spectrometry. A serine protease inhibitor belonging to the Kunitz family was identified, and using primers based on the sequence encoding this protein found in the M. sexta genome sequence, we cloned its cDNA from larval fat body RNA. The cDNA encodes a protein with a secretion signal sequence followed by the mature secreted inhibitor protein of 56 amino acid residues. This sequence is highly similar to a Kunitz inhibitor previously purified from M. sexta (Ramesh, N., Sugumaran, M., Mole, J.E. 1988. J Biol Chem. 263:11523‐7). Using a synthesized and codon‐optimized DNA encoding the protease inhibitor, we are currently producing the recombinant inhibitor in E. coli for further biochemical characterization. We will study its inhibitory activity as a potential regulator of proteases in hemolymph that function in the innate immune system of M. sexta. The genome of M. sexta contains nine genes for single‐domain Kunitz proteins closely spaced on a single scaffold. The ultimate goal of this project is to understand the biological functions of these Kunitz protease inhibitors in innate immunity.Grant Funding Source: Supported by NIH grant GM41247.

Sitagliptin does not inhibit the M1 alanyl aminopeptidase from Plasmodium falciparum.

The M1 alanyl aminopeptidase from Plasmodium falciparum has been shown to be an essential hemoglobinase enzyme, catalyzing the final stages of hemoglobin break-down within intra-erythrocytic parasites 1. Recently there has been much interest in this protease as a potential drug target for the development of novel antimalarials [1–13]. In a recent report, Krishnamoorthy and Achary propose that Sitagliptin may serve as a potent competitive inhibitor of the M1 alanyl aminopeptidase enzyme from Plasmodium falciparum [14]. The molecule of interest, the M1 alanine aminopeptidase or PfA-M1, has been well studied and characterized by our group and others (For examples of recent papers, see [1–13]). To date, multiple published X-ray crystal structures and models are available of inhibitors bound to the active site of PfA-M1. Krishnamoorthy and Achary report a docking analysis of the enzyme with “about 100 low molecular weight protease inhibitors …”. The docking results were validated by inclusion of specific substrate (Ala-β-naphthylamide) from which they calculated an in silico Km value that was closely correlated with experimental data. Unfortunately, this correlation with experimental data was not observed with the selected positive inhibitor control, Bestatin. The interaction / inhibition of PfAM1 by Bestatin has been reported previously [1, 9, 12, 15] and the dipeptide analog has an in vitro Ki in the nM range for PfAM1. The in silico value calculated was ~ 100 μM, indicating that the parameters defined for docking were likely inadequate for the cation-occupied active site. The article states that Sitagliptin is the most potent in silico inhibitor with a Ki(avg) of 2 uM (however only 8/100 docking results were provided in Supplementary Table 1). We have completed an in vitro analysis of the effect of Sitagliptin on the activity of both PfA-M1 and the second neutral aminopeptidase, PfA-M17. Aminopeptidase activity assays were carried out in 200 µl total volume in 50 mM Tris-HCl pH 8.0, at 37 °C (with the addition of 2 mM CoCl2 for PfA-M17). Following a 10 min incubation of enzyme and Sitagliptin (0 – 0.5 mM), reactions were initiated by addition of fluorigenic substrate (L-Leucine-7-amido-4-methylcoumarin). Progress curves were monitored using a spectrofluorimeter until a final steady-state velocity was reached. We determined the inhibitory kinetics via Ki values from Dixon plots of 1/υs versus inhibitor concentration when [S]<<KM. We report here that this compound had no effect on the aminopeptidase activity of either enzyme. in silico drug docking suffers from several problems, including modeling the physics of the system, solvent effects, dynamics, and the difficulty in accurately ranking the docked results, and therefore relies critically on validation by experiment. It is vital that we rigorously test hypotheses generated from webservers to ensure that these algorithms continue to improve in their accuracy and hence usefulness. Blanket statements about efficacy from untested in silico studies will confound the literature and waste precious resources by following up on false positives identified in poorly controlled in silico studies. Computational biology has an important role to play in research and drug design; however, it is absolutely vital that we apply critical evaluation of results obtained to ensure that it becomes a robust method in the future.

Exploration of Sitagliptin as a potential inhibitor for the M1 Alanine aminopeptidase enzyme in Plasmodium falciparum using computational docking.

Plasmodium falciparum has limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin to maintain protein metabolism and synthesis of proteins. M1 alanine aminopeptidase enzyme of the parasite involved in the terminal degradation of host hemoglobin was subjected to in silico screening with low molecular weight protease inhibitors. The km (avg) of the enzyme M1 alanine aminopeptidase for the substrate DL – Alanine β Napthylamide Hydrochloride was estimated as 322.05µM. The molecular interactions between the enzyme and the substrate and the mechanism of enzyme action were analyzed which paved way for inhibition strategies. Among all the inhibitors screened, Sitagliptin was found to be most potent inhibitor with ki of 0.152 µM in its best orientation whereas the ki(avg) was 2.0055 µM. The ki of Sitagliptin is lower than the km of M1 alanine aminopeptidase for the substrate DL – Alanine β Napthylamide Hydrochloride (322.05 µM) and Ki of the known inhibitor Bestatin. Therefore Sitagliptin may serve as a potent competitive inhibitor of the enzyme M1 alanine aminopeptidase of Plasmodium falciparum.

Diffusion of Active Proteins into Fish Meat To Minimize Proteolytic Degradation

Proteases in fish muscle often cause undesired softening of intact meat pieces during refrigerated storage or slow cooking. Several food-grade proteinaceous inhibitors can overcome this softening if properly delivered to the intracellular sites where proteases are located. Fluorescence recovery after photobleaching (FRAP) and laser scanning confocal microscopy (LSCM) were used to measure the translational diffusion of fluorescein isothiocyanate (FITC)-labeled protease inhibitors into intact muscle fibers of halibut. Diffusion coefficients (D) of alpha-2-macroglobulin (720 kDa), soybean trypsin inhibitor (21 kDa), and cystatin (12 kDa) were measured in both muscle fibers and dilute aqueous solutions. On the time scale of the observation (35 min), cystatin and soybean trypsin inhibitor diffused through the cell membrane (sarcolemma) and sarcoplasm, but at a considerably slower rate (>10-fold difference) than in dilute aqueous solution. alpha-2-Macroglobulin did not diffuse into muscle cells within the time frame of the experiment, but did completely penetrate the cell during overnight exposure. The present study thus shows a clear dependence of D on protein inhibitor size when moving within intact skeletal muscle fibers. Low molecular weight protease inhibitors such as cystatin can be effectively diffused into intact fish muscle cells to minimize proteolytic activity and meat softening.

EXPRESSION PROFILES OF POTATO PROTEINASE INHIBITORS POTENTIALLY USEFUL FOR THE CONTROL OF PHYTOPATHOGENIC FUNGI

Proteinase inhibitors (PIs) are small ubiquitous proteins showing a variety of biological functions in plants, such as stabilization of proteins, modulation of apoptosis and defence against pathogens. They are particularly abundant in storage organs, such as seeds and tubers, and can accumulate in other plant tissues following a variety of different stimuli. Besides patatin, the major protein constituents of potato (Solanum tuberosum) tubers are low molecular weight PIs, such as protease inhibitors I (PI-1), II (Pin2) and Kunitz-type (PKPIs). PI-1 and PKPI inhibitors are coded in potato plants by relatively small gene families and synthesized as pre-pro-proteins (signal peptide-propeptidemature proteins). A minimum of 21 Kunitz inhibitor genes (subdivided, on the basis of their sequence homology, into group A, B, and C) has been found to be present in the potato genome, while contrasting data have been reported for the number of genes belonging to the PI-1 family. In addition, PKPI and PI-1 accumulate in different plant tissues in response to biotic or abiotic stresses. However, little information is today available about the regulation (e.g. after nematode infection) of individual PI-1 genes or PKPI groups. In the present work, a PI mixture, obtained from potato (cv.. Desiree) sprouts and showing a strong inhibitory activity against Botrytis cinerea fungal proteases, spore germination, hyphal elongation and necrotic activity, was purified and sequenced. Five novel full length PI-1 cDNAs (PPI3B2, PPI3A2, PPI3A4, PPI2C4 and PPI2C1A) and two PKPI genes (PKI1 and PKI2) were isolated, sequenced and characterized. Southern analysis, performed to confirm the presence of PKI1 and PPI3B2 genes and to determine the number of PI-1 genes in the genome, indicated that the PKI1 gene is present as a single copy in the highly polymorphic family of the Kunitz inhibitors of the cultivar Desiree. On the other hand, the hybridizing bands obtained with the PPI3B2 probe (at least nine) indicate that this gene could be part of a small multigene family (~ 10 members). Alignment of cloned and database retrieved PI inhibitor genes was used to design family, group or gene specific primers to individually analyze target transcripts by semiquantitative RT-PCR. Expression profiles of the three PKPI groups (A, B and C), Pin2 and PR1 (pathogenesi related 1) gene families, and the PI-1 family genes PPI3A2, PPI3B2, PPI2C4 were studied in various tissues of Desiree, LB7/4/c-I-7 and P40 potato genotypes, after wounding and infection by Globodera rostochiensis. PI, but not PR1 genes, were found to be involved in the plant response triggered by the nematode G. rostochiensis, indicating an activation of the defense pathway via jasmonic acid. Moreover, individual PI-1 genes and PKPI groups were expressed in a tissue and genotype specific manner after biotic and abiotic stresses. The differences in the PI expression patterns concerned the intensity, type of inhibitors and rapidity of induction.

Cytokine-mediated induction of the human elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB.

Elafin, a low molecular-weight proteinase inhibitor, is a member of the recently described trappin gene family. These proteins are thought to play important roles in the regulation of inflammation and are expressed in multiple epithelia. Elafin is found within the lung, and its expression can be induced by inflammatory mediators. The molecular mechanisms that mediate its induction are not understood. In this study we investigated the transcriptional regulation of the elafin gene in pulmonary epithelial cell lines. Transfection of elafin promoter constructs into the elafin-expressing pulmonary epithelial cell line A549 identified a number of positive-acting elements. Cytokine-mediated inducibility of the elafin gene promoter was shown to occur through a nuclear factor (NF)-kappaB site present within the minimal promoter. This site was shown to bind to NF-kappaB proteins within nuclear extracts from cytokine stimulated cell lines as well as to in vitro-translated RelA. Cotransfection with both RelA and NF-kappaB-inducing kinase induced reporter gene activation via this site, and mutagenesis experiments confirmed that it was crucial for induction of elafin gene activity. These results clearly identify a role for elafin in the inflammatory response of the airway epithelium to pathogenic insult and show that this response is mediated by an NF-kappaB site within the proximal promoter.

A Functional Proteomics Screen of Proteases In Colorectal Carcinoma

Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.

Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences

A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

Methionine and Sulfate Increase a Bowman−Birk-Type Protease Inhibitor and Its Messenger RNA in Soybeans

A soybean cotyledon cDNA library was screened for clones representing RNAs that were more abundant in cotyledons grown on methionine-supplemented medium than in those grown on unsupplemented (basal) medium. Three isolated clones encoded the Bowman−Birk protease inhibitor (BBPI). RNA gel blot hybridization confirmed that the BBPI mRNA level is increased by methionine (Met). Met and/or sulfate supplementation increased the amount of protease inhibitor activity in cultured cotyledons. In seeds of greenhouse-grown plants, protease inhibitor activity was increased by the addition of exogenous sulfate to the nutrient solution. Since BBPI and related low molecular weight proteinase inhibitors have an unusually high cyst(e)ine content, these compounds have been hypothesized to function in the storage of reduced sulfur. The data presented here support this hypothesis by providing evidence that additional sulfur as Met or sulfate increases the quantity of these proteins in seeds by increasing their mRNA levels. Keywords: Bowman−Birk; Glycine max; protease inhibitor; soybean; sulfate

Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain.

A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish, Pacifastacus leniusculus, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper, Locusta migratoria. The nine light chain domains and the Locusta inhibitors share a characteristic cysteine array (Cys-Xaa9-12-Cys-Xaa2-Cys-Xaa-Cys-Xaa6-8-Cys-Xaa4++ +-Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor.

Distribution of a synthetic protease inhibitor in rat pancreatic acini after supramaximal secretagogue stimulation.

Protease inhibitors may have a beneficial effect in acute pancreatitis. The effects of E3123, a new low molecular weight protease inhibitor, on the ultrastructure of isolated pancreatic acini were examined using transmission electron microscopy. Acini supramaximally stimulated with cerulein (10(-8) M) formed large cytoplasmic vacuoles similar to those generated in the cerulein-induced in vivo model of pancreatitis. Pretreatment of isolated acini with E3123 significantly reduced the size and number of vacuoles associated with cerulein treatment. The distribution of 3H-E3123 in acinar cells was examined using a pulse-chase protocol and electron microscopic autoradiography. Cellular levels of 3H-E3123 increased about 30-fold in acinar cells treated with cerulein (10(-8) M) compared to unstimulated controls. In cerulein-treated acini examined after a 5-min chase, 47.4% of the autoradiographic grains were associated with the rough endoplasmic reticulum and 13.2% were associated with zymogen granules. After 30 min of incubation, the grains associated with the endoplasmic reticulum decreased to 18.5% but increased to 26.3% over zymogen granules. Thus, E3123 is taken up by the acinar cell and follows a cellular itinerary similar to that of newly synthesized secretory proteins. One potential conclusion from these studies is that the ability of E3123 to reduce the formation of vacuoles in supra-maximally stimulated acini may be due to its inhibition of proteases within the secretory pathway.

The Role of the Calpain-Calpastatin System in Thyrotropin-releasing Hormone-induced Selective Down-regulation of a Protein Kinase C Isozyme, nPKCε, in Rat Pituitary GH4C1 Cells

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.

Cell surface binding of TIMP-2 and pro-MMP-2/TIMP-2 complex

Tissue inhibitor of metalloproteinases (TIMP-2) is a low molecular weight proteinase inhibitor capable of inhibiting activated matrix metalloproteinases (MMPs). TIMP-2 is found both free and in a 1:1 stoichiometric complex with the pro-enzyme form of MMP-2 (pro-MMP-2/TIMP-2 complex). We have measured the binding of recombinant TIMP-2 to intact HT-1080 and MCF-7 cells. HT-1080 cells in suspension bound 125I-labeled rTIMP-2 with a K d of 2.5 nM and 30,000 sites/cell. Monolayers of MCF-7 cells were similarly found to bind [ 125I]rTIMP-2 with a K d of 1.6 nM and 25,000 sites/cell. Specific binding of MMP-2 alone to HT-1080 cells was not observed; however, pro-MMP-2/TIMP-2 complex was capable of binding to the surface of HT-1080 cells in a TIMP-2-dependent manner. Binding of rTIMP-2 was not competed by the presence of TIMP-1. These results suggest that rTIMP-2 alone binds directly to the cell surface of HT-1080 and MCF-7 cell lines, and TIMP-2 is capable of localizing MMP-2 to the surface of HT-1080 cells via interaction with a specific binding site.

Anesthetic Agents Modify Tissue Proteinase Inhibitor Content and Tumor Behavior

Anesthetic agents may modify the tissue content of low molecular weight proteinase inhibitors (mol wt less than 50,000) and affect the colonization and proliferation of B16-F10 melanoma cells in lungs. Lungs of female mice exposed to halothane in oxygen had significantly greater low molecular weight proteinase inhibitor activity than those from unexposed female mice. The amount of activity in male mice similarly exposed did not differ from unexposed controls. Lungs of exposed female mice also had greater activity as compared with males similarly exposed. No differences occurred in identical experiments with ketamine. High-performance liquid chromatography revealed that the inhibitor in all groups of both sexes shared a major peak with a molecular weight similar to that of the trypsin inhibitor aprotinin. Female mice bearing B16-F10 melanoma cells and exposed to halothane in oxygen had significantly more small lung tumor nodules than tumor-bearing female mice injected with ketamine. However, the total number of nodules did not differ between the two groups. These observations support the hypothesis that stimulation of proteinase inhibitory activity by halothane in oxygen may be responsible for an inhibition of tumor cell proliferation, resulting in smaller tumor nodules and no effect on the incidence of colonization.

Gabexate mesilate in human acute pancreatitis

Background: A multicenter controlled study was performed to evaluate the effect of high doses of the low molecular weight protease inhibitor gabexate mesilate on mortality and complications associated with moderate and severe acute pancreatitis. Methods: Two hundred twenty-three patients from 29 hospitals were entered in the randomized, double-blind trial. Admission to the study was based on strict criteria excluding mild acute pancreatitis. The patients received placebo or 4 g gabexate mesilate per day intravenously for 7 days. All patients were followed up for 90 days after randomization. The analysis was based on 14 complications, including death. Results: There was no statistical difference in either mortality or complications associated with acute pancreatitis between the placeboand gabexate mesilate groups. Conclusions: The results show that gabexate mesilate was not effective in preventing complications and mortality in acute pancreatitis.

Cytoprotective Effects of Prostaglandins and a New Potent Protease Inhibitor in Acute Pancreatitis

The redistribution of cathepsin B, a lysosomal enzyme, from the lysosomal pellet to the zymogen pellet in the subcellular fractionation, the colocalization of cathepsin B with digestive enzyme, and increased cellular, lysosomal, and mitochondrial fragility within acinar cells have been found during the early stages of caerulein-induced acute pancreatitis in rats. In the present study, the authors investigated the protective effects of prostaglandin E1 and E2, a combined therapy of these prostaglandins, and a new, synthetic, low molecular weight protease inhibitor, ONO3307, on the exocrine pancreas in this noninvasive model of experimental pancreatitis in vivo and in vitro. Prostaglandin E2, but not E1, prevented hyperamylasemia, congestion of amylase and trypsinogen in the acinar cells, redistribution of cathepsin B, and amylase and lactate dehydrogenase discharge from the dispersed acini. It also prevented cathepsin B leakage from the lysosomes and malate dehydrogenase leakage from the mitochondria in an almost dose-dependent manner, particularly at the dose of 100 micrograms/kg/hr continuous infusion. Furthermore, the combined therapy of prostaglandin E2 with ONO3307 strongly inhibited all the parameters tested in this study. This combination therapy seems to be the most effective against secretagogue-induced pancreatic injuries. These results indicate that cellular and subcellular organellar fragility seem to be closely involved in the pathogenesis of acute pancreatitis. Prostaglandin E2 seems to have important cytoprotective effects on the biologic membranes, such as a stabilizer of lysosomal or mitochondrial membranes. In addition, these findings also suggest the crucial roles of some unknown proteases in the etiology of acute pancreatitis, and indicate the clinical effectiveness of prostaglandins and this type of low molecular weight protease inhibitor for acute pancreatitis.

A new synthetic protease inhibitor, E-3123, prevents lysosomal and mitochondrial fragility in rat caerulein-induced pancreatitis.

The study investigated the protective effect of a new synthetic protease inhibitor, E-3123, a 4-guanidinobenzoate methanesulphonate, on the exocrine pancreas in caerulein-induced pancreatitis of rats both in vivo and in vitro. Hyperamylasaemia, pancreatic oedema and congestion of amylase, as well as cathepsin B leakage from lysosomes and malate dehydrogenase leakage from mitochondria, were prevented by infusion of 5 mg/kg.h E-3123 particularly when infused for 2 h before and during 5 micrograms/kg.h caerulein infusion for 3.5 h. The results indicate that E-3123 plays its protective roles against pancreatitis in the subcellular compartments such as lysosomes and mitochondria, and that such a low molecular weight protease inhibitor as E-3123 may be clinically useful in the treatment of acute pancreatitis.

Effect of a synthetic protease inhibitor (Fut-175) on coagulation abnormalities during experimental acute pancreatitis in dogs

The coagulation disturbance observed during severe acute pancreatitis before and after the infusion of a new synthetic low molecular weight protease inhibitor (Fut-175) was compared. The coagulo-fibrinolytic changes after acute pancreatitis was induced by the intraductal injection of an autologous bile and trypsin mixture showed decreased platelet counts, decreased plasma fibrinogen levels, prolonged partial prothrombin time and increased fibrinogen degradation products. In addition, markers of hypercoagulation showed increased fibrin-peptide A and decreased antithrombin III. The two markers of fibrinolysis showed increased B beta 15-42 immunoreactive peptide and decreased alpha 2 antiplasmin. After the infusion of Fut-175, the coagulo-fibrinolytic abnormalities, which were observed during severe acute pancreatitis without infusion of Fut-175, were improved. Furthermore, Fut-175 could suppress the rise in fibrino-peptide A and B beta 15-42 immunoreactive peptide and decrease in antithrombin III and alpha 2 antiplasmin. Thus, Fut-175 seems to be an effective inhibitor of protease-mediated hypercoagulation and fibrinolysis in severe acute pancreatitis.

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Previous studies with rats have shown that a single oral dose of the proteinase inhibitor Camostate (FOY-305) induces release of cholecystokinin (CCK) into the circulation, which lasts for 3 to 6 h. This transient endogenous release of hormone results in a depletion of pancreatic enzyme stores within 1 h and an increase in total rate of protein synthesis, which peaks at 6 to 9 h. At the level of individual enzyme biosynthesis a transient decrease in amylase and an increase in trypsinogen and chymotrypsinogen is observed. In the present study the time course of DNA synthesis and the labeling index of 5 populations of pancreatic cells have been analysed following a single oral dose of 50 or 100 mg/kg proteinase inhibitor, using in vivo labeling with 12 microCi/g body weight 3H-thymidine 1 h prior to sacrifice of the animals. DNA synthesis did not change during the initial 12 h following inhibitor feeding and then showed a phasic increase with a peak (20-fold) at 24 h and intermediate increases (4- to 5-fold) at 18 and 36 h, respectively. From the 5 pancreatic cell populations studied by autoradiography the labeling indices of interlobular duct cells and islet cells did not change over the entire observation period. Acinar cells, intralobular duct cells and interstitial cells showed a marked increase in labeling index with peak values at 24 h, which were 20-fold in acinar cells and 5.5- and 8.5-fold in intralobular duct cells and interstitial cells, respectively. The data demonstrate a significant growth response of pancreatic acinar tissue after a single episode of endogenous CCK-release, which is similar in extent, time course and cellular source as previously demonstrated during persistent stimulation of the pancreas by prolonged infusion of the CCK-analogue caerulein.

Camostate (FOY-305) improves the therapeutic effect of peritoneal lavage on taurocholate induced pancreatitis.

The effect of peritoneal lavage with the addition of camostate to the lavage fluid on the outcome of taurocholate pancreatitis in rats was studied. Camostate is a low molecular weight protease inhibitor which has been developed recently. Peritoneal lavage was performed for 12 hours and camostate was added to the lavage fluid in five concentrations. At 0.1 mg/ml the survival rate increased significantly (11 of 20 v controls 4 of 20, p less than 0.05); the maximal effect was observed at 0.2 mg/ml, and no effect was seen at 0.01 and 0.05 mg/ml. Adverse effects occurred at 0.5 mg/ml. The addition of 0.1 mg/ml camostate significantly reduced the acidosis in arterial blood: mean (SD) pH 7.30 (0.035) v controls 7.23 (0.054) (n = 9, p less than 0.01) and arterial base excess: -15.4 (1.26) mmol/l v controls: -17.4 (2.51) mmol/l (n = 9, p less than 0.05). There was no difference, however, in plasma amylase activity and in the histological degree of specific tissue damage to the pancreas. A combination of intravenous camostate and lavage with the addition of camostate to the lavage fluid yielded a significantly improved survival compared with treatment with intravenous camostate alone (10 out of 16 animals v intravenous camostate alone, two out of 16, p greater than 0.01). We conclude that lavage with camostate significantly improves the prognosis of severe necrotising pancreatitis in rats.