Specific Trypsin Inhibitor Research Articles

Determination of the proteolytic profile in Balaustium murorum (Hermann, 1804) (Acari: Prostigmata: Erythraeidae)

ABSTRACT Balaustium murorum (Acariformes: Erythraeidae) is a pollen feeder and predatory mite that occasionally causes dermatitis. We sought to determine the protease profile in the whole-mite extract of B. murorum using general and specific substrates as well as inhibitors. The pH profiles using two general substrates, azacasein and haemoglobin, revealed an optimal pH of 6 for both substrates. Specific trypsin inhibitor TLCK showed no influence activity towards BApNA; however, TPCK and AEBSF inhibited chymotrypsin and elastase, respectively. Specific cysteine protease inhibitors Cystatin and E-64 influenced both types of cathepsins B and L, while DTT as a specific activator increased the enzymatic activities. Only aminopeptidase showed significant activity and inhibition using negative control and phenanthroline. Chymotrypsin and elastase had the highest activity at pH of 9–10 and 10, respectively, by using both substrates alone and substrates along with specific inhibitors. Cathepsins B and L had pH optima of 6. Aminopeptidase revealed a broad pH optimum of 5–7 on both substrates HA and HPA alone and substrate along with inhibitor phenanthroline. Finally, different concentrations of each inhibitor were used to find IC50 values of TPCK, AEBSF, cystatin, E-64, DTT and phenanthroline required to inhibit half of enzyme activity.

The secretion of the pancreas upon intraduodenal administration of trypsin and trypsin with its specific synthetic substrate

Earlier in our laboratory, conclusions were drawn regarding the specific substrates alleviating the selective inhibitory effects of enzymes by interacting at the catalytic center of the enzyme with the substrate. This was evidenced by data using a specific trypsin inhibitor that blocked the inhibitory effect of intraduodenally and intravenously administered trypsin (1), along with our laboratory's findings of alleviating the selective inhibition of amylase secretion by intraduodenal administration of a specific pancreatic amylase inhibitor (2). However, both protein and lipid emulsion, as substrates, exhibited elements of nonspecific enzyme sorption in their alleviating action

Physicochemical and Biochemical Properties of Trypsin-like Enzyme from Two Sturgeon Species.

This work aimed to determine the physicochemical and biochemical properties of trypsin from beluga Huso huso and sevruga Acipenser stellatus, two highly valuable sturgeon species. According to the results obtained from the methods of casein-zymogram and inhibitory activity staining, the molecular weight of trypsin for sevruga and beluga was 27.5 and 29.5 kDa, respectively. Optimum pH and temperature values for both trypsins were recorded at 8.5 and 55 °C by BAPNA (a specific substrate), respectively. The stability of both trypsins was well-preserved at pH values from 6.0 to 11.0 and temperatures up to 50 °C. TLCK and SBTI, two specific trypsin inhibitors, showed a significant inhibitory effect on the enzymatic activity of both trypsins (p < 0.05). The enzyme activity was significantly increased in the presence of Ca+2 and surfactants and decreased by oxidizing agents, Cu+2, Zn+2, and Co+2 (p < 0.05). However, univalent ions Na+ and K+ did not show any significant effect on the activity of both trypsins (p > 0.05). The results of our study show that the properties of trypsin from beluga and sevruga are in agreement with data reported in bony fish and can contribute to the clear understanding of trypsin activity in these primitive species.

Selective Measurement Of Activated Contact Activation Factor XII In The Matrix Of Intravenous Immunoglobulin

Activated factor XII (FXIIa) has been identified as a possible impurity in intravenous immunoglobulin preparations. Chromogenic peptide substrates are used for measuring FXIIa. Absolute selectivity, however, can be hardly obtained in such direct chromogenic assays due to the similar cleavage preferences of related proteases including kallikrein (KK) and activated factor XI (FXIa). Our highly selective method for the FXIIa measurement combines the binding of FXIIa to its solid phase-immobilized, specific inhibitor corn trypsin inhibitor (CTI) followed by immunological detection of the resulting complex via immunological detection of the protease.

Production Enhancement of an Anticoagulant Trypsin Inhibitor from Oceanimonas sp. BPMS22 and Its Anti-cancer Activity

The significant role of proteases in the cancer progression exposed them as potential targets for drug discovery. Regulating protease activities using protease inhibitors is one of the major therapeutic strategies for the treatment of cancer. The proteolysis activities of serine proteases in different stages of metastasis can be regulated by serine protease inhibitors. In the present work, the application of a peptide trypsin inhibitor (TI) from Oceanimonas sp. BPMS22 in cancer treatment was exploited. Initially, the process parameters such as initial medium pH, glucose, yeast extract, and sodium chloride concentrations were optimized for the maximum inhibitor activity using Response surface methodology. The maximum specific trypsin inhibitor activity observed was 40.16 ± 1.33 IU/mg protein at predicted critical conditions, glucose, 19.51 g/L; yeast extract, 23.04 g/L; sodium chloride, 28.29 g/L; and pH 7.26. Further, the apoptotic cell death of purified trypsin inhibitor-treated human chronic myeloid leukemia (K562) cell lines was demonstrated by MTT assay, AO/EtBr staining, and DNA ladder assay. The cellular oxidative stress in K562 cells was confirmed by glutathione levels and fluorescence imaging of ROS generation.

Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI.

Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel β-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by ‘red shift’ in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.

Structural and functional relationship of Cassia obtusifolia trypsin inhibitor to understand its digestive resistance against Pieris rapae

Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic β-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa. The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH.

Trypsin from the digestive system of carp Cirrhinus mrigala: Purification, characterization and its potential application

Trypsin was purified 35.64-fold with 4.97% recovery from the viscera of carp Cirrhinus mrigala (mrigal) by ammonium sulfate precipitation, ion exchange and affinity chromatography. The purified enzyme was active at a wide range of pH (7.0–9.2) and temperature (10–50°C). The purified enzyme exhibited high thermal stability up to 50°C for 1h. The enzyme activity was stabilized by Ca+2 (2mM) up to 7h at 40°C. The Km and kcat values of purified enzyme were 0.0672mM and 92.09/s/mM, respectively. Soybean trypsin inhibitor and phenylmethylsulphonylflouride completely inhibited the enzyme activity. The specific inhibitor of trypsin, N-α-p-tosyl-l-lysine chloromethyl ketone inhibited 99.67% activity. Na+, K+ and Li+ inhibited 20.99±5.25%, 16.53±4.80% and 18.99±1.42% of enzyme activity, respectively. Divalent ions Mg+2, Zn+2, Co+2, Hg+2 and Cd+2 inhibited 21.61±2.22%, 31.62±1.78%, 31.62±1.96%, 85.68±1.51% and 47.95±2.13% enzyme activity, respectively. SDS–PAGE showed that the molecular mass of purified enzyme was 21.7kDa. MALDI-TOF study showed a peptide sequence of AFCGGSLVNENKMHSAGHCYKSRIQV at the N-Terminal. This sequence recorded 76–84% identity with trypsin from Thunnus thynnus and other fish species. This confirmed that the purified protein was trypsin. The purified enzyme has potential applications in detergent and food industry because of its thermal stability and alkaline nature.

Purification and Characterization of Cold Adapted Trypsins from Antarctic krill (Euphausia superba)

Three trypsins (TRY-ES) were purified from Antarctic krill (Euphausia superba) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography, with relative molecular mass of 28.7, 28.8 and 29.2 kDa respectively. The TRY-ES was inhibited by specific trypsin inhibitors (benzamidine, STI, CHOM and TLCK), with optimum temperature at 40 (Trypsin I), 45 (Trypsin II) and 40 °C (Trypsin III) repetitively. The TRY-ES was stabled between 5 and 40 °C, which was consistent with the red shift in fluorescence intensity peak at 40 °C (Trypsin I) and 45 °C (Trypsin II and Trypsin III) and blue shift at 40 °C (Trypsin II and Trypsin III). The K cat/K m values of the TRY-ES was 14.28, 9.46 and 5.93 mM−1s−1 respectively, 1.1–10.2 folds higher than trypsins from other crustacean and mammal, which was supported by the differences in thermodynamics parameters, the free energy, enthalpy, and entropy of benzamidine and the TRY-ES system.

Generation and Analysis of Serine Protease Inhibitor Kazal Type 3-Cre Driver Mice

Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue Spink3) was initially discovered as a trypsin-specific inhibitor in the pancreas. However, previous studies have suggested that SPINK1/Spink3 is expressed in a wide range of normal tissues and tumors, although precise characterization of its gene expression has not been described in adulthood. To further analyze Spink3 expression, we generated two mouse lines in which either lacZ or Cre recombinase genes were inserted into the Spink3 locus by Cre-loxP technology. In Spink3lacZ mice, β-galactosidase activity was found in acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in the lung, but not in the gastrointestinal tract or liver. Spink3cre knock-in mice were crossed with Rosa26 reporter (R26R) mice to monitor Spink3 promoter activity. In Spink3cre;R26R mice, β-galactosidase activity was found in acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in endoderm-derived tissues, and that Spink3cre knock-in mice are a useful tool for establishment of a conditional knockout mice to analyze Spink3 function not only in normal tissues, but also in tumors that express SPINK1/Spink3.

English

Alkaline proteases from the digestive tract of anchovy were partially purified by ammonium sulfate fractionation, dialysis and Sephadex G-75 gel filtration. The purification fold and yield were 6.23 and 4.49%, respectively. The optimum activities of partially purified alkaline proteases were observed at 60°C and at pH 11.0. The alkaline proteases were stable within the temperature range of 40 to 50°C and pH range of 9.0 to 11.0. They were inhibited by the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and trypsin specific inhibitor benzamidine, but were not inhibited by the β-mercaptoethanol. The enzymes were slightly activated by metal ions such as Na + and Ba 2+ and inhibited by Cu 2+ , Zn 2+ , K + and Mn 2+ at different degrees. The molecular weight of the partially purified enzyme was 24 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Key words : Alkaline proteases, Engraulis encrasicholus , purification, characterization, digestive tract.

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb2+, Ca2+, Mg2+, Cu2+, and Mn2+ could active the enzyme certainly when Zn2+ and Hg2+ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.

PURIFICATION AND CHARACTERISTICS OF TRYPSIN FROM THE PANCREAS OF TIBETAN SHEEP

ABSTRACT Trypsin from the pancreas of Tibetan sheep was purified to 7.4-fold by ammonium sulphate and acetone precipitation, followed by Sephacryl S-200 (Whatman, Maidstone, England) and Sephadex G-75 (Whatman) gel filtration, with an 23.2-fold increase in specific activity and 13.6% yield. The final enzyme preparation was nearly homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of the enzyme was estimated to be approximately 27 kDa by SDS-PAGE. Trypsin-like enzymes had maximal activities at around pH 9.0 and 60C for the hydrolysis of Nα-ρ-Tosyl–L–arginine methyl ester hydrochloride (TAME). Trypsin was unstable above 60C and below pH 4.0, and was stabilized by calcium ions. Purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (Kcat) of 0.53 mM and 206 s−1, respectively, when TAME was used as the substrate. The specific trypsin inhibitors, soybean trypsin inhibited, Nα-ρ-tosyl-L-lysine chloromethyl ketone and phenyl methyl sulfonyl fluoride, strongly inhibited the activity of trypsin, while other protease inhibitors exhibited negligible inhibition. The result suggests that major proteinase in the pancreas of Tibetan sheep was trypsin-like serine proteinase. PRACTICAL APPLICATIONS Tibetan sheep is one of the unique livestock resources in western China. It was found that the trypsin extraction and purification from waste pancreas have the biological characteristics of the heat resistance, alkali resistance, high specific activity and high hydrolysis efficiency. It does not only serve as a better research tool enzyme, but is also widely applied in the food, pharmaceutical, textile and other industries. This paper provides the scientific research basis of the development of the waste pancreas for the Tibetan sheep, therefore reducing environmental pollution.

Mackerel trypsin purified from defatted viscera by supercritical carbon dioxide.

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and 60°C, respectively, using N α-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above 40°C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO2 defatting process is useful as a substitute for organic solvent defatting process.

A trypsin homolog in amphioxus: expression, enzymatic activity and evolution

Trypsin has been documented in a variety of species including both vertebrates and invertebrates, but little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a trypsin gene in Branchiostoma japonicum. The cDNA was 978 bp long with an ORF encoding a deduced protein of 272 amino acids. The deduced protein had an N-terminal signal peptide of 15 amino acids, a 16 activation peptide with the typical cleavage site Arg/Ile, a Tryp_SPc domain with the catalytic triad His(72)-Asp(118)-Ser(215) and the S1 substrate binding residue Asp(209), which are all characteristic of trypsinogens. The recombinant trypsin protein was able to hydrolyse the trypsin prototypic substrate BAEE, which was inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. Both northern blotting and tissue-section in situ hybridization demonstrated that trypsin gene was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum, mid-gut and ovary. And the whole mount in situ hybridization showed that it began to express in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Phylogenetic analysis indicated that both amphioxus and ascidian trypsins are more closer to each other than to vertebrate trypsins, suggesting a continuous evolutionary divergence of vertebrate trypsins after split from protochordate/vertebrate common ancestor.

Serine proteases mediate inflammatory pain in acute pancreatitis

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.

The Tick Protein Ir-Cpi Efficiently Delays Contact Pathway Induced Thrombin Generation and Displays In Vivo Antithrombotic Activity

AbstractAbstract 3336 Introduction:The search for new anticoagulants is a major challenge in medicine. Contact factors have never been considered as interesting targets for the development of new anticoagulant agents since they are not required for in vivo coagulation (i.e. deficiencies affecting these factors do not cause excessive bleeding). The discovery that FXI and FXII deficiency protects against thrombosis without causing spontaneous bleeding in mice makes FXII a unique and ideal target for drug design. We demonstrated in vitro that Ir-CPI, a 67 amino acids recombinant Kunitz-type protein from Ixodes ricinus, specifically interacted with activated human contact phase factors (FXIIa, FXIa, and kallikrein). Aims:The goal of this study was to investigate the potential anticoagulant and antithrombotic efficacy of Ir-CPI. Methods:The effects of Ir-CPI were investigated on the thrombin activity during the coagulation process in human plasma using the CAT method. Three different inducers were employed to specifically trigger the coagulation pathways and to generate thrombin. A standard concentration of 5 pM TF with 4 μM PL and 16.7 mM CaCl2 was used to activate the TF pathway whereas a lower TF concentration of 1 pM with 4 μM PL and 16.7 mM CaCl2 was selected for stimulating the TF pathway in combination with the contact phase pathway through a thrombin-mediated positive feedback. The contact phase pathway was also triggered alone by ellagic acid, PL and 16.7 mM CaCl2 (25-fold diluted APTT reagent Actin FS). The effect of Ir-CPI on venous thrombus formation was assessed using a rat thrombosis model induced by complete venous stasis in the posterior vena cava and FeCl3 topical application on the outer vessel wall. Results:When stimulating plasma coagulation trough the contact pathway, Ir-CPI caused a concentration-dependent prolongation of the lag time and the Tmax and a concentration-dependent decrease in the Cmax compared to the control curve (i.e. without inhibitor). When the coagulation cascade was triggered by the TF pathway (5 or 1 pM TF), only a slight concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were observed. For comparison purpose, the effects of the specific competitive FXIIa inhibitor corn trypsin inhibitor (CTI) were also investigated using the three same inducers than for Ir-CPI. For the contact pathway, a concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were found. When stimulating the TF pathway (5 or 1 pM TF), no modification of the thrombin generation curves was observed with the tested concentrations of CTI. When comparing the results of the two inhibitors acting trough the delay of the contact pathway, we found that Ir-CPI was about 30-fold more potent than CTI.We further evaluated the antithrombotic effect of Ir-CPI on a rat venous thrombosis model induced by endothelial damage and vessel ligation, close to the physiological venous thrombus formation in humans. We showed that Ir-CPI reduced thrombosis in a dose-dependent manner with an ED50 close to 65 μg/kg. A maximum effect starting from 0.5 mg/kg was observed with a mean reduction in the clot weight/body weight of 75 ± 7%. This antithrombotic effect of Ir-CPI was exclusively mediated through the inhibition of thrombin generation since it did not interference with collagen-induced platelet aggregation. This is the first time that an inhibitor of the coagulation contact phase was shown to protect against the formation of venous thrombi. The antithrombotic effect of Ir-CPI was also confirmed using other venous and arterial thrombosis models. We also showed that the effective antithrombotic dose of Ir-CPI in these tests did not promote bleeding or impair blood coagulation parameters. Conclusions:The present study demonstrated that Ir-CPI, a recombinant protein from the tick Ixodes ricinus targeting the contact factors (FXII, FXI and kallikrein), displayed an anticoagulant activity mainly through the delay of the contact pathway induced thrombin generation. This drug also exhibited an antithrombotic activity in our venous and arterial thrombosis model. This drug may thus provide an interesting and innovative therapeutic tool for the prevention and the treatment of thromboembolic diseases with a minimal risk of therapy-associated bleeding. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Isolation of Potent and Specific Trypsin Inhibitors from a DNA-Encoded Chemical Library

Collections of chemical compounds, individually attached to unique DNA fragments serving as amplifiable identification bar codes, are generally referred to as "DNA-encoded chemical libraries". Such libraries can be used for the de novo isolation of binding molecules against target proteins of interest. Here, we describe the synthesis and use of a DNA-encoded library based on benzamidine analogues, which allowed the isolation of a trypsin inhibitor with an IC(50) value of 3.0 nM, thus representing a >10 000-fold potency improvement compared to the parental compound. The novel trypsin inhibitor displayed an excellent selectivity toward other serine proteases. This study indicates that DNA-encoded libraries can be used for the facile "affinity maturation" of suboptimal binding compounds, thus facilitating drug development.

Hereditary pancreatitis – managing patient clinical information and sequence variants in the CFTR, PRSS1 and SPINK1 genes

Hereditary pancreatitis is a rare inherited disorder, characterized by recurrent episodes of acute pancreatitis usually within the first two decades of life progressing to chronic pancreatitis. Disease progression includes pancreatic calcifications, exocrine insufficiency, and diabetes mellitus in most patients. Some cases progress to pancreatic cancer. The mode of inheritance suggests an autosomal dominance trait with incomplete penetrance. PRSSI is a one of eight trypsinogen genes. Mutations in the PRSS1 gene are believed to be mostly gain-of-function leading to increased activity or stability of trypsin and / or decreased trypsin inactivation resulting in autolysis of the pancreatic tissue. Almost all the PRSS1 pancreatitis-associated mutations discovered to date seem to cluster in exons 2 and 3. The two most common c.365G>A, p.R122H and c.86A>T, p.N29I are missense mutations and are reported to have a penetrance of 80% for hereditary pancreatitis. Two other genes, SPINK1 (serine protease inhibitor Kazal type 1) and CFTR are risk factors for pancreatitis. Mutations in these two genes may be inherited in an autosomal recessive or multifactorial fashion. SPINK1, is a specific trypsin inhibitor expressed within the pancreas, providing an important defense against prematurely activated trypsinogen. 15% of adults, and 25% of children, with idiopathic pancreatitis have SPINK1 mutations. The primary mutation found in the SPINK1 gene, c.101A>G, p.N34S, increases the risk for pancreatitis by 14 fold. Approximately 30% of individuals with idiopathic pancreatitis have at least one CFTR mutation. Hereditary pancreatitis is an autosomal disease with variable expression caused by mutations in the PRSS1 gene. Two other genes, SPINK1 and CFTR, are risk factors for pancreatitis and can be inherited in an autosomal recessive or multifactorial fashion. Gene sequencing of all three genes aids in the diagnosis of hereditary pancreatitis which can be difficult to distinguish from a mild cystic fibrosis phenotype. Manageability of data derived from the patient’s clinical presentation and sequencing results is vital to understand genotype/phenotype correlation, to help classify variants and to assure quality in laboratory reporting. To this end, we have employed the clinical data management software, Progeny, to track genotypic and phenotypic information on individuals and pedigrees. The database was customized for pancreatitis patients. Data entered includes patient demographics and clinical presentation, laboratory sequencing results (nucleotide and amino acid changes for CFTR, PRSS1 and SPINK1; mutation type and effect; and significance of the variant) and evidence for reclassification of variants. Analysis of 59 pancreatitis cases (classified as idiopathic, acute or chronic) found 10.2% of the patients carried one CFTR mutation, 5.1% carried one CFTR and one SPINK1 mutation, 5.1% carried one PRSS1 mutation, 1.7% carried two CFTR mutations, 1.7% carried one CFTR mutation and two SPINK1 mutations and 6.8% carried one SPINK1 mutation. No mutations were found in 69.5% of the cases. Progeny software allows customized databases for combining clinical and laboratory information, providing a means for accessible collection and querying of data. Samples Patient samples were sent to ARUP Laboratories (Salt Lake City, Utah) for clinical testing. Sequencing Patient samples requesting an idiopathic pancreatitis panel (IP panel) were sequenced in the clinical laboratory. An IP panel composed of full gene sequencing (FGS) for all three genes CFTR, PRSS1 and SPINK1 was ordered on 45 samples. Seven patient samples had FGS ordered only for PRSS1, 5 had FGS for SPINK1 only and 2 patient samples had FGS ordered both for PRSS1 and SPINK1. Thirty-three samples sent in for only CFTR FGS were selected for additional analysis due to their singular diagnosis of pancreatitis. The samples were de-identified and sequencing for PRSS1 and SPINK1 was performed in the R&D laboratory.