Kunitz-type Serine Protease Inhibitor Research Articles

The Diversity of Bioactive Proteins in Australian Snake Venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Tissue factor pathway inhibitor‐2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma†

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.

Identification of a human TFPI-2 splice variant that is upregulated in human tumor tissues

BackgroundPrevious studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter. In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2).ResultsNucleotide sequence analyses indicated that asTFPI-2 consists of complete exons II and V, fused with several nucleotides derived from exons III and IV, as well as six nucleotides derived from intron C. 5'- and 3'-RACE analyses of total RNA amplified exclusively the wild-type TFPI-2 transcript, indicating that asTFPI-2 lacks either a 5'-untranslated region (UTR) or a 3'-poly (A)+ tail. Quantitative real-time RT-PCR analyses revealed that several human tumor cells contain 4 to 50-fold more copies of asTFPI-2 in comparison to normal cells. In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells.ConclusionOur studies reveal the existence of a novel, aberrantly-spliced TFPI-2 transcript predominantly expressed in tumor cells and provides suggestive evidence for an additional mechanism for tumor cells to down-regulate TFPI-2 protein expression enhancing their ability to degrade the extracellular matrix.

Characterization of TFPI and TFPI-beta in Mouse Tissues.

Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor. The first two Kunitz domains efficiently inhibit factor VIIa and factor Xa and thereby abrogate tissue factor initiated blood coagulation. Two isoforms of TFPI have been identified. Full-length TFPI is thought to indirectly associate with endothelium via binding to a GPI-anchored co-receptor. TFPI-beta is produced by alternative splicing of full-length TFPI. It consists of the first two Kunitz domains followed by a unique C-terminal region with a direct GPI-anchor attachment site. The physiological significance of TFPI-beta is not known. We performed 3′RACE of mouse placental cDNA to determine if additional isoforms of TFPI exist. These studies identified only TFPI and TFPI-beta. Of importance, a full-length form of TFPI containing a direct GPI-anchor attachment site was not identified. This finding supports the hypothesis that full-length TFPI binds to endothelium indirectly through association with a GPI-anchored co-receptor. In order to identify potential physiological roles for TFPI and TFPI-beta, we analyzed mRNA expression and protein production in mouse tissues. Message for both TFPI and TFPI-beta was present in all tissues examined. Real time PCR showed TFPI message 2.5- to 65-fold higher than TFPI-beta message in the different tissues (heart (2.5)>lung (5.1)>testicle (5.8)>spleen (9.3)>thymus (10)>uterus (11)>kidney (26)> liver (35)> placenta (37)>brain (65)). The high relative amount of TFPI-beta in heart, lung and testicle suggest that it may have an important physiological role in these tissues. In situ hybridization studies did not identify vascular bed specific expression of TFPI or TFPI-beta. These studies demonstrated that TFPI and TFPI-beta localize predominantly within endothelial cells with identical staining patterns in all tissues studied. Western blot analysis for TFPI and TFPI-beta in detergent lysates of the different tissues demonstrated the presence of full-length TFPI in all tissues in amounts proportional to the vascularization of the tissue (placenta>>heart>lung>spleen>kidney>liver>>>brain).TFPI-beta was not observed by western blot analysis in any of the tissues indicating that TFPI-beta message may be transcriptionally repressed. Brain contains much less TFPI than other tissues, yet has high amounts of tissue factor. The low TFPI in the brain could be due to either low numbers of endothelial cells within the brain or to low expression of TFPI by brain endothelial cells. We used real time PCR to compare the level of TFPI and TFPI-beta mRNA expression to that of the endothelial specific proteins VE-cadherin and PECAM. When compared to these markers, the relative amount of TFPI mRNA brain is similar to that present in the heart and lung, indicating that TFPI likely has an important role in prevention of thrombosis within the cerebral vasculature.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is essential for the integrity of basement membranes in the developing placental labyrinth

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serine protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodeling and tumorigenesis, such as HGFA, matriptase, prostasin and hepsin. We generated HAI-1-deficient mice, which died in utero due to placental defects. The HAI-1 −/− placental labyrinth exhibited a complete failure of vascularization and a compact morphology of the trophoblast layer. Immunofluorescent staining of collagen IV and laminin and electron microscopy analysis revealed that this aberrant labyrinth architecture was associated with disrupted basement membranes located at the interface of chorionic trophoblasts and allantoic mesoderm. Unlike the placental labyrinth, basement membranes and vasculogenesis were normal in embryo and yolk sac. Therefore, basement membrane defects appear to be the underlying cause for the greatly impaired vascularization and trophoblast branching in HAI-1 −/− placentas. In wild-type placentas, the expression of matriptase and prostasin co-localized with their physiological inhibitor HAI-1 to the labyrinthine trophoblast cells in proximity to basement membranes. In HAI-1 −/− placentas, both the localization and expression of the two proteases remained unchanged, implying uncontrolled proteolytic activities of the two enzymes. Our study demonstrates the important role of HAI-1 in maintaining the integrity of basement membrane most likely by regulating extracellular proteolytic activities during placental development.

Matriptase inhibition by hepatocyte growth factor activator inhibitor-1 is essential for placental development

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor that forms inhibitor complexes with several trypsin-like serine proteases and is required for mouse placental development and embryo survival. Here we show that the essential function of HAI-1 in placentation and all other embryonic processes is to restrict the activity of the type II transmembrane serine protease, matriptase. Enzymatic gene trapping of matriptase combined with HAI-1 immunohistochemistry revealed that matriptase is co-expressed with HAI-1 in both extraembryonic and embryonic tissues. As early as embryonic day 8.5, matriptase and HAI-1 were expressed in a population of chorionic trophoblasts. Ablation of HAI-1 disrupted the epithelial integrity of this cell population, causing disorganized laminin deposition and altered expression of E-cadherin and beta-catenin. This led to a complete loss of undifferentiated chorionic trophoblasts after embryonic day 9.5 and prevented the formation of the placental labyrinth. Genetic ablation of matriptase activity in HAI-1-deficient embryos, however, restored the integrity of chorionic trophoblasts and enabled placental labyrinth formation and development to term. Furthermore, matriptase/HAI-1 double-deficient mice were phenotypically indistinguishable from matriptase single-deficient littermates.

Diagnostic utility of aberrant methylation of tissue factor pathway inhibitor 2 in pure pancreatic juice for pancreatic carcinoma

The tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI-2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI-2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation-specific polymerase chain reaction (MSP) and quantitative MSP (Q-MSP) assay, we investigated the aberrant methylation of TFPI-2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI-2 methylation was seven (77.8%) of nine PCa cell lines by Q-MSP. In cell lines, the expression of TFPI-2 mRNA by quantitative reverse transcription-polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI-2. The incidence of aberrant TFPI-2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut-off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI-2 methylation in the PPJ by real-time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI-2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI-2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q-MSP assay. These results suggest that promoter methylation of TFPI-2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach.

Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro

Objective Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial ( Ea.hy 926) and in chondrosarcoma ( SW 1353) cells. Methods We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24 h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. Results Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. Conclusion Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Expression of hepatocyte growth factor activator inhibitor type 1 in endothelial cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is an integral membrane Kunitz-type serine proteinase inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). HGFA is a serum proteinase that is critically involved in the activation of hepatocyte growth factor/scatter factor (HGF/SF) in injured tissue. Previous studies have shown that HAI-1 is expressed on the basolateral surface of various epithelial cells. In this study, we analyzed the expression of HAI-1 in human endothelial cells. Immunohistochemically, HAI-1 protein was observed in the endothelial cells of capillaries, venules and lymph vessels. On the other hand, arterial endothelial cells were poorly stained for HAI-1. Mesothelial cells on the serous surface were also positively immunostained. The endothelial expression of HAI-1 was also examined in cultured human endothelial cells of various origins, such as umbilical vein, microvessels and aorta. Notably, in accordance with the results of immunohistochemistry, HAI-1 mRNA and protein levels were high in the endothelial cells derived from umbilical vein and were hardly detectable in those derived from aorta. A low but distinct level of HAI-1 expression was also observed in endothelial cells from microvessels. As these HAI-1-positive endothelial cells also expressed MET tyrosine kinase, the specific receptor of HGF/SF, it is conceivable that HAI-1 might have an important regulatory role in the HGF/SF-MET signaling axis of endothelial cells, which could be involved in the process of angiogenesis.

Restoration of tissue factor pathway inhibitor inhibits invasion and tumor growth in vitro and in vivo in a malignant meningioma cell line

Tissue factor pathway inhibitor 2 (TFPI-2) is a 32-kDa extracellular matrix-associated kunitz-type serine proteinase inhibitor. It is secreted by all vascular cells and plays a role in tumor invasion and metastasis, presumably by plasmin-mediated matrix remodeling. Previous studies have shown high expression of TFPI-2 by benign tumors and low or absent expression in highly malignant tumors. Malignant meningiomas constitute 10-15% of all meningiomas and our previous studies revealed loss of expression of TFPI-2 in malignant gliomas. To investigate the role of TFPI-2 in the invasiveness of malignant meningiomas, we stably transfected the human meningioma cell line, IOMM-Lee, with a vector capable of expressing a transcript complementary to the full length of TFPI-2 mRNA in a sense orientation. Restoration of TFPI-2 led to decreased invasiveness of transfected cells compared to parental and vector controls in Matrigel and spheroid assays and inhibition of angiogenesis in in vitro co-cultures with human umbilical vein endothelial cells (HUVEC) and in vivo dorsal skin assay studies. As assessed by Western blotting, we also observed increased expression of BAX, cytochrome c and caspase 3 as well as decreased expression of XIAP (X-linked inhibitor of apoptosis). Finally, TFPI-2 overexpression inhibited intracranial tumor formation in nude mice. Our data substantiate our previous observation that TFPI-2 plays an important role in tumor progression and has potential in anti-cancer therapy.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstroms resolution, while crystals of free rTxln-1 diffracted to 1.63 angstroms resolution.

Hepatocyte growth factor activation inhibitors (HAI‐1 and HAI‐2) regulate HGF‐induced invasion of human breast cancer cells

Hepatocyte growth factor (HGF) plays a plethora of roles in cancer metastasis and tumour growth. The interaction between tumour cells and their surrounding stromal environment is a crucial factor regulating tumour invasion and metastasis. Stromal fibroblasts are the main source of HGF in the body, and release HGF as an inactive precursor (pro-HGF). HGF activator (HGFA), matriptase, urokinase-type plasminogen activator and hepsin are the main factors responsible for converting pro-HGF into active HGF. HAI-1 and HAI-2 are 2 novel Kunitz-type serine protease inhibitors that regulate HGF activity through inhibition of HGFA, matriptase and hepsin action. Recent studies demonstrate that HAI-1 and HAI-2 may also potently inhibit a number of other pro-metastatic serine proteases and therefore have direct bearing on the spread of tumours. Our study examined the potential of these HAI's to suppress the influence of HGF and regulate cancer metastasis. We generated a retroviral expression system that induced HAI expression in a human fibroblast cell line. Forced expression of either HAI-1 or HAI-2 in these fibroblasts resulted in a dramatic decrease in the production of bioactive hepatocyte growth factor (HGF). This reduction in HGF activity subsequently suppressed HGF's metastatic influence on breast cancer cells. To further assess the anti-cancer properties of HAI-1 and HAI-2 we generated recombinant HAI proteins. These recombinant HAI proteins possessed the ability to potently quench HGF activity. We also demonstrate that these recombinant HAI's suppressed fibroblast-mediated breast cancer invasion. An additional ribozyme transgenes study revealed that elimination of HAI-1 and HAI-2 expression, in an MDA-MB-231 breast cancer cell line, significantly enhanced the migratory, proliferative and invasive nature of these breast cancer cells. Overall, our data demonstrates the important roles of HAI-1 and HAI-2 in cancer metastasis, and reveals that these serine protease inhibitors display strong therapeutic potential.

Protein Interaction Analysis of ST14 Domains and Their Point and Deletion Mutants

ST14 (suppression of tumorigenicity 14) is a transmembrane serine protease that contains a serine protease catalytic (SP) domain, an SEA domain, two complement subcomponent C1r/s (CUB) domains, and four low density lipoprotein receptor class A domains. Glutathione S-transferase fusion proteins with SP, CUB, and low density lipoprotein receptor domains and their corresponding mutants were generated to analyze protein interactions with these domains. Modified glutathione S-transferase pull-down assays demonstrated the interaction between the SP domain and hepatocyte growth factor activator inhibitor-1. With the same method, a CUB domain-interacting protein was isolated and turned out to be the transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1). Quantitative real time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. Our results also suggested that ST14 and TMEFF1 were co-expressed in the human breast cancer cell line MCF7, human placenta, kidney, and liver tissues. Interestingly, these two genes were co-up-regulated in kidney tumors versus normal tissues, consistent with our results that showed the dependence of TMEFF1 expression on ST14 in RKO cells. Finally, homology modeling studies suggested that TMEFF1 might form a complex with ST14 by an interaction between epidermal growth factor and CUB domains.

Hyposecretion, Not Hyperabsorption, Is the Basic Defect of Cystic Fibrosis Airway Glands

Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na(+) channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.

Advances in the research on human tissue factor pathway inhibitor

人类组织因子途径抑制因子属于库尼(Kunitz)型丝氨酸蛋白酶抑制剂家族蛋白,分为组织因子途径抑制因子-1(tissue factor pathway inhibitor-1,TFPI-1)和组织因子途径抑制因子-2(tissue factor pathway inhibitor-2,TFPI-2).TFPI-1以抗凝血作用为主,而TFPI-2是广谱丝氨酸蛋白酶抑制剂.二者在结构上有部分同源性,均由3个重复的Kunitz结构域组成.但二者在基因序列、组织来源、分布和作用机理上的很大差异,导致二者在多种生理和病理过程中发挥的作用也大不相同.就有关研究进展做一综述。

Expression and characterization of wild-type TFPI and the [P151L]TFPI mutant in insect cells

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation. In earlier studies we could identify the [P151L]TFPI mutant, and we could also demonstrate that heterozygous carriers of this mutant show a nine-fold increased risk for deep venous thrombosis (DVT). To express greater amounts of both proteins and to enable their characterization, we expressed wild-type TFPI as well as [P151L]TFPI in High Five insect cells with expression rates of up to 215 ng/ml for wild-type TFPI and 214 ng/ml for [P151L]TFPI. The specific inhibitory activities for the recombinant proteins were determined as 11.3 and 11.5 mU/ng, respectively. Both proteins were detected via Western blot analysis and ELISA. The recombinant proteins' inhibitory activities were characterized by a chromogenic assay and by the determination of a modified activated thromboplastin time (aPTT) in which both of them proved to be inhibitorily active. We also examined both recombinant proteins' binding properties to glycoproteins, glycosaminoglycans, lipoproteins and tissue factor. Our results show that we have developed an efficient model system for the recombinant expression of inhibitorily active wild-type TFPI as well as [P151L]TFPI in insect cells, and we were able to characterize both proteins' inhibitory properties by determination of their influence on the aPTT and also their binding properties. Although both recombinant proteins did not show a significant difference in their effect on the aPTT, their binding properties differed significantly between the wild type and mutant protein.

An unexpected inhibitory activity of Kunitz-type serine proteinase inhibitor derived from Boophilus microplus trypsin inhibitor on cathepsin L

Several BPTI-Kunitz-type serine proteinase inhibitors were described in tick Boophilus microplus and Rhipicephalus sanguineus species. In this work, we present a synthetic gene based on two tick BPTI-Kunitz-type serine proteinase inhibitors, the first domain of B. microplus trypsin inhibitor-A (BmTI-A) and the carrapatin, the inhibitors were named BmTIsint and BmTIsint Mut. Our present results showed that BmTIsint and BmTIsint Mut inhibited trypsin ( K i 3.3 and 1.0 nM) and human plasma kallikrein ( K i 16.5 and 35 nM), but in contrast to BmTI-A, the inhibitors did not inhibit human neutrophil elastase. BmTIsint was able to produce immunological response in mice but not in bovines. In addition, it is the first description of a BPTI-Kunitz-type inhibitor as a cysteine proteinase inhibitor, BmTIsint apparent dissociation constant ( K i) for cathepsin L was 108 nM. Our findings open the possibility up to obtain new molecules as potent serine or cysteine proteinase inhibitors using BmTIsint as a model.

P.3.a.021 Examination of specific genetic aspects of the dopaminergic neurotransmission and neuronal plasticity in neurocognitive subgrouping of schizophrenia

Composition of Indian Russell's viper (Daboia russelii russelii) venom, a medically important snake and member of “Big Four” snakes of India was done by gel filtration chromatography followed by tandem mass spectrometry. The MS/MS analyses of tryptic digested gel filtration peaks divulged the presence of 63 different proteins belonging to 12 families. Phospholipase A2 (PLA2), serine proteases, metalloproteases, cysteine-rich secretory proteins, l-amino acid oxidase, C-type lectin-like proteins, kunitz-type serine protease inhibitor, disintegrin, nucleotidase, phosphodiesterase, vascular endothelial growth factor and vascular nerve growth factor families were identified. PLA2 enzymes with isoforms of N-, S- and H-type based on their first N-terminal amino acid residue were observed. The venom is also found to be rich in RVV-X, RVV-V and thrombin-like enzymes. Homologues of disintegrins with RGD and RTS motifs were also observed. The high percentage of PLA2 and proteases in the venom proteome could be responsible for the observed coagulopathy, haemorrhage and edema which can be correlated with the clinical manifestations of Russell's viper envenomation. This is the first proteomic analysis of Indian D. russelii venom which might assist in understanding the pathophysiological effects of viper envenomation. Such study will also be important for developing more effective antivenom for viper bite management.

P.3.c.016 Diabetes and impaired glucose tolerance in the treatment of schizophrenia - interim assessment of prevalence and investigation of genetic predictors

Composition of Indian Russell's viper (Daboia russelii russelii) venom, a medically important snake and member of “Big Four” snakes of India was done by gel filtration chromatography followed by tandem mass spectrometry. The MS/MS analyses of tryptic digested gel filtration peaks divulged the presence of 63 different proteins belonging to 12 families. Phospholipase A2 (PLA2), serine proteases, metalloproteases, cysteine-rich secretory proteins, l-amino acid oxidase, C-type lectin-like proteins, kunitz-type serine protease inhibitor, disintegrin, nucleotidase, phosphodiesterase, vascular endothelial growth factor and vascular nerve growth factor families were identified. PLA2 enzymes with isoforms of N-, S- and H-type based on their first N-terminal amino acid residue were observed. The venom is also found to be rich in RVV-X, RVV-V and thrombin-like enzymes. Homologues of disintegrins with RGD and RTS motifs were also observed. The high percentage of PLA2 and proteases in the venom proteome could be responsible for the observed coagulopathy, haemorrhage and edema which can be correlated with the clinical manifestations of Russell's viper envenomation. This is the first proteomic analysis of Indian D. russelii venom which might assist in understanding the pathophysiological effects of viper envenomation. Such study will also be important for developing more effective antivenom for viper bite management.

P.3.a.012 Low concentration of plasma nitric oxide metabolites in schizophrenic patients

Composition of Indian Russell's viper (Daboia russelii russelii) venom, a medically important snake and member of “Big Four” snakes of India was done by gel filtration chromatography followed by tandem mass spectrometry. The MS/MS analyses of tryptic digested gel filtration peaks divulged the presence of 63 different proteins belonging to 12 families. Phospholipase A2 (PLA2), serine proteases, metalloproteases, cysteine-rich secretory proteins, l-amino acid oxidase, C-type lectin-like proteins, kunitz-type serine protease inhibitor, disintegrin, nucleotidase, phosphodiesterase, vascular endothelial growth factor and vascular nerve growth factor families were identified. PLA2 enzymes with isoforms of N-, S- and H-type based on their first N-terminal amino acid residue were observed. The venom is also found to be rich in RVV-X, RVV-V and thrombin-like enzymes. Homologues of disintegrins with RGD and RTS motifs were also observed. The high percentage of PLA2 and proteases in the venom proteome could be responsible for the observed coagulopathy, haemorrhage and edema which can be correlated with the clinical manifestations of Russell's viper envenomation. This is the first proteomic analysis of Indian D. russelii venom which might assist in understanding the pathophysiological effects of viper envenomation. Such study will also be important for developing more effective antivenom for viper bite management.