A novel coagulation inhibitor from Schistosoma japonicum

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

BACKGROUND: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.

METHODS: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.

RESULTS: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.

CONCLUSIONS: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Rotary moulding of ceramic hollow wares

A novel processing method for the fast and economic production of hollow ceramic components has been developed by combining in situ coagulation moulding with a modified version of the technique of rotary moulding[Binner, J. G. P., Al-Dawery, I. A., Tari, G. and Yan, Y., Rotary casting technique. UK Patent application No. 0506349.0, March 2005], the latter being adapted from the polymer industry. The process was found to require a high solids content suspension, hence development work was performed in this direction though in the end a new, commercial suspension was utilised. Of the three forming routes of gel casting, direct coagulation casting and in situ coagulation moulding, the latter was found to be the most promising for the new process of rotary moulding of ceramics. Due to the low value of clay-based ceramics, a new low cost coagulant was identified and the effect of lactone concentration and temperature on setting time determined. Following substantial optimisation work, it was found that a two-speed approach to multi-axial rotation was the most successful; medium sized cream jugs could be produced in just 7 min. With respect to mould materials, the porous resin normally used for pressure casting of sanitary ware was found to be the best option, though since this is quite expensive conventional plaster-of-paris moulds were found to be a suitable material to enable companies, particularly SMEs, to become familiar with the technology whilst avoiding high costs for trials. The processed articles could be successfully fired and glazed using gas-fired kilns with no sign of any black cores. Major advantages of the process include the ability to precisely calculate the amount of ceramic slip required, eliminating either slip wastage or the need to pour used slip back into the virgin material as currently happens with slip casting. In addition, since the precursor suspension has a very high solids content, the time and energy required to dry the green product and associated moulds has been considerably reduced. © 2008 Elsevier Ltd. All rights reserved.