Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram‐negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.
Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacrylamide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.
SUMMARY The concentration of the elastase-a 1 proteinase inhibitor complex (E-a I PI) in a meningococcal infection in an index case with severe changes in haemostasis was measured. The concentration of the E-al PI complex was increased throughout the duration of the illness, although concentrations of the blood clotting factors were severely decreased. The release of polymorphonuclear elastase activity may contribute to the depletion in clotting factors.Disseminated intravascular coagulation is often associated with meningococcal infection.1 Recently data have been presented, which suggest that the depletion in blood clotting factors, observed in blood testing, could be the direct result of clotting factor degradation by the action of systemically released polymorphonuclear leucocyte elastase activity.2 With the development of a sensitive immunoassay for the elastase-a 1 proteinase inhibitor complex (E-a 1 PI), its quantitation in plasma provides an indirect but clear indication of elastase release.3 We describe a case of disseminated intravascular coagulation caused by Neisseria meningitidis. Material and methodsClotting factor assays were performed using methods previously described.4 Total white cell count, platelet count, one stage prothrombin times (PT), and partial thromboplastin (kaolin) times (PTTK) were estimated using routine tests available in the department.The estimation of the elastase-c 1 proteinase inhibitor complex (E-al PI) was performed as previously described,3 but with the following changes: standard, test, and control plasmas were diluted 1/1000 in 0 05 M phosphate buffer (pH 7 4) containing 0-05% Tween and 1% chick serum. The second antibody in the assay was conjugated to peroxidase and used at a dilution of 1/800. CASE REPORTA five year old girl was admitted to hospital with an 18 hour history of malaise, fever, and vomiting followed by the abrupt and rapid appearance of an extensive generalised purpuric and ecchymotic rash.Accepted for publication 30 June 1986 Culture of cerebrospinal fluid and blood yielded group B meningococci. The child was immediately treated with intravenous penicillin. Ecchymotic spots continued to develop over the next few hours, and at 15 hours (T15) this was followed by the abrupt and rapid appearance of extensive generalised purpuric and ecchymotic rash. The clotting screen repeated at 30 hours (T30) showed a pronounced deterioration in the clotting factor activity and platelet count. The coagulation screen at 34 hours (T34) showed a considerable improvement. Thereafter she made a slow steady improvement, her toxaemia settling within 48 hours. About five days after admission all coagulation changes had returned to normal. ResultsBacterial analysis identified the organism as Neisseria meningitidis. The results show a time dependent decrease in blood clotting factors (table 1). The concentration of the E-a 1 PI complex showed a sustained increase throughout the duration of the illness. One hundred and twenty hours (T120) after admission all coagulation variabl...
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