The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific ...pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXaQ61A interacts with antithrombin alone with a nearly normal kass; however, we observe only a 4-fold increase in kass in the presence of H5. The x-ray crystal structure of fXa reveals that Gln61 forms part of the S1′ and S3′ pocket, suggesting that the P′ region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.
Viral oncolytic therapy for malignant brain tumors involves local intratumoral delivery of a genetically engineered virus with tumor cell-specific lytic activity. Promising preliminary results have ...been achieved in preclinical models with G207, a replication-competent herpes simplex virus type 1 constructed with multiple directed mutations. Although the safety of G207 has been demonstrated in adults, application of viral oncolytic therapy to children with brain tumors has been delayed because of previous lack of data concerning the impact of a replication-competent oncolytic virus on the developing mammalian brain. In this study there was no significant difference in long-term physical development, cognitive performance, or exploratory behaviors between mice that received intracerebral inoculation of G207 or control saline at 4 days of age. However, histological examination and magnetic resonance imaging revealed frequent unilateral ventriculomegaly ipsilateral to the site of injection in only the G207 group. These results suggest that although it is unlikely that G207 will have significant adverse effects on neurodevelopmental outcomes of pediatric patients with brain tumors, an initial study of G207 in children should exclude those patients with tumors in or near the ventricular system as well as patients less than 2 years of age. Furthermore, patients in such a study will need to be closely monitored for the development of hydrocephalus.
The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates ...representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P1‘−P4‘ substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P1‘−P4‘ positions had been substituted by different classes of amino acids. This revealed that the P1‘ position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P1‘−P4‘ positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.
The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region ...are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in α1-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 °C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and α1-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.
Objective The purpose of this analysis was to evaluate a novel strategy for reporting adverse events in the Pediatric Heart Network’s randomized surgical trial of systemic–pulmonary artery shunt ...versus right ventricle–pulmonary artery conduit in infants with hypoplastic left heart syndrome. The strategy was developed to align the reporting process with the needs of a surgical trial while maintaining participant safety. Methods Adverse event reporting was analyzed for 2 groups of study subjects: those randomized to a trial arm during a period in which a standard adverse event reporting system was used (period 1) and those randomized after institution of a system that focused serious adverse event reporting on 6 sentinel events (period 2). The analysis encompassed the period from randomization (Norwood surgery) to hospital discharge from stage II surgery. Adverse event rates were compared using a Poisson regression model for the number of events per subject. Results From period 1 to period 2, the rate of serious adverse events requiring expedited reporting decreased as expected (0.42 vs 0.14/subject/month of follow-up; P < .001). Subjects with a serious (sentinel) adverse event in period 2 had a significantly higher rate of death and cardiac transplantation. Conclusions The new adverse event reporting system successfully targeted subjects at highest risk, while decreasing the administrative burden associated with adverse event reports. This methodology may be of benefit in trials evaluating surgical or device-based interventions and in critically ill populations where many common clinical events would qualify as serious adverse events in the context of a drug trial.
Transport in vitro fertilisation (IVF) is an important development in assisted conception. We report our experience of transport IVF treatment from May 1993 to April 1996 at Arrowe Park Fertility ...Centre. A total of 74 patients were treated during this period. The main indications of treatment were tubal damage, unexplained infertility of more than 3 years duration, polycystic ovarian disease and endometriosis. Total number of simulated ovarian cycles were 101. Thirteen cycles were abandoned. Eighty-eight transport IVF cycles led to 29 pregnancies, giving a live birth rate and on-going pregnancy rate per patient of 31% and per cycle rate of 23%. There was one case of severe ovarian hyperstimulation syndrome. Of the 74 patients, 70 (95%) patients preferred to have treatment at the local hospital. Transport IVF is an effective, efficient and economic way of providing assisted conception at district general hospital. The success rate and safety of transport IVF are comparable with conventional IVF treatment.
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