Labor induction with oxytocin in pregnant rats is not associated with oxidative stress in the fetal brain

Despite the widespread use of oxytocin for induction of labor, mechanistic insights into fetal/neonatal wellbeing are lacking because of the absence of an animal model that recapitulates modern obstetric practice. Here, we create and validate a hi-fidelity pregnant rat model that mirrors labor induction with oxytocin in laboring women. The model consists of an implantable preprogrammed microprocessor-controlled infusion pump that delivers a gradually escalating dose of intravenous oxytocin to induce birth at term gestation. We validated the model with molecular biological experiments on the uterine myometrium and telemetry-supported assessment of changes in intrauterine pressure. Finally, we applied this model to test the hypothesis that labor induction with oxytocin would be associated with oxidative stress in the newborn brain. Analysis of biomarkers of oxidative stress and changes in the expression of associated genes were no different between oxytocin-exposed and saline-treated pups, suggesting that oxytocin-induced labor was not associated with oxidative stress in the developing brain. Collectively, we provide a viable and realistic animal model for labor induction and augmentation with oxytocin that would enable new lines of investigation related to the impact of perinatal oxytocin exposure on the mother-infant dyad

Structural pathology and functional analysis of vitamin K-dependent protein S

Protein S deficiency is an autosomal dominant trait affecting around 10% of families thrombophilic families. The high affinity interaction of approximately 60% of protein S with C4b -binding protein (C4BP) has raised a complicated situation for the diagnosis of deficiency states. Because only free protein S functions as cofactor in the protein C anticoagulant, it is important to measure free protein S level precisely. In this study a free protein S assay was developed using its natural ligand C4BP to capture free protein S from plasma for routine clinical purpose. Although interference by C4BP is so far considered as the major hindrance for developing ELISA, the new approach has utilized this phenomenon of high affinity interaction between protein S and C4BP, thereby reversing the adverse effect of C4BP to one of good use. (1) Mutations in a variety of human genes are now known to predispose to venous thrombosis. Characterization of the wide spectrum of gene mutations causing thrombosis may allow us to relate specific gene lesions to the probability of thromboembolism as well as to the severity of thrombotic episodes, beside providing the molecular mechanism of deficiency states. To establish the relationship between genetic abnormalities of protein S and their phenotypic expressions, four naturally occurring missense mutations were chosen to analyze thier in vitro secretion profiles and functional characteristics, which illustrated the importance of in vitro experimental characterization in each and individual cases of naturally occurring missense mutations before marking them as the underlying genetic defect. (2) Since early nineties, the suggestion about the synergistic effect between two thrombotic risk factors when associated in one patient has been lacking a biochemical basis. The now observed deficient APC-cofactor activity of protein S Heerlen in the degradation of FVa Leiden suggests a possible synergistic pathogenic mechanism between these two genetic traits resulting in increased risk of thrombosis. (3) In addition, using two monoclonal antibodies as probes the structure-function relationship studies on protein S and APC interaction were carried out. R49, Q52 in TSR was found to be part of the epitope of monoclonal HPS 67 and K97, T103 in EGF1 for monoclonal HPS 54. These data implicated indirectly as the key amino acids for APC interaction, based on the fact that these two antibodies could completely block the APC-cofactor activity of protein S. The observation that HPS 67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface, suggesting that TSR is free to interact with membrane-bound APC. (4) Also, evaluation of the importance of amino acid residues 447-460 in protein S for binding to C4BP was performed. One amino acid Y456 was found to be important residue for C4BP interaction in this region. However, blocking this region by monoclonal antibody HPS 34 was not sufficient enough to inhibit protein S-C4BP interaction completely, suggesting that the interaction site constitute a fairly large binding surface

Placental dermatan sulfate: isolation, anticoagulant activity, and association with heparin cofactor II

Pregnancy is associated with hemostatic challenges that may lead to thrombosis. Heparin cofactor II (HCII) is a glycosaminoglycan-dependent thrombin inhibitor present in both maternal and fetal plasma. HCII activity increases during pregnancy, and HCII levels are significantly decreased in women with severe pre-eclampsia. Dermatan sulfate (DS) specifically activates HCII and is abundant in the placenta, but the locations of DS and HCII in the placenta have not been determined. We present evidence that DS is the major anticoagulant glycosaminoglycan in the human placenta at term. DS isolated from human placenta contains disaccharides implicated in activation of HCII and has anticoagulant activity similar to that of mucosal DS. Immunohistochemical studies revealed that DS is associated with fetal blood vessels and stromal regions of placental villi but is notably absent from the syncytiotrophoblast cells in contact with the maternal circulation. HCII colocalizes with DS in the walls of fetal blood vessels and is also present in syncytiotrophoblast cells. Our data suggest that DS is in a position to activate HCII in the fetal blood vessels or in the stroma of placental villi after injury to the syncytiotrophoblast layer and thereby inhibit fibrin generation in the placenta

A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen

A new method to determine the concentration of the free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specifity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies

Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Heparin cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo