Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic ...TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean ± SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 ± 0.02. A progressive and significant (P < .0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 ± 0.1, +1 hour: 1.3 ± 0.9, +2 hours: 4.1 ± 0.9), peaking at +3 hours (10 ± 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge.
Familial thrombophilia, the hereditary predisposition to venous thromboembolic disease, is associated with a protein S deficiency in approximately 8% of the cases. Laboratory measurements of total ...protein S antigen in affected families have indicated that heterozygotes, ie, individuals carrying both a normal and a defective protein S gene, are severely at risk of developing venous thrombosis at a young age. The recent isolation of protein S cDNA has enabled us to start a search for genetic defects in the protein S gene of heterozygotes. Using Southern blotting on probands of six unrelated families with hereditary protein S deficiency, one proband was found to have a grossly abnormal gene pattern. The abnormality appears to involve at least the deletion of the middle portion of the protein S coding sequence. Family analysis showed that the defect cosegregates with the protein S deficiency. These data agree with the notion that hereditary thrombophilia associated with protein S deficiency is indeed directly the result of a defect in the protein S gene.
Near-physiologic hemodynamic conditions for several hours were needed to study cardiovascular physiology in a murine model. We compared two commonly used anesthetic treatments, urethane ...alpha-chloralose (U-alphaCh; 968 mg U and 65 mg alphaCh/kg) and 2,2,2-tribromoethanol (TBE; 435 mg/kg) and fentanyl fluanisone midazolam (FFM; 3.313 mg fentanyl, 104.8 mg fluanisone, and 52.42 mg midazolam/kg) with respect to mean arterial blood pressure (MAP) and heart rate (HR) for 100 min at similar levels of surgical anesthesia. Assessed every 10 to 15 min, the U-alphaCh+TBE group maintained a significantly (P < 0.001) lower mean MAP (49 4 mmHg) than did the FFM group (78 5 mmHg). Mean HR in the U-alphaCh+TBE group significantly (P < 0.001) increased from 308 34 bpm at the beginning to 477 43 bpm at the end of the experiment. In comparison, the FFM group showed a stable HR of 431 37 bpm. The MAP and HR of the U-alphaCh+TBE group were extremely unstable, with sudden and unpredictable changes in MAP when examined at 1-min intervals. The results of our study show that U-alphaCh+TBE anesthesia should not be used in murine models in which stable, near-physiologic hemodynamics are needed for cardiovascular studies.
Summary
Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and Villa.
Protein S, migrates as a ...doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment.
All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity. Finally, all variants bind to C4b-binding protein with an affinity similar as the wild type recombinant molecule.
Summary
Mouse C127 epithelioid cells were genetically engineered to produce biologically active ³-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma ...virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of ³-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully ³-carboxylated and fully p-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gin, respectively and this variant was designated C variant. The binding of these variants to C4BP was the same as wild type recombinant protein S. This suggests that amino acid residues leu-608, asp-609, asp-611 and glu-612 are not essential for binding of the intact full length protein to C4BP.
Objective. The 20210 G-A prothrombin gene variant and the Factor V Leiden mutation are mutations associated with venous thrombotic risk. The aim of our study was to assess the prevalence of these ...specific mutations in women with a history of preeclampsia or hemolysis elevated liver enzymes, and low platelet count (H)ELLP syndrome and their influence on perinatal outcome. In addition, the association with venous thromboembolism was assessed.
Methods. One hundred fourteen (114) women with a history of preeclampsia or (H)ELLP syndrome were investigated at least 3 months' postpartum for the presence of 20210 G-A prothrombin gene variant and Factor V Leiden mutation.
Results. Four (3.5%) of the women had the 20210 G-A prothrombin gene variant, four (3.5%) women had the Factor V Leiden mutation, and one of these women carried both mutations. The prevalence of prothrombin 20210 G-A prothrombin and Factor V Leiden mutation in women with severe preeclampsia or (H)ELLP syndrome were comparable with the prevalence in the general Dutch population. The odds ratio for thromboembolism for carriers versus noncarriers was 22 (95% confidence interval: 1.7-303). Perinatal mortality was not significantly higher in women with any mutation odds ratio: 1.5 (0.2-9.5).
Conclusions. Although our study confirms that prothrombin 20210 G-A mutation and Factor V Leiden mutation are important genetic risk factors associated with thrombotic risk, these mutations appear not related to perinatal outcome in women with preeclampsia or (H)ELLP syndrome. It is unlikely that these mutations are of major importance for the development of severe preeclampsia or (H)ELLP syndrome.
Summary
Although normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide ...(LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.
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