Clinical usefulness of global assays of the anticoagulant pathway of protein C

Abnormalities of the protein C anticoagulant pathway i.e. protein C and protein S deficiencies and factor V Leiden-related activated protein C resistance are among the most frequent risk factors for thrombosis in the Caucasian population. Until now, their screening is based upon the use of individual assays for each component. However, normal results are found in more than 70% of the tested patients. So, there was a rational behind the development of global assays to evaluate the functionality of the protein C pathway, as this would rationalize the use of costly specific assays. Such global assays are based on the ability of endogenous activated protein C generated by activation of protein C by a snake venom extract to prolong a clotting time. Up to now, two assays are commercially available, the ProC Global assay (Dade Behring) and the Protein C Pathway Test (Gradipore). Both were found to be highly sensitive for the factor V Leiden and for protein C deficiency, as confirmed by different studies in patients with a history of venous thrombosis. However, sensitivity of both global assays for protein S deficiency was found to be only moderate and highly variable depending of the cut-off level used. So, these global assays can be validly used for the screening of the factor V Leiden-related APC resistance and for protein C deficiency, but protein S have to be measured in all the cases. The overall sensitivity of these assays for abnormalities of the protein C pathway was dramatically different, since 40% of the patients without any known abnormality have a decreased response to the ProC Global assay versus less than 13% for the Protein C Pathway Test. These characteristics suggest that the clinical usefulness of these two global assays might be different.     

Antiphospholipid antibodies and the protein C pathway

Among the mechanisms suggested for the prothrombotic activity of lupus anticoagulant and antiphospholipid antibodies is the direct inhibition of the anticoagulant activated protein C (APC) pathway. Although some pathological antibodies may be directed towards the proteins involved, we hypothesize that populations exist which selectively inhibit the APC complex as a result of differences in the phospholipid requirements of this complex as compared to those of the procoagulant complexes. The most prominent feature is the requirement for the presence of phosphatidylethanolamine in the membrane for APC anticoagulant function. This mimics the requirements for inhibitory activity of at least a subset of autoantibodies associated with thrombosis. The role of oxidation of the phospholipid in APC function and antibody reactivity is also discussed.     

The effect of lupus anticoagulant in the second-generation assay for activated protein C resistance

The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V-deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.     

Automated assay method for protein C anticoagulant activity]

We developed an automated assay method for protein C anticoagulant activity to prolong the APTT measured with automated coagulation apparatus. The automated assay method required following three reagents. One was 0.025 mol/l calcium chloride solution. The other two were a protein C deficient plasma and a phospholipid plus protein C activator both of which constituted a recomposed protein C assay kit (BMY). 0-50% activities of protein C and clotting times were regressed on normal linear curve, while 25-150% activities and clotting times were regressed on logarithmic curve. The coefficients of variation were 2.9-3.4% (intra-assay) and 3.6-3.9% (inter-assay). A correlation of protein C activities between measured by automated assay method and by ordinary manual method was good (r = 0.954). Our data suggest that this automated assay is a useful method for measuring the protein C anticoagulant activities utilizing a coagulation apparatus.     

Antithrombin III, protein C, and protein S. Naturally occurring anticoagulant proteins

Protein C, protein S, and antithrombin III are naturally occurring anticoagulant proteins. Activated protein C and protein S act by inhibiting the action of the cofactors factor Va and factor VIIIa. Antithrombin III inhibits the serine proteases (factors II, IX, X, XI, and XII); its anticoagulant action is dramatically enhanced by heparin. Deficiencies of these proteins are associated with thromboembolic disease. Antigen and activity assays practical for the clinical laboratory are available for protein C and antithrombin III. Protein S antigen assays are also available. Diagnosis of an anticoagulant protein deficiency has serious implications for a patient and his kindred, and should therefore only be made on the basis of repeated abnormal determinations of a single protein in an individual patient, or documentation of the same protein deficiency in other family members.     

Inherited abnormalities in the protein C activation pathway

The protein C (PC) anticoagulant pathway plays a crucial role in the regulation of fibrin formation via proteolytic degradation of the procoagulant cofactors factor Va and VIIIa by activated PC (APC). PC circulates in plasma as a zymogen, which is activated, on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another endothelial cell-specific protein, the endothelial cell PC/APC receptor (EPCR), binds PC on the endothelial cell surface and further enhances the rate of PC activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of at least four proteins: thrombin, thrombomodulin (TM), PC and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced APC generation and an increase in the risk of thrombosis. In the last years, several reports have suggested the association between mutations in TM and EPCR genes and venous and arterial thrombosis. Surprisingly, no studies have been reported linking mutations with levels of circulating APC, the final product of the interaction between thrombin, TM, PC and EPCR. Here, we describe the previously reported mutations in the TM and EPCR genes, and present the design and evaluation of a new strategy to investigate TM, EPCR, PC and prothrombin gene mutations in arterial and venous thrombosis.     

The effect of the cholinergic anti-inflammatory pathway on experimental colitis

Inflammatory bowel diseases (IBD) are characterized by proinflammatory cytokines, tissue damage and loss of neuron in inflamed mucosa, which implies the cholinergic anti-inflammatory pathway may be destroyed during the process of inflammatory response. In the study, we identified the effect of cholinergic agonist as anabaseine (AN) and nicotinic receptor antagonist as chlorisondamine diiodide (CHD) on trinitrobenzene sulfonic acid (TNBS)-induced colitis, to investigate the potential therapeutic effect of the cholinergic anti-inflammatory pathway on IBD. Experimental colitis was induced by TNBS at day 1, 10 mug AN or 1.5 mug CHD was injected i.p. to mouse right after the induction of colitis, and repeated on interval day till the mice were sacrificed at day 8. Colonic inflammation was examined by histological analysis, myeloperoxidase (MPO) activity, and the production of tumour necrosis factor (TNF)-alpha in tissue. Lamina propria mononuclear cells (LPMC) were isolated, and NF-kappaB activation was detected by western blot. The mice with colitis treated by AN showed less tissue damage, less MPO activity, less TNF-alpha production in colon, and inhibited NF-kappaB activation in LPMC, compared with those mice with colitis untreated, whereas the mice with colitis treated by CHD showed the worst tissue damage, the highest MPO activity, the highest TNF-alpha level, and enlarged NF-kappaB activation in LPMC. Agonist of the cholinergic anti-inflammatory pathway inhibits colonic inflammatory response by downregulating the production of TNF-alpha, and inhibiting NF-kappaB activation, which suggests that modulating the cholinergic anti-inflammatory pathway may be a new potential management for IBD.     

The regulatory roles of C1q

C1q binds to immune complexes to elicit complement-dependent microbial killing and enhance phagocytosis. Besides this classical role, C1q also opsonizes apoptotic cells for clearance by phagocytes. C1q deficiency increases susceptibility to microbial infections and is also associated with elevated autoimmunity as characterized by increased apoptotic bodies in tissues. Most complement proteins are of liver origin, but C1q is predominantly synthesized by peripheral tissue macrophages and dendritic cells. Besides being found in the blood, C1q has also been found deposited in extracellular tissues around these cells. In vitro, immobilized C1q inhibits monocyte, macrophage and T-cell production of inflammatory cytokines. It also regulates T-cell activation. Therefore, mounting evidence suggest a major regulatory role for C1q in inflammation and autoimmunity.     

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development.

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian-specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers.     

Cellular interactions in the thymus regulate the protein kinase C signaling pathway

We provide evidence that thymocytes receive signals from the thymic microenvironment which regulate the protein kinase C (PKC) signaling pathway. Thus, phorbol 12-myristate 13-acetate (PMA) causes a PKC-dependent down-regulation of CD4 expression and induces apoptosis in isolated thymocytes but has little effect on thymocytes maintained within intact thymic lobes or in reaggregate lobes containing purified thymocytes with either thymic or non-thymic stromal cells. Moreover, compact pellets of thymocytes alone are protected from the effects of PMA. This protection is maintained when the compacted thymocytes are rigorously depleted of MHC class II-expressing cells. We conclude that signals arising from thymocyte-thymocyte contact control the utilization of the PKC cascade. These observations have implications for thymocyte signaling in general as well as for the interpretation of studies carried out on thymocyte suspensions.     

胆碱能抗炎通路的机制及其在脓毒症的应用

胆碱能抗炎通路是近年来发现的对全身性炎症反应有调节作用的通路,可通过迷走神经抑制促炎细胞因子的合成从而抑制机体炎症反应.核因子-κB和Janus激酶/信号转导与转录激活子是该通路细胞内信号转导中最重要的两条信号通路.脓毒症以全身炎症反应为特点,是危重症患者的常见死因.根据胆碱能抗炎通路作用迅速有效的特点,推测其可应用于...     

Dicer蛋白在RNA干涉中的作用研究进展

近年来，对功能基因组的研究使人们对基因功能的认识不断深入。后基因时代的来临，又使基因功能研究备受瞩目。RNA干涉对基因表达的抑制为我们提供了新的研究工具。RNA干涉(RNAi)是将双链RNA(dsRNA)导入细胞引起特异基因mRNA降解的一种细胞反应过程。它是转录后基因沉寂(PTGS)的一种。本文简要介绍RNAi的发生机制及该过程中重要分子Dicer的研究进展。     

The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to ±80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.     

The emerging roles of the STING adaptor protein in immunity and diseases

DNA that gains access to the cytoplasm generally serves as a danger signal for the hosts. An emerging paradigm for responding to cytosolic DNAs centres on the endoplasmic reticulum‐resident protein stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS), the hub adaptor of the recently identified DNA sensors. Dynamic regulations of STING action are critical for shaping innate immune responses against microbial infections, as well as for preventing autoimmune diseases. STING is also indispensable for the detection of immunogenic tumours. A deeper understanding of STING modulations could be instrumental for developing novel immunotherapeutic strategies against infectious, autoimmune and cancerous diseases. In this review, we summarize the latest advances on the role of STING in the DNA‐triggered immune reactions, and underscore the critical issues that remain to be resolved in future studies.     

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.     

Wnt 信号通路和微生物在结直肠癌中的作用及意义

结直肠癌是临床上常见的恶性肿瘤之一,发病率在所有恶性肿瘤中排第3位,死亡率排第4位,对人类的健康造成了极大的威胁。近年来,在中国结直肠癌的发病率和死亡率均呈上升趋势,成为威胁人民健康的主要恶性肿瘤之一。结直肠癌发生和发展的机制非常复杂,涉及到的因素多种多样。例如,饮食、遗传、身体活动水平、环境等。目前具体致病机制还不十分清楚,这为结直肠癌的预防及治疗带来了极大的困难。随着分子生物学和分子遗传学的发展,已经明确 Wnt／β-catenin 信号通路在结直肠癌发生和发展中的重要作用。此外,越来越多的证据表明结直肠癌患者的肠道微生物群落与正常人存在明显不同,暗示了肠道微生物群与结直肠癌的关系。本文以 Wnt 信号通路和微生物为主线,总结它们之间的相互作用及在结直肠癌发生、发展和治疗中的作用。