Shiga toxin 2a binds antithrombin and heparin,but does not directly activate platelets

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism.The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP^® system. ROTEM^® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry.We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM^® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated.In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.     

Novel SERPINC1 missense mutation (Cys462Tyr) causes disruption of the 279Cys-462Cys disulfide bond and leads to type Ⅰ hereditary antithrombin deficiency

BackgroundAntithrombin (AT) is the primary physiological anticoagulant of normal hemostasis. Hereditary AT deficiency, an autosomal dominant thrombotic disease caused by mutations in the AT gene (SERPINC1), is associated with venous thromboembolism.ObjectiveWe investigated the phenotypes, genotypes, and pathogenesis of hereditary AT deficiency in a 12-year-old boy (proband) who developed a pulmonary embolism and a subsequent deep vein thrombosis.MethodsThe AT activity and AT antigen level of the proband and his family members were measured. Mutation sites in all seven exons of SERPINC1 were identified. Analysis of conserved regions around codon 462 of the SERPINC1 gene and functional predictions were performed using bioinformatics tools.ResultsThe proband, his father, and his paternal grandmother demonstrated reduced AT activity and antigen levels consistent with Type I AT deficiency. A novel heterozygous missense mutation, c.1385G>A (Cys462Tyr) was identified in all three symptomatic family members. This missense mutation causes disruption of the 279Cys-462Cys disulfide bond and leads to type Ⅰ hereditary AT deficiency.ConclusionA SERPINC1 missense mutation (Cys462Tyr) causing damage to the 279Cys-462Cys disulfide bond of the AT protein appears to be the cause of Type I AT deficiency in this family. These findings indicate one pathological mechanism associated with hereditary AT deficiency.     

Hyperglycaemia impairs antithrombin secretion: Possible contribution to the thrombotic risk of diabetes

Recent data support that diabetes might be a conformational disease. Certainly, hyperglycaemia causes a broad range of deleterious effects that might facilitate protein aggregation. We have evaluated the effects of hyperglycaemia on antithrombin, a conformationally sensitive serpin with a potent anticoagulant role. Moreover, these studies might also help to understand the thrombotic risk associated to diabetes. We incubated in vitro plasma and purified antithrombin and human hepatoma cells (HepG2) with methyl-glyoxal and glucose. Moreover, a mouse model of acute diabetes was generated with streptozotocin. Antigen, anti-FXa activity, heparin affinity and conformational features of antithrombin were analysed. Histological and intracellular features and distribution of antithrombin in HepG2 and livers of mice were also evaluated. Hyperglycaemia in vitro induced a transition of antithrombin to a form with low heparin affinity that explained the loss of anticoagulant activity, without generation of abnormal conformers (polymers or latent antithrombin). However, these effects were not observed on circulating antithrombin from diabetic mice. In contrast, hyperglycaemia in vivo had significant effects on intracellular antithrombin, which was retained, forming microaggregates within the lumen of dilated cisterns of the endoplasmic reticulum. These effects explained the moderate type I deficiency observed in diabetic mice. Similar intracellular consequences were observed for another hepatic serpin, α1-antitrypsin. Our data further support that diabetes has conformational effects on structurally sensitive proteins. These effects on antithrombin, the main natural anticoagulant, might contribute to the hypercoagulable status of diabetic patients.     

Neonatal renal vein thrombosis and prothrombotic risk

A case of extensive deep venous thrombosis in a four a day old infant was presented. Unusually this patient was shown to be heterozygous for three thrombophilia genes; Factor V Leiden, prothrombin and antithrombin gene mutations, the latter being novel. Conclusion:  There are no randomized controlled trials to guide management in deep venous thrombosis in the newborn but knowledge of the prothrombotic risk factors may help direct treatment.     

Heparin versus prostacyclin in continuous hemodiafiltration for acute renal failure: Effects on platelet function in the systemic circulation and across the filter

Continuous venovenous hemodiafiltration (CVVHDF) is the treatment of choice for critically-ill patients suffering from acute renal failure (ARF). One major problem of extracorporeal circuits is their thrombogenicity, which requires pharmacological blockade of primary (platelet-dependent) or secondary (plasmatic) haemostasis, increasing the patient's bleeding risk.Our study assessed platelet function during CVVHDF, comparing anticoagulant versus antiplatelet pharmacological strategies, commonly used to avoid circuit clotting. Twenty-three critically-ill patients with ARF, requiring CVVHDF were randomized to a prostacyclin analogue (PGI) or to unfractionated heparin (UFH). Ex vivo platelet function, assessed by optical aggregometry (OPA) induced by collagen or ADP, was studied in peripheral blood at baseline, 4 and 24 hrs after starting CVVHDF, and at 4 hrs within the circuit, before and after the filter (n = 9). Coagulation was also monitored.PGI significantly inhibited ADP-induced OPA of peripheral platelets: maximal aggregation (T_max) was reduced at 4 and 24 hrs by 20%, while collagen-induced T_max was significantly reduced at 4 hrs only. In the UFH group, collagen-induced OPA in peripheral platelets was significantly inhibited: slopes of OPA tracings were decreased by 25%, lag time was prolonged by 22%, T_max decreased by 10% already at 4 hrs. ADP-induced OPA showed a similar, but non-significant trend. UFH expectedly prolonged aPTT. In the UFH group, platelet responsiveness to collagen was significantly increased by 30% in post-filter versus pre-filter samples. This effect was blunted in the PGI group.UFH does not protect platelets from filter-induced activation and is associated with a reduced function of systemic platelets. Platelet-inhibiting agents might better prevent the activatory effect of the filter.     

Use of antithrombin III in critical patients

Objective To evaluate the effect of the AT III concentrates upon the clinical evolution and hemostatic parameters.Design Prospective, open, randomized trial.Patients and participants Septic and multiple trauma patients admitted to our Intensive Care Unit.Setting Levels of AT III below 70% were used as criteria to choose 36 patients, 20 of whom received treatment with AT III and 16 did not.Interventions AT III concentrates were administered at an initial dose of 60 U/kg followed by 10 U/kg every six hours.Results The administration of AT III neither contributes to alterations in haemostasis, nor the clinical evolution (evaluated according to Apache II score).Conclusions The results suggest that the administration of AT III concentrates to critical patients with acquired low levels, but without manifest DIC, may not be justified; although further studies on a larger population are required to establish definite conclussions.     

脓毒症患者血管性血友病因子、抗凝血酶和D二聚体的变化及其临床意义

目的研究脓毒症患者血管性血友病因子(vWF)、抗凝血酶(AT)和D二聚体(D-D)变化,探讨其临床意义。方法 58例脓毒症患者为研究对象,41例健康体检者为对照组。检测血浆vWF、AT和DD水平,同时计算患者的急性生理和慢性健康评分(APACHEⅡ)。结果脓毒症组患者血浆vW F和D-D水平明显高于对照组,血浆AT水平明显低于对照组(P0.01);脓毒症患者中死亡组vW F和D-D水平明显高于存活组,AT水平明显低于存活组(P0.01);AT与APACHEⅡ评分呈负相关,相关系数r为-0.839;vWF和D-D与APACHEⅡ呈正相关,相关系数r分别为0.812和0.749。结论 vW F、AT和D-D在脓毒症的发病中起重要的作用,与病情严重程度相关,可作为反映脓毒症预后的指标。     

Revisiting antithrombin in health and disease,congenital deficiencies and genetic variants,and laboratory studies on α and β forms

Antithrombin [AT] is the main inhibitor for activated plasma coagulation serine esterases, inhibiting thrombin, Factors Xa and IXa, but also Factors XIIa, XIa, VIIa, kallicrein, and plasmin. Its activity is highly enhanced by heparin, through binding to the pentasaccharide sequences, for inhibition of all coagulation proteases, except thrombin, which inhibition requires its additional binding to the heparin polysaccharide chain. However, AT is the major inhibitor of thrombin in the blood circulation. Congenital or acquired deficiencies of AT expose affected patients to an increased risk of developing unprovoked and recurrent thrombo-embolic diseases. Antithrombin can be measured with various laboratory techniques, by either immunological or functional methods. Earlier, a radial immunodiffusion immunoassay allowed measurement of the protein antigenic content. Functional assays are mainly designed with Anti-Thrombin or Anti-Factor Xa chromogenic methods and are useful for detecting genetic molecular mutations with decreased inhibitory activity and contributed to study the conformational changes of antithrombin and its variants, which potentially regulate the activity of this serine protease inhibitor. These assays are not equivalent in terms of diagnosing protein abnormalities, associated with increased thrombotic incidence, and they have variable performance for reflecting impaired antithrombin binding capacity for heparin, reduced progressive inhibition of serine proteases, or accelerated switch rates to the latent and less active forms. A small proportion of AT (<10%) is present in blood in the β-form, with a lower oligosaccharide content, a lower Molecular Weight, a higher binding rate to endothelial glycosaminoglycans, and a higher anticoagulant activity, hence requiring specific laboratory methods for its measurement. The β-AT form is then of critical importance for controlling blood activation by tissue injury and preventing development of thrombo-embolic diseases. This article reviews the performance characteristics of the currently available assays, and their usefulness for monitoring the use of AT concentrates in intensive care units, disseminated intravascular coagulation or severe infections, to restore the anticoagulant protective effect of heparin by supplementing the requested AT concentration. The issues of automation, harmonization and standardization are also revisited and discussed.     

危重病患者并发弥散性血管内凝血的诊治

目的探讨危重病患者并发弥散性血管内凝血（DIC）的临床表现及有效治疗措施。方法对我院2005年1月至2006年5月危重病并发DIC患者的临床表现及治疗措施进行回顾性分析和总结;用免疫法、发色底物法和凝固法分别测定36例危重病并发DIC患者血浆D二聚体（D—dimer,D—D）、抗凝血酶活性（AT：A）、凝血酶原时间（PT）、活化部分凝血活酶时间（aPTT）、凝血酶时间（TT）、纤维蛋白原含量（Fbg）等凝血及抗凝指标。结果本组36例危重病患者并发DIC,治愈15例,好转9例,死亡12例;DIC患者的各项指标与正常对照组间差异有统计学意义,特别是D—D含量明显增高,AT：A明显减低,差异具有显著性意义（P〈0．01）,其中AT：A检测具有较高的阳性率（94．4％）,且12例死亡病例均发生明显减低（阳性率达100％）。结论危重病患者DIC发生率高,死亡率高。治疗关键是祛除原发病,密切观察早期D1C诊断指标,早发现、早治疗。对危重病患者的早期综合治疗至关重要。     

肝、肾及肝肾联合移植患者围手术期血浆AT活性检测及其临床意义

目的 探讨肝、肾及肝肾联合移植患者围手术期及恢复期血浆AT活性测定的动态变化及其临床意义。方法观察了176例各类终末期肝病患者、69例。肾病患者、16例肝肾综合征患者在围手术期间血浆AT活性的动态变化。均于术前、术中、术后10d每天抽取静脉血,用发色底物法测定AT活性,并给予分析。结果3组患者术中、术后早期血浆AT活性降低,恢复期AT的活性逐渐升高;肾脏移植组AT活性的恢复最快,与其它两组有显著差异性,而肝移植组与肝肾联合移植组AT的恢复无显著差异性。结论AT活性的恢复是衡量移植物成功的较好指标之一;术后早期受者血液的抗凝作用减弱,处于危险的血栓形成高凝状态之中。