Heparanase neutralizes the anticoagulation properties of heparin and low-molecular-weight heparin

BACKGROUND: Heparanase is a mammalian endo-D-glucuronidase that cleaves heparan sulfate (HS) in the extracellular matrix and cell surface. It is preferentially expressed by cells of the immune system and tumor cells. Heparanase overexpression in experimental tumor models results in increased angiogenesis and metastasis. Heparin and low-molecular weight heparin (LMWH) inhibit HS degradation by heparanase. OBJECTIVE: To investigate whether heparanase cleaves heparin and LMWH, and elucidate its effect on blood coagulation. METHODS: Heparin and LMWH were incubated with recombinant heparanase and subjected to measurements of molecular size (size exclusion chromatography) and anticoagulant activity (plasma APTT-activated thromboplastin time, and anti-Xa activity). APTT was also measured in plasma samples of transgenic mice overexpressing heparanase, in comparison with control mice. RESULTS: Incubation of heparin and LMWH with heparanase resulted in degradation of these substrates, as revealed by a significant decrease in their molecular weight. This was correlated with a marked suppression of the anticoagulant activity of heparin and LMWH, as indicated by a decreased effect on APTT and anti-Xa activity, respectively, when human plasma was added. Transgenic mice overexpressing heparanase exhibited a significantly shorter APTT than control mice. CONCLUSION: Heparanase is capable of degrading heparin and LMWH, so that its overexpression by tumor cells may contribute to heparin resistance, commonly occurring in cancer patients. In view of the complexity of the currently available heparanase activity assays, we propose an indirect approach to quantify heparanase activity by measuring the decrease in plasma APTT or anti-Xa activity exerted by the enzyme under the defined conditions.     

Anticoagulation monitoring part 2: Unfractionated heparin and low-molecular-weight heparin

OBJECTIVE: To review the availability, mechanisms, limitations, and clinical application of point-of-care (POC) devices used in monitoring anticoagulation with unfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs). DATA SOURCES: Articles were identified through a MEDLINE search (1966-August 2004), device manufacturer Web sites, additional references listed in articles and Web sites, and abstracts from scientific meetings. STUDY SELECTION AND DATA EXTRACTION: English-language literature from clinical trials was reviewed to evaluate the accuracy, reliability, and clinical application of POC monitoring devices. DATA SYNTHESIS: The activated partial thromboplastin time (aPTT) and activated clotting time (ACT) are common tests for monitoring anticoagulation with UFH. Multiple devices are available for POC aPTT, ACT, and heparin concentration testing. The aPTT therapeutic range for UFH will vary depending upon the reagent and instrument employed. Although recommended by the American College of Chest Physicians Seventh Conference on Antithrombotic and Thrombolytic Therapy, establishing a heparin concentration-derived therapeutic range for UFH is rarely performed. Additional research evaluating anti-factor Xa monitoring of LMWHs using POC testing is necessary. CONCLUSIONS: Multiple POC devices are available to monitor anticoagulation with UFH. For each test, there is some variability in results between devices and between reagents used in the same device. Despite these limitations, POC anticoagulation monitoring of UFH using aPTT and, more often, ACT is common in clinical practice, particularly when evaluating anticoagulation associated with interventional cardiology procedures and cardiopulmonary bypass surgery.     

Effects of low-molecular-weight heparin on platelets as compared with commercial heparin

Five subjects were injected with 5,000 IU of commercial heparin and low-molecular-weight heparin at an interval of 20 days after each injection. Both heparins produced the same platelet factor 4 release immediately after administration (commercial heparin 114.6 +/- 21.6 ng/ml, low-molecular-weight heparin 113.1 +/- 22.1 ng/ml). However, commercial heparin induced a more evident potentiating effect on ADP-induced platelet aggregation and was still present 60 min after injection. Low-molecular-weight heparin had a higher anti-Xa-specific activity than that determined by activated partial thromboplastin time. The opposite was true for the commercial preparation.     

不同肝素溶媒对血仿膜吸附法无肝素透析吸附稳定性及临床应用效果的影响

目的在前期研究应用血仿膜吸附法无肝素透析(HCHD)对高危出血患者行血液透析(HD)基础上,深入探讨不同肝素溶媒对此方法安全性和临床应用效果的影响。方法急、慢性HD高危出血患者14例。自身交叉对照设计,分别以生理盐水(NS)和5%葡萄糖(GS)为肝素溶媒,每一患者分别先后经历两次不同方式HCHD治疗。观察指标分3组进行自身和组间对照,同时观察不同溶媒对肝素吸附量和吸附稳定性的影响。结果①HCHD期间,14例患者未有加重出血的情况,3例活动性出血患者出血停止。②肝素平均吸附量GS组(4513±1446)U,NS组(4011±444)U;仅在NS冲洗液中检测到少量肝素的释放。③血活化部分凝血活酶时间(APTT)水平HCHD期间基本无变化,而同期HD组延长了274%（P<0.01）。其余指标3组间无显著性差异（P>0.05）。结论极性溶媒NS对HCHD肝素吸附量、体内凝血系统无明显影响,对于高危出血、尤其是活动性出血患者,HCHD是一种简便、安全、有效的无肝素透析方法。     

无肝素、低分子肝素及改良无肝素透析在肝硬化病人血液透析中的应用

[目的]观察无肝素、低分子肝素及改良无肝素透析在肝硬化病人血液透析中的安全性。[方法]选取88例肝硬化伴出血倾向病人,按采取透析方式的不同分为3组,A组为无肝素透析;B组为低分子肝素透析;C组为改良无肝素透析,观察病人透析前后凝血功能、透析器凝血情况及临床出血情况。[结果]3组病人透析前后凝血功能比较差异无统计学意义,A组发生凝血率显著高于B组、C组;透析后A组、C组病人出血倾向无加重表现,B组2例病人出现透析后出血倾向加重。[结论]改良无肝素透析在肝硬化病人血液透析中的应用安全性最高。     

应用抗Xa活性测定肝素、低分子肝素及利伐沙班的性能验证

目的评价CS5100全自动血凝仪(简称CS5100)应用抗Xa活性检测血浆肝素、低分子肝素(LMWH)水平及利伐沙班的性能验证。方法参照美国临床和实验室标准协会文件对CS5100应用抗Xa活性检测血浆肝素、LMWH及利伐沙班水平的精密度(批内精密度及日间精密度)、准确度、线性、参考区间、携带污染率性能进行验证。结果CS5100检测血浆肝素、LMWH及利伐沙班水平的批内精密度及日间精密度符合厂家说明书给定的标准;准确性验证结果偏倚在生物学变异系数(CV)要求内;应用试剂配套稀释液时血浆肝素水平的线性验证试验未达标;应用体检健康者血浆作稀释液时肝素、LMWH及利伐沙班水平的线性验证试验理论值和实测值的回归方程均符合要求;携带污染率各参数CV<10%;参考区间验证通过。结论CS5100检测血浆肝素、LMWH及利伐沙班水平的精密度、准确度、携带污染率、线性结果均符合质量控制要求,能够保证检验质量。但要注意检测结果超过线性时,要使用体检健康者血浆作稀释液手动上机检测。     

构建动脉球囊损伤大鼠模型中低分子肝素与普通肝素的作用比较

背景:球囊对血管的损伤容易导致血栓形成,所以适当的抗凝治疗是动脉球囊损伤模型建立成功的关键. 目的:对比低分子肝素与普通肝素在建立大鼠颈动脉球囊损伤模型中的效果及作用.设计、时间及地点:随机对照动物实验,于2006-11/2007-03在深圳市人民医院临床实验中心完成.材料:雄性SD大鼠60只,体质量250～300 g;2.0 mm×20 mm经皮冠状动脉球囊成形导管及压力泵为Medtronic公司产品.方法:60只雄性SD大鼠随机分为2组,低分子肝素组与普通肝素组,每组30只,分别行左颈总动脉PTCA球囊内膜剥脱术建立动脉球囊损伤模型,低分子肝素组于术前12 h及术后24 h分别皮下注射低分子肝素600 U/kg,普通肝素组于术前经尾静脉注射普通肝素100 U/kg.主要观察指标:分别于术后即刻,3,7,14,21及28 d取5只大鼠颈动脉损伤段和对侧正常动脉行苏木精-伊红染色病理观察.结果:球囊导管损伤使大鼠颈总动脉内膜剥脱和新生内膜增生,管腔狭窄.术后7 d内膜开始增生,14～28 d增生最明显.低分子肝素组建模成功率高于普通肝素组,血栓形成几率低于普通肝素组.结论:在建立大鼠颈动脉球囊损伤模型过程中,低分子肝素较普通肝素进一步降低血栓形成的几率,提高建模成功率.     

小剂量肝素钠与低分子肝素钙对脓毒症治疗作用的对比研究

目的观察小剂量肝素钠与低分子肝素钙对脓毒症的治疗效果差异。方法将90例脓毒症患者随机分为A、B、C三组,A组给予常规治疗包括早期广谱抗生素,液体复苏等,B组常规治疗基础上持续给予小剂量肝素5~10U/(kg·h)静脉泵入,C组在常规治疗基础上加低分子肝素钙0.6 ml皮下注射。对比三组患者治疗前及治疗后第1、3、5、7 d急性生理学慢性健康状况Ⅱ(APACHEⅡ)评分,治疗前和治疗后的凝血指标、炎症因子指标、播散性血管内凝血(DIC)发生率、28 d病死率和不良反应发生率。结果 B、C两组患者治疗后各时点APACHEⅡ显著低于A组(P0.05),C组患者治疗5 d后APACHEⅡ分值显著低于B组(P0.05)。B、C两组患者治疗后血小板(PLT)、纤维蛋白原(Fib)、凝血活酶时间(APTT),凝血酶原时间(PT)水平显著优于A组(P0.01),C组患者各指标显著优于B组(P0.05)。B、C两组患者治疗后血清肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、C反应蛋白(CRP)水平显著低于A组(P0.05),C组患者TNF-α水平显著低于B组(P0.05)。B、C两组患者DIC发生率、28 d病死率显著低于A组(P0.01,P0.05),C组患者DIC发生率显著低于B组(P0.05)。结论低分子肝素钙对脓毒症的治疗更为有效,可以有效改善患者的凝血功能及炎症水平,具有临床应用价值。     

Alternatives to heparin infusion.

This article offers an overview of the pathophysiology, diagnosis, and treatment of heparin-induced thrombocytopenia (HIT). This disorder is an immune-mediated reduction of platelets with subsequently increased generation of thrombin and increased risk of arterial and venous thrombosis. Heparin-induced thrombocytopenia can occur as an isolated incident or with acute thrombosis (HIT with thrombosis syndrome [HITTS]). Proper recognition, cessation of all forms of heparin (and compounds that cross-react with heparin), and rapid initiation of nonheparin anticoagulation are essential steps in reducing the risk of death, limb amputation, and new thrombotic events. Current alternatives to heparin are reviewed.     

Structure-activity relationships of heparin. Independence of heparin charge density and antithrombin-binding domains in thrombin inhibition by antithrombin and heparin cofactor II.

To better understand how heparin structure affects its activity the relationships between the functional domains for inhibitor binding and charge density were investigated to determine how these domains affect heparin-mediated thrombin inhibition by two different heparin-dependent protease inhibitors, antithrombin (AT) and heparin cofactor II (HC II). A series of heparins, fractionated systematically by charge density, was further fractionated on antithrombin agarose to isolate more homogeneous subfractions that were either inactive or highly active with respect to thrombin inhibition by AT. With AT, the activities of the AT-active subfractions increased sharply with heparin charge density, while those with little or no affinity for AT were virtually inactive. In contrast, with HC II inhibitor, the activities of the heparins depended only upon their charge densities and were independent of AT affinity. At any given charge density, the heparin before fractionation by AT affinity and the fractions that were highly active and inactive with AT were all equally active with HC II. The two inhibitors also differed in their reactivity with heparan sulfate and dermatan sulfate. A charge-density effect with the subfractions having similar high affinity for AT demonstrates that charge density represents a heparin functional domain that is independent of the AT-binding domain. The behavior of the AT-inactive heparins, being fully active with HC II, demonstrates the functional domain necessary for AT binding is not needed to produce HC II activity.     

Erythrocytes as carriers for heparin

Encapsulation of heparin into resealed carrier erythrocytes may be useful in the prevention of thromboembolism because heparin may be released locally during retraction of fresh thrombi. Heparin encapsulation in human and canine erythrocytes was achieved by hypo-osmotic dialysis with 44% and 36% encapsulation, respectively. Encapsulated heparin did not leak from carrier erythrocytes in vitro. In vivo survival of heparin-loaded carrier erythrocytes in dogs was biphasic: After an initial phase of cell loss the carrier cells survived in circulation with a half-life of 28 h. The present results seem to warrant clinical studies with heparin-loaded carrier erythrocytes.