Mechanisms responsible for the failure of protamine to inactivate low-molecular-weight heparin

Protamine is unable to completely reverse the anticoagulant effect of the low-molecular-weight heparins (LMWH), a fact of clinical importance given the rapid increase in use of LMWH in clinical practice. This investigation sought to determine the mechanism by which LMWH were able to resist protamine-mediated inactivation. Affinity fractionation of LMWH by passage through a protamine column, with subsequent determination of molecular mass and sulphate charge density, demonstrated that the protamine-resistant fraction in LMWH is an ultra-low-molecular-weight fraction with low sulphate charge density. This group of molecules was not found in unfractionated heparin, even when species of similar molecular mass were compared. We then determined that different commercially available LMWH varied in their ability to be neutralized by protamine, and that this variability correlated with the total sulphate content of the LMWH. We conclude that reduced sulphate charge, not molecular mass, is the principle reason that protamine is unable to fully inactivate LMWH. Furthermore, different LMWH vary in their ability to be neutralized by protamine, suggesting that product-specific recommendations for neutralization might be developed.     

Influence of protamine on adhesion, chemotaxis and proliferation of human vascular smooth muscle cells

Summary It has been shown that, in streptozotocin diabetic rats, protamine-retarded insulin administered in vivo stimulates intimal hyperplasia in balloon-injured carotid artery. The aim of this study was to evaluate the influence of protamine on cultured human vascular smooth muscle cells (hVSMC), by observing its effects on adhesion, chemotaxis and proliferation. hVSMC were isolated during abdominal surgery, cultured and utilized at passages 6–10. We observed that protamine stimulates: 1) cell adhesion in the concentration range 0.04–20 μg/ml (analysis of variance, ANOVA, p < 0.0001); 2) cell chemotaxis in the absence of fetal calf serum (FCS) in the concentration range 1–200 μg/ml (ANOVA, p < 0.0001) and in the presence of 1 % FCS in the concentration range 5–200 μg/ml (ANOVA, p < 0.0001), further enhancing the chemotaxis induced by 10 % FCS in the concentration range 20–200 μg/ml (ANOVA, p < 0.0001); 3) cell proliferation and ³H-thymidine incorporation from 1 to 5 μg/ml (ANOVA, p < 0.0001); 4) cell c-fos oncoprotein nuclear expression. We also observed that protamine effects on chemotaxis, proliferation and c-fos expression are inhibited by heparin that human insulin stimulates cell proliferation and ³H-thymidine incorporation (ANOVA, p < 0.0001) at concentrations equal to or greater than 480 pmol/l and that these effects of insulin persist in the presence of protamine. In conclusion, protamine influences hVSMC behaviour by interfering with biological functions involved in atherogenesis. The concentrations used in this short-term in vitro study were higher than those probably occurring in vivo in patients chronically treated by protamine-retarded insulin preparations: further studies, therefore, are needed to evaluate the safety of protamine as a retardant of insulin action in vivo. [Diabetologia (1997) 40: 67–75] Received: 23 May 1996 and in revised form: 19 September 1996     

Randomized comparison of enoxaparin with unfractionated heparin following fibrinolytic therapy for acute myocardial infarction.

AIMS: To compare the efficacy and safety of low molecular weight heparin with unfractionated heparin following fibrinolytic therapy for acute myocardial infarction. METHODS AND RESULTS: Three-hundred patients receiving fibrinolytic therapy following acute myocardial infarction were randomly assigned to low molecular weight heparin as enoxaparin (40 mg intravenous bolus, then 40 mg subcutaneously every 8 h, n=149) or unfractionated heparin (5000 U intravenous bolus, then 30 000 U. 24 h(-1), adjusted to an activated partial thromboplastin time 2-2.5x normal, n=151) for 4 days in conjunction with routine therapy. Clinical and therapeutic variables were analysed, in addition to use of enoxaparin or unfractionated heparin, to determine independent predictors of the 90-day composite triple end-point (death, non-fatal reinfarction, or readmission with unstable angina). The triple end-point occurred more frequently in patients receiving unfractionated heparin rather than enoxaparin (36% vs. 26%; P=0.04). Logistic regression modelling of baseline and clinical variables identified the only independent risk factors for recurrent events as left ventricular failure, hypertension, and use of unfractionated heparin rather than enoxaparin. There was no difference in major haemorrhage between those receiving enoxaparin (3%) and unfractionated heparin (4%). CONCLUSION: Use of enoxaparin compared with unfractionated heparin in patients receiving fibrinolytic therapy for acute myocardial infarction was associated with fewer recurrent cardiac events at 90 days. This benefit was independent of other important clinical and therapeutic factors.     

Differential effect of unfractionated heparin and low molecular weight heparin on intravascular tissue factor pathway inhibitor: evidence for a difference in antithrombotic action

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation, which is increased several-fold in post-heparin plasma and thought to contribute significantly to the antithrombotic action of heparin. In the present study we investigated whether subcutaneous (s.c.) administration of a low molecular weight heparin (LMWH), enoxaparin, had a different effect on intravascular pools of TFPI compared with continuous i.v. infusion of unfractionated heparin (UFH). 18 healthy male volunteers were randomly assigned to continuous i.v. infusion with UFH (initially 450 U/kg/24 h, n  = 6) or to s.c. treatment with LMWH once daily (enoxaparin, 1.5 mg/kg, n  = 12) for 72 h. A bolus injection of 5000 IU UFH i.v. caused an 8–13-fold increase in plasma-free TFPI antigen (TFPI Ag), followed by a progressive decrease (81 ± 4%, P  < 0.001) during the 72 h infusion with UFH. 4 h after discontinuation of the infusion, basal free TFPI Ag and heparin-releasable TFPI were significantly decreased compared with the concentrations before the infusion (30 ± 9% and 27 ± 7%, respectively). In contrast, LMWH treatment did not reduce either basal or heparin-releasable TFPI Ag. The changes in plasma TFPI Ag by UFH and LMWH were statistically different between groups both in pre- ( P  < 0.001) and post-heparin ( P  < 0.0001) plasma. The differential effect of UFH and LMWH on intravascular pools of TFPI may contribute to the understanding of the apparent superior efficacy of LMWHs in the treatment of both arterial and venous thrombosis.     

Optimizing adjunctive antithrombotic therapy in the treatment of acute myocardial infarction: a role for low-molecular-weight heparin

Thrombotic complications account for a large proportion of in-hospital deaths from acute myocardial infarction (MI). Although thrombolytic therapy has greatly improved clinical outcomes following MI, thrombin released during clot lysis has a prothrombotic effect, and the thrombolytic agents themselves may directly activate platelets. Antithrombotic therapy as an adjunct to thrombolysis improves the speed and extent of artery recanalization and reduces the incidence of secondary ischemic complications. The current treatment standard is unfractionated heparin (UFH) administered intravenously for 24-48 h. However, UFH has not been unequivocally shown to improve outcomes in large-scale, randomized clinical trials, and shows no evidence of benefit when used as an adjunct to streptokinase-based thrombolysis. Unfractionated heparin also has several clinical and practical disadvantages, such as the need for coagulation monitoring, difficulties attaining a stable and reliable anticoagulant effect, and the risk of hemorrhagic side effects. Low-molecular-weight heparin (LMWH) represents a safe and effective alternative antithrombotic therapy, with a stable and predictable anticoagulant effect, potential for use in combination with either fibrin-specific or streptokinase-based thrombolysis, no need for anticoagulation monitoring, and a low risk of hemorrhagic and other heparin-related complications. Several randomized clinical trials have shown that adjunctive LMWH is at least as effective as UFH in the acute phase of MI, is associated with fewer in-hospital recurrent ischemic events, and has an acceptable safety profile.     

84例肺血栓栓塞症的抗凝治疗分析

目的 探讨低分子肝素（low molecular weightheparin,LMWH）与普通肝素（unfractionatal heparin,UFH）在治疗肺血栓栓塞症（pulmonary thromboembolism,PTE）的疗效和安全性差异。方法 将收治的84例肺血栓栓塞症患者,随机分成两组,42例为治疗组,42例为对照组,治疗组应用低分子肝素抗凝治疗,对照组应用普通肝索抗凝治疗,观察其疗效及副作用。结果 治疗组总有效率90．5％,对照组总有效率69．1％,副作用出现率治疗组为7．1％,对照组为14．3％。结论 低分子肝素和普通肝素均为有效的抗凝药物,综合副作用和治疗效果考虑,低分子肝索更适合于临床。     

Non‐immediate heparin and heparinoid cutaneous allergic reactions: a role for fondaparinux

Non‐immediate allergic cutaneous reactions to heparins have been increasingly reported, typically manifesting as large, eczematous plaques at sites of subcutaneous injection. Patients may demonstrate cross‐reactivity between unfractionated heparin, low molecular weight heparin and semi‐synthetic heparinoids, making finding an alternative difficult. Fondaparinux has been identified as a useful alternative in such patients; here we present the first two documented cases in Australia and a literature review.     

低分子肝素与普通肝素治疗不稳定型心绞痛的对比分析

目的 比较低分子肝素（LMWH）与普通肝素（UFH）治疗不稳定型心绞痛（UA）的有效性及安全性：方法 65例UA病人，随机分为LMWH组（33例）和UFH组（32例），对比观察风组病人心绞痛改善程度、心电图变化情况及不良反应：结果 LMWH组和UFH组心绞痛改善程度比较，两组总有效率比较有统计学意义（93．9％和75．0％，P〈0．05），心电图变化情况总有效率比较无统计学意义（66．7％和62．5％，P〉0．05），不良反应发生率比较有统计学意义（9．1％和15．6％，P〈0、05）。结论 LMWH治疗UA较UFH治疗可显著减少心绞痛的发作，且不良反应少。     

Partial depletion of tissue factor pathway inhibitor during subcutaneous administration of unfractionated heparin, but not with two low molecular weight heparins

Tissue factor pathway inhibitor (TFPI) is released to circulating blood after intravenous (i.v.) and subcutaneous (s.c.) injections of heparins, and may thus contribute to the antithrombotic effect of heparins. We have recently shown that total TFPI activity, plasma free TFPI antigen, and heparin releasable TFPI were partially depleted during repeated and continuous i.v. infusion of unfractionated heparin (UFH), but not during s.c. treatment with a low molecular weight heparin (LMWH). The difference may be attributed to a different mode of action or the different mode of administration. In the present randomized cross-over study, s.c. administration of therapeutic doses of UFH was compared with s.c. administration of two LMWHs. 12 healthy male volunteers were treated for 3 d with UFH, 250 U/kg twice daily, dalteparin, 200 U/kg once daily, and enoxaparin, 1.5 mg/kg once daily. Six participants were also treated with UFH, 300 U/kg once daily. On day 5 a single dose of either drug was given. Peak levels of total TFPI activity and free TFPI antigen were detected 1 h after injection, whereas maximal prolongation of activated partial thromboplastin time (APTT) and peak levels of anti-factor Xa activity and anti-factor IIa activity were detected after 4 h. On UFH administered twice daily, free TFPI antigen decreased by 44% from baseline level before the first injection on day 1 to pre-injection level on day 5. On UFH administered once daily, basal free TFPI antigen decreased by 50%, 56% and 27% on day 2, 3 and 5 respectively, compared with day 1. Minimal depletion of TFPI was detected during treatment with LMWHs. The study demonstrates the different modes of action of LMWHs and UFH and may help to explain the superior antithrombotic efficacy of LMWHs.     

Biological and clinical features of low-molecular-weight heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of unfractionated heparin (UFH) therapy. In contrast, only a few patients have been reported with HIT following low-molecular-weight heparin (LMWH) therapy (LMW-HIT). To define the clinical and biological characteristics of LMW-HIT, 180 patients treated for suspected HIT at 15 French centres were investigated. Clinical history was recorded and HIT was confirmed in 59 patients with positive serotonin release assay results: 57 of them had high levels of antibodies (Abs) to heparin-platelet factor 4 complexes (H/PF4) and two had Abs to interleukin 8. Eleven patients were treated exclusively with LMWH (LMW-HIT) and 48 with UFH either alone (UF-HIT, n = 34) or combined with LMWH (UF/LMW-HIT, n = 14). The LMW-HIT and UF-HIT groups were similar with respect to sex, age, platelet count before heparin therapy, frequency of bleeding and occurrence of disseminated intravascular coagulation. The interval to onset of HIT was longer in LMW-HIT patients compared with UF-HIT patients (P = 0.03). Severe thrombocytopenia (platelets < 15 x 10(9)/l) was more frequent in the LMW-HIT group (P = 0.04). Thrombosis occurred in three of 11 LMW-HIT patients, i.e. as frequently as in UF-HIT patients. LMW-HIT is potentially severe and may be observed after longer heparin treatment compared with UF-HIT. It is highly recommended, therefore, that platelet counts be monitored carefully whenever LMWH is administered.     

Hirudin-Based Anticoagulant Strategies for Patients with Suspected Heparin-Induced Thrombocytopenia Undergoing Percutaneous Coronary Interventions and Bypass Grafting

Multiple changes in the coagulation system occur during pregnancy and account for the hypercoagulable state of pregnancy. Consequently, pregnant women are five times more likely to experience venous thromboembolism than non-pregnant women. Although the estimated rates of such events are low, venous thromboembolic disease is a leading cause of maternal death. The administration of intravenous or subcutaneous unfractionated heparin is the treatment and prophylaxis of choice. Warfarin is safe and efficacious following delivery, but should be avoided during pregnancy. LMWH is a promising alternative for treatment and prophylaxis, but further clinical experience is required.     

Comparison of two treatment durations (6 days and 14 days) of a low molecular weight heparin with a 6-day treatment of unfractionated heparin in the initial management of unstable angina or non-Q wave myocardial infarction: FRAX.I.S. (FRAxiparine in Ischaemic Syndrome)

Aim To assess the benefit of short-term low molecular weightheparin nadroparin compared with unfractionated heparin in unstableangina or non-Q wave myocardial infraction patients and to determinewhether a longer, 2-week low molecular weight heparin regimenwould offer additional clinical benefit. Patients, Methods and Results This was a multicentre, prospective,randomized, double-blind study in three parallel groups, involving3468 patients. Patients received one of three treatment regimens:the unfractionated heparin group received an intravenous bolusof unfractionated heparin 5000IU, followed by an activated partialthromboplastin time adjusted infusion of unfractionated heparinfor 6±2 days; the nadroparin 6 group received an intravenousbolus of nadroparin 86 anti-Xa IU.kg^–1, followed by twicedaily subcutaneous injections of nadroparin 86 anti-Xa IU.kg^–1for6±2 days, and the nadroparin 14 group received an intravenousbolus of nadroparin 86 anti-Xa IU.kg^–1, followed by twicedaily subcutaneous injections of nadroparin 86 anti-Xa IU.kg^–1for14 days. No statistically significant differences were observedbetween the three treatment regimens with respect to the primaryoutcome (cardiac death, myocardial infarction, refractory angina,or recurrence of unstable angina at day 14). The absolute differencesbetween the groups in the incidence of the primary outcome were:–0·3% (P=0·85) for the nadroparin 6 groupvs the unfractionated heparin group and +1·9% (P=0·24)for the nadroparin 14 group vs the unfractionated heparin group.Furthermore, there were no significant intergroup differencesregarding any of the secondary efficacy outcomes. However, therewas an increased risk of major haemorrhages in the nadroparin14 group compared with unfractionated heparin (3·5% vs1·6%;P=0·0035). Conclusions Treatment with nadroparin for 6±2 days providessimilar efficacy and safety to treatment with unfractionatedheparin, for the same period, in the therapeutic managementof acute unstable angina or non-Q wave myocardial infarction,and may be easier to administer. A prolonged regimen of nadroparin(14 days) does not provide any additional clinical benefit.     

Fibrinogen determination in a series of proficiency studies

Plasma heparin levels measured using several different methods showed a wide variation in results. In particular an anti-factor Xa clotting technique gave very much higher results than thrombin clotting time and partial thromboplastin time techniques. This was less marked when a synthetic chromogenic substrate assay was used. Possible reasons for these observations and their relevance in the selection of therapeutic ranges are discussed.     

Once-Daily Enoxaparin in The Outpatient Setting Versus Unfractionated Heparin in Hospital for the Treatment of Symptomatic Deep-Vein Thrombosis

Background: Once- and twice-daily low-molecular-weight heparin administered in hospital have been shown to be effective and safe for treating deep-vein thrombosis. The aim of this study was to compare the efficacy and safety of deep-vein thrombosis treatment using once-daily subcutaneous enoxaparin in the outpatient setting with intravenous unfractionated heparin in hospital.Methods: This randomized, parallel-group, open-label study was conducted in 18 centers in 4 countries. In total, 298 patients with symptomatic deep-vein thrombosis who were eligible for home treatment were randomized to treatment with enoxaparin in the outpatient setting (1.5 mg/kg subcutaneously once-daily) or unfractionated heparin in hospital (5000 IU bolus and 1250 IU/hour intravenous infusion) for ≥5 days. Clinical endpoints were assessed during a 6-month follow-up period.Results: Among all patients treated with enoxaparin, there was a trend towards fewer recurrent deep-vein thromboses (1.3% vs. 5.4%; p = 0.060) and pulmonary emboli (1.3% vs. 4.1%; p = 0.17) compared with patients treated with unfractionated heparin. When considering a post-hoc combined endpoint of deep-vein thrombosis and pulmonary embolism, significantly fewer events occurred in the enoxaparin group than in the unfractionated-heparin group (2.7% vs. 8.8%; p = 0.026). The incidences of bleeding events and adverse events in the enoxaparin and unfractionated-heparin groups were similar.Conclusions: Once-daily subcutaneous enoxaparin in the outpatient setting is at least as effective and as well tolerated as in-hospital intravenous unfractionated heparin for treatment of deep-vein thrombosis.     

Incidence and outcomes of protamine reactions in patients undergoing catheter ablation of atrial fibrillation

Background  Aggressive anticoagulation with heparin to maintain an activated clotting time (ACT) >300 s is required during catheter ablation of atrial fibrillation (AF) to reduce the risk of systemic thromboembolism. The purpose of this study is to describe the incidence and outcome of protamine reactions and analyze the risk factors in patients undergoing catheter ablation of AF. Methods  The patient population included 242 consecutive patients (193 men, age 57.6 ± 10.8 years) with drug refractory AF who underwent catheter ablation and received protamine immediately following catheter ablation to reverse the effects of heparin. Fifty eight of these patients had prior exposure to protamine. Results  Three of the 242 patients in our study developed an adverse reaction to protamine (1.2%). Although each of the three protamine reaction presented in a dramatic fashion with profound hypotension, all three patients responded to medical treatment and did not experience clinical sequelae. Age, gender, type of AF, number of ablations, prior exposure, diabetes mellitus, and ejection fraction did not predict the occurrence of these reactions. Conclusion  This study reports, for the first time, the incidence and outcomes of protamine reaction in patients undergoing catheter ablation of AF. The results of this study reveal that protamine reactions present in a dramatic fashion often with profound hypotension. Although the incidence of protamine reactions in this setting is low (1.2%), they do occur. Electrophysiologists who use protamine need to be aware of this reaction and the appropriate therapeutic interventions. Dr. Chilukuri was supported by The Norbert and Louise Grunwald Cardiac Arrhythmia Research Fund. Dr. Scherr was supported by the FWF—Austrian Science Fund.     

Haemorrhagic Effect of Enoxaparin,a Low Molecular Weight Heparin: Comparison with Unfractionated Heparin in Humans

The haemorrhagic effects of unfractionated heparin (UFH) and the low molecular weight heparin (LMWH) enoxaparin were investigated and compared in the gastric mucosa (haemorrhage induced by biopsy) and skin (haemorrhage induced by Simplate) of 12 healthy volunteers. Administration of UFH and LMWH (given in a dose of 75 anti-Xa U/kg intravenously) increased median gastric bleeding time (3.5 min) and geometric mean blood loss (11.5 l) to 19 min (p = 0.00003) and 54.1 l (p = 0.0021) after UFH and to 13 min (p = 0.008) and 29.0 l (p = 0.275) after LMWH. Median skin bleeding time (4.25 min) increased to 6.0 min after UFH (p = 0.003) and to 6.75 min after LMWH (p = 0.0008). Mean heparin activity in plasma was 20% higher after LMWH than after UFH. The calculated gastric bleeding time to heparin activity ratio was significantly lower for LMWH than for UFH (0.05).