Partial reversal of low molecular weight heparin (PK 10169) anti-Xa activity by protamine sulfate: in vitro and in vivo study during cardiac surgery with extracorporeal circulation

Neutralization of a low molecular weight (LMW) heparin fraction by protamine sulfate was evaluated in vitro and in vivo. Anti-Xa and anti-IIa activities were measured by amidolytic and coagulation methods (activated partial thromboplastin time, APTT). Fifteen patients (4 males and 11 females) underwent surgery with extracorporeal circulation. In vitro, anti-Xa and anti-IIa activities and APTT of unfractionated heparin were neutralized with a protamine/heparin (P/H) gravimetric ratio of 1.6, 1.33 and about 2, respectively. Anti-IIa activity and APTT induced by PK 10169 were completely corrected at a P/H ratio of 1 and 2, respectively, while anti-Xa activity was incompletely neutralized at a ratio of 5. In vivo, in 9 patients who did not receive intravenous protamine sulfate, a good correlation was found between doses of PK 10169 infused, anti-IIa plasma level and blood loss. In 3 patients who were treated prophylactically with protamine, bleeding was normal or only slightly increased. In 3 patients who received protamine because of hemorrhage, mean anti-Xa and anti-IIa were 2.3 and 0.54 U before and 1.32-0.06 U after neutralization. Bleeding was stopped by a second dose of protamine in 1 patient, but blood loss was abnormal in the other patients. However, a correlation between bleeding and anti-Xa or anti-IIa activities was not clearly evident.     

Clinical effects of different protamine doses after cardiopulmonary bypass

The optimal dose of protamine needed to reverse the anticoagulant effect of heparin after cardiopulmonary bypass is still not known. In this retrospective cohort study, we investigated 3 different dose regimes in 300 patients undergoing coronary artery bypass grafting. Group A patients (n = 100) were given protamine in the ratio of 1.3 mg to 1 mg heparin, group B patients (n = 100) were given 0.75 mg protamine to 1 mg heparin, and group C patients (n = 100) were given protamine in fractionated doses of 1 mg + 0.15 mg + 0.15 mg to 1 mg heparin. The groups were comparable in all major clinical and operative variables. The heparin dose was almost identical in the groups. The rate of red cell transfusion was significantly higher in group B than in the other groups. A similar but nonsignificant trend was observed in the incidence of resternotomy for postoperative bleeding, mediastinal drainage, and postoperative hemoglobin loss. The study demonstrates that a single bolus dose of 1.3 mg protamine to 1 mg heparin is safe and efficient for neutralizing heparin after cardiopulmonary bypass.     

Use of Organon, a synthetic heparinoid, in two cardiopulmonary bypass procedures in the same patient sensitive to heparin

We report the case of a patient who underwent two cardiopulmonary bypass (CPB) procedures with Orgaran because of heparin-induced thrombocytopenia. A 38 years-old man with ischemic mitral insufficiency was operated for coronary artery bypass and valvular replacement. The CPB was carried out with heparin. Heparin-induced thrombocytopenia occured and was proven immunologically. Two months later, a new valvular replacement was performed because of paravalvular leak due to endocarditis. The Orgaran-CPB protocol was as follows: 5,000 units before cardiopulmonary bypass, 5,000 units in the priming volume, anti-Xa level between 0.9 and 1.1 units/mL, with injection of 1,500 units if necessary, no administration of protamine. One month later, a new valvular replacement was necessary and performed with the same protocol using Orgaran. No bleeding or thrombotic complication occurred. Orgaran is a safe and reliable anti-thrombotic substitute if anti-Xa activity is closely monitored.     

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 microliters) to 19 min (p = 0.00003) and 54.1 microliters (p = 0.0021) after UFH and to 13 min (p = 0.008) and 29.0 microliters (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 (p < 0.05).     

Heparin rebound phenomenon--much ado about nothing?

Significant postoperative bleeding following open-heart surgery is often ascribed to the so-called heparin 'rebound' phenomenon and as such is treated with additional empiric doses of protamine sulphate. However, inappropriate protamine administration has been reported to be associated with acute pulmonary hypertension. The efficacy of heparin reversal was investigated in 42 patients undergoing open-heart surgery. The standard heparin bolus of 3 mg/kg body weight (4.1 IU/ml blood) administered before cardiopulmonary bypass was countered at the end of bypass using an empirical equivalent (3 mg/kg) of protamine. This regimen resulted in complete heparin neutralization (measured by the Hepcon HMS [Hemotec Inc., Englewood, CO, USA]) 15 min after protamine administration in all 42 patients, but heparin levels (0.4 IU/ml) were transiently detectable (duration less than 1 h) in six (14%) of the 42 cases 2 h later. Twenty-four hour postoperative bleeding in these patients did not differ significantly from that seen in patients who did not exhibit heparin rebound. Similarly, the thrombelastographic profiles (at 15 min and 2 h post-operation) and coagulation screen (prothrombin time, activated partial thromboplastin time, activated clotting time and platelets) did not differ significantly from those of non-rebound patients. The significance, if any, of the phenomenon of heparin rebound following cardiac surgery remains to be elucidated, and, until such time, conservative administration of protamine in response to 'rebound' is recommended.     

Relationship between biological activity and concentration of a low-molecular-weight heparin (PK 10169) and unfractionated heparin after intravenous and subcutaneous administration

The pharmacokinetics of unfractionated heparin and the low-molecular-weight (LMW) heparin PK 10169 after intravenous and subcutaneous injection were compared by crossover study in 8 healthy volunteers. The heparin concentrations in plasma were measured by a competitive binding assay, and anti-IIa and anti-Xa activities were also assayed. Unfractionated heparin was cleared after intravenous administration with a half-life of 35 min irrespective of assay method. However, the concentration of PK 10169 declined with the longer half-life of 60 min, and its anti-IIa and anti-Xa activities had half-lives of 40 and 275 min, respectively. Some of this anti-Xa activity may be mediated by a compound released by PK 10169 rather than by the LMW heparin itself. The bioavailability of PK 10169 was 3-fold greater than that of unfractionated heparin, due to more effective absorption after subcutaneous administration.     

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).     

Kinetics of intravenously administered heparin in normal humans

Heparin of five commercially available brands was used to study the disappearance of heparin anticoagulant activity in normal humans. The drug was administered intravenously by bolus injection and by continuous infusion. Heparin anticoagulant activity was determined by two assays: a diluted activated partial thromboplastin time (APTT) and an assay based on inactivation of bovine factor Xa, using a clotting system. After a bolus injection, the data fitted neither single exponential nor zero-order clearance. In semilogarithmic plots, heparin anticoagulant activity disappeared according to a slightly convex curve almost always preceded by a rapid initial loss of heparin anticoagulant activity. This disappearance profile was observed with all heparin regardless of the brand or assay system. Heparin anticoagulant activity estimated by the APTT disappeared faster than heparin anticoagulant activity estimated by the anti-Xa activity in the first phase. As expected, higher anticoagulant levels with the anti-Xa assay than with the APTT were also found on continuous infusion in normals as well as in patients treated for deep vein thrombosis or pulmonary embolism. The experimental data suggested a model based on the combination of a saturable and a linear clearance mechanism. These experimental data provide reliable guidelines for adjustment of the dose of heparin in single patients.     

Neutralization of Heparin in Plasma by Platelet Factor 4 and Protamine Sulphate

The neutralization of heparin by protamine sulphate and platelet factor 4 (PF₄) has been studied using kaolin cephalin clotting time (KCCT), thrombin clotting time (TCT) and anti-Xa assays to measure residual heparin levels. Protamine sulphate and purified PF₄ had almost equivalent neutralizing ability on a weight basis regardless of which assay system was used. Plasma samples that were heparinized in vitro could be totally and readily neutralized by both agents in all of the assay systems used. However, when heparinized plasma samples were obtained following intravenous injection of the drug different results for neutralization were obtained depending on the heparin assay used. When residual heparin was measured by anti-Xa assay partial neutralization of heparin was observed even in the presence of a large excess of neutralizing agent. The same in vivo derived heparinized plasma samples had no heparin activity following neutralization if residual heparin was measured by KCCT and TCT assays. Further ion exchange chromatography experiments demonstrated that the heparin-like activity that could not be neutralized in the anti-Xa assay was not adsorbed by the resin ECTEOLA-cellulose, and therefore could not be removed from plasma by this technique. These results suggest, therefore, that part of the plasma anti-Xa activity produced following intravenous injection of heparin differs from the anti-Xa activity obtained following addition of the drug to plasma in vitro .     

In vitro coagulant and amidolytic methods for evaluating the activity of heparin and a low molecular weight derivative (PK 10169)

In summary, the following points have been presented. PK 10169 produced somewhat weaker effects on the coagulant tests in comparison to heparin in various whole blood and citrated plasma assays. In the synthetic substrate assays, PK 10169 produced a pronounced inhibition of various serine proteases in the AT III supplemented system. No significant inhibition was noted in the non-AT III systems. Preliminary data show that PK 10169-AT III complex is capable of producing direct inhibition of the generation of factors Xa and XIIa. In all platelet function tests studied, this agent failed to produce any modulating effects. PK 10169 did not produce an effect on the fibrinolytic system in vitro. However, analysis of blood samples obtained from animals treated in vivo with this agent suggests activation of fibrinolysis. Thus, the mechanism of action must involve certain cellular components or endogenous modulation of the heparin fraction. The newly developed FPA generation test can be modified by various activators or blood systems to mimic closely in vivo physiology. PK 10169 produces a dose response that is more sensitive and more global by this method than the amidolytic anti-Xa or anti-IIa. In contrast to heparin, larger amounts of platelet factor 4 and protamine sulfate are needed to neutralize the anti-Xa and anti-IIa actions of this agent. Additionally, the anti-IIa component is more susceptible to neutralization than the anti-Xa component. Our studies also suggest that PK 10169 is resistant to the action of certain heparin digestive systems, such as heparinase.     

Effects of tranexamic acid and autotransfusion in coronary artery bypass

The aim of this study was to compare the effects of intraoperative autotransfusion and tranexamic acid on postoperative bleeding and the need for allogeneic transfusion. In a prospective randomized study, 200 patients undergoing coronary artery bypass were divided into two groups: 100 patients received 1-2 units of autologous blood after termination of cardiopulmonary bypass; and 100 patients were given tranexamic acid 15 mg x kg(-1) before injection of heparin and again before injection of protamine. Postoperative bleeding was significantly lower in the tranexamic acid group (600 mL) than the autotransfusion group (1,100 mL). The percentage of patients transfused in the autotransfusion and tranexamic acid groups was 70% and 65%, respectively. Patients in the autotransfusion group received significantly more whole blood (2.82 vs 1.93 units). Intensive care and hospital stays were shorter in the tranexamic acid group. There was no hospital mortality and no difference in thrombotic complications between groups. Tranexamic acid was more effective than autotransfusion in reducing postoperative blood loss and allogeneic transfusions after coronary bypass.     

Preliminary study of the use of low-molecular weight heparin to prevent thrombotic risk

This study involved two stages: In vitro study. Three LMWHs, CY 216-CY 222 (Choay) and PK 10169 (Pharmuka), were compared to standard heparin, SH (Choay), and to a high-molecular-weight heparin, HAF (Choay), which particularly revealed a dissociation of anti-Xa (++) and anti-IIa (+) activities for the LMWHs: (table: see text) In vivo study. Healthy volunteers. Subcutaneously injections of PK 10169 or CY 222 according to various protocols (rythm of injections, dosage). For both LMWHs, the dissociation of anti-Xa and anti-IIa activities reoccurs in vivo. A peak of anti-Xa effect was observed 3 to 4 hours after the injections. The duration period of this activity is about 12 hours. The importance of the global platelet tests HT and TEG (modified for the highest doses) should be noted. Study of haemorrhagic tendencies in patients with slight thrombotic risk. From this preliminary study, protocols with CY 216, CY 222 and PK 10169 are proposed for the prevention of thrombotic risk in orthopaedic surgery (injections at 12-hour intervals).     

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.     

不同酸碱度对鱼精蛋白中和肝素的影响

目的:探讨心脏手术后患者不同酸碱度对鱼精蛋白中和肝素的影响。方法:我院2009年10月至2010年5月期间对352例心脏病患者行体外循环手术,麻醉后即测正常活化凝血时间(ACT)值,在打开心包后按2.5 mg/kg从中心静脉给予肝素,在体外循环结束时,鱼精蛋白的剂量从肝素的1.5倍开始在升主动脉根部给药,测量中和后的ACT值,并依此作为追加鱼精蛋白量的参考。按体外循环结束时其酸碱度不同分为3组,观察比较各组肝素与鱼精蛋白总量之比、中和后ACT值,并观测3组术后6 h、12 h及24 h引流量及术后总引流量。结果:在酸性环境下,鱼精蛋白中和肝素剂量及术后引流量高于其他2组,差异有统计学意义(P<0.05)。在非酸性环境下,鱼精蛋白和肝素比平均值为1.63∶1;在pH<7.31或剩余碱(BE)<-6时,鱼精蛋白的用量显著增加,鱼精蛋白和肝素比平均值为2.33∶1。结论:不同酸碱度对鱼精蛋白拮抗肝素有较大影响,酸性环境下鱼精蛋白的中和作用会减弱,鱼精蛋白用量增加,术后引流量也会增多。在体外循环结束时,测得pH<7.31或BE<-6时,建议纠正酸碱平衡的同时适量追加鱼精蛋白。     

Pharmacodynamic effects on blood coagulation of a new low molecular weight heparin (alfa-LMWH) in healthy volunteers and gynecological surgery patients

The pharmacodynamic properties of a new LMWH (alfa-LMWH) were investigated in 8 healthy volunteers after single subcutaneous administrations of 7,500, 15,000 and 30,000 anti-XaU doses at weekly intervals. Anti-Xa and anti-IIa heparin activities were monitored together with aPTT, thrombin time, bleeding time and euglobulin lysis time. No relevant changes in bleeding time or major side-effects were ever recorded. A group of 26 patients submitted to gynaecological surgery were then investigated to determine the dosage schedule for prophylaxis of post-operative deep vein thrombosis. Two subgroups received daily subcutaneous doses of 7,500 and 15,000 anti-XaU alfa-LMWH respectively, beginning 2 h before surgery; the third subgroup received 5,000 IU calcium heparin three times daily over the seven postoperative days. The following tests were peri-operatively monitored: anti-Xa heparin activity, aPTT, PT, fibrinogen, Antithrombin III. No differences in intra-operative bleeding or side-effects were recorded. On the basis of the levels of anti-Xa heparin activity and the negligible effects on aPTT, the dose of 7500 anti-XaU was selected at single daily administration for thromboprophylaxis in gynecological surgery.     

Anticoagulant Activities of High and Low Molecular Weight Heparin Fractions

The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti-Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti-Xa assays, but in APTT assays using synthetic substrate S-2222 gave about 20% lower levels than anti-Xa clotting assays for both heparins. Complete protamine neutralization of the post-injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti-Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three assays in vitro. This non-neutralizable activity probably accounts for the difference between the anti-Xa clotting and synthetic substrate assays. Studies by crossed immunoelectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.     

PGE1治疗鱼精蛋白中和肝素诱发肺动脉高压的疗效观察

本研究的目的观察PGE1对鱼精蛋白中和肝素所致肺动脉高压的治疗作用.7例体外循环心内直视手术患者,心内操作结束主动脉根部注射鱼精蛋白对抗肝素后肺动脉平均压≥30mmHg即行PGE1 0.02～0.04蘥*min-1*Kg-1持续输注,并于注射前后不同时间分别监测动脉收缩压(SABP)、舒张压(DABP)、平均动脉压(MABP)、中心静脉压(CVP)、平均肺动脉压(MPAP)、肺毛细血管嵌压(PCWP),采用热稀释法测定心输出量(CO),并计算心指数(CI)、每搏指数(SI)、左心室和右心室做功指数(LVSWI,RVSWI)、肺循环阻力(PVR)及体循环阻力(SVR).结果:PGE1持续输注后,SABP、DABP、CVP、CO、SI未发生变化.肺动脉压、右室做功指数和肺血管阻力在持续PGE1 0.02～0.04μg*min-1*Kg-1注射后30min降低为注射前水平(P＜0.05).结论:持续静脉输注PGE1 0.02～0.04μg*min-1*Kg-1可以逆转体外循环心内直视手术鱼精蛋白中和肝素所致的肺动脉高压,降低肺血管阻力,对动脉压和心排血量无明显影响.     

Antithrombotic properties of dermatan sulphate in a rat venous thrombosis model

It has been suggested that glycosaminoglycans (GAG) such as heparan sulphate (HS), dermatan sulphate (DS), chondroitin-4-sulphate and chondroitin-6-sulphate contribute to the nonthrombogenic properties of the vascular wall. We have investigated the potential role of DS and HS as antithrombotic agents in an experimental model of stasis-induced venous thrombosis in rats. We utilized a range of doses of both DS and HS (0.25-4 mg/kg BW) to test both their antithrombotic activity and potential bleeding effects. The results were evaluated with reference to an unfractionated heparin (0.5-2 mg/kg BW). We report that the antithrombotic activity of DS is not related to its anticoagulant activity as measured by the activated partial thromboplastin time (APTT), thrombin time (TT) and anti-Xa tests. The dose of DS which was able to inhibit thrombus formation by 70% did not prolong the bleeding time measured using two techniques (template and tail transection); in contrast, with HS a prolongation of both times could clearly be seen. On the other hand, standard unfractionated heparin, at a dose which is equipotent to that of DS in preventing thrombus formation, significantly prolonged the bleeding time. These results suggest that DS may be a useful antithrombotic agent with a lower haemorrhagic effect than heparin, unlike HS which expresses a haemorrhagic risk similar to heparin.     

Low molecular weight heparin fraction PK 10169: a new therapeutic means for anticoagulant therapy?

To test the efficacy and safety of low molecular weight heparin (LMWH) fractions in thromboembolic diseases, 2 pilot studies were performed. The aim of the first was to test the antithrombotic efficacy of the LMWH PK 10169 (Pharmuka Laboratories) in 9 patients (group I) with acute pulmonary embolism documented by pulmonary angiography and scintiscan. The second study was intended to test the safety of the same LMWH fraction in a group (group II) of 30 patients at high risk of bleeding but also requiring antithrombotic therapy. In the 2 groups, the antithrombotic efficacy of PK 10169 was found to be excellent: no recurrence of pulmonary embolism was noted in group I and no thrombotic event occurred in group II. Neither side effects nor major bleeding complications occurred in either group. However, 1 patient in group II had a moderate bleeding in relation to a transient overdosage, and 3 others exhibited minor bleeding which did not necessitate dosage adjustment. In these studies, PK 10169, at the dosage used, proved an effective antithrombotic therapy with few side effects even in a group of patients considered to be at high risk of bleeding.