Neutralization of enoxaparine-induced bleeding by protamine sulfate

It has been suggested that protamine sulfate is a poor antidote for the bleeding side-effects of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effects of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin +/- protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effects of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.     

Neutralization of a low molecular weight heparin (LHN-1) and conventional heparin by protamine sulfate in rats

The neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized. In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.     

Comparative antithrombotic and hemorrhagic effects of dermatan sulfate, heparan sulfate and heparin

Dermatan sulfate and heparan sulfate are currently under development as potential antithrombotic drugs. In our studies we have evaluated the relative antithrombotic and bleeding effects of these two agents in comparison to heparin, the commonly used anticoagulant. In a rabbit model of stasis thrombosis, a 500 micrograms/kg IV dose of dermatan or heparan produced 50-60% inhibition of induced in vivo thrombosis. At 750 micrograms/kg, both agents produced greater than 75% inhibition of thrombosis. Ex vivo measurement of plasma samples obtained from these animals demonstrated variable clotting effects at the lower dose and a proportional increase in the clotting activity at the higher dose. No anti-Xa or anti-IIa activity was observed in any sample. In contrast, animals treated with only 100 micrograms/kg heparin showed complete inhibition of induced thrombosis with significant anti-Xa and anti-IIa activities as well as prolongation of the clotting assays (APTT, TT and HeptestR). In the hemorrhagic studies utilizing a rabbit ear blood loss model, a 5.0 mg/kg IV dose of dermatan or heparan produced much less blood loss than heparin. On a gravimetric basis, dermatan and heparan were 10 fold less hemorrhagic than heparin. These results indicate that the relative contribution of plasmatic and cellular sites to the mediation of the antithrombotic action of heparin, dermatan and heparan differ. Although the antithrombotic dosages of dermatan and heparan are higher than heparin, due to the different mechanisms of action of each agent, a better safety index may be provided by dermatan and heparan than heparin.     

Neutralization of dermatan sulfate in vitro and in vivo by protamine sulfate and polybrene

The neutralization of the anticoagulant, anti-thrombin, and bleeding effects of dermatan sulfate (DS), a potential antithrombotic agent, was investigated. Protamine sulfate (PS) and hexadimethrine bromide (Polybrene), which reverse the anticoagulant effect of heparin, also neutralized DS in vitro. In human plasma, polybrene was approximately 3 times more active on a weight basis than PS for neutralizing DS (1.5 micrograms polybrene inhibits 1 microgram DS). Intravenous administration of polybrene to rabbits pretreated with DS in a 1:1 weight ratio immediately neutralized 90% of DS and this effect was stable with time. In contrast, PS in a weight ratio of 6:1 (PS to DS) only neutralized 50% of DS injected. When plasma DS concentrations were maintained by continuous infusion between 3 and 15 micrograms/ml, a bolus of polybrene 0.25 mg/kg induced an immediate drop of about 4 micrograms/ml but initial values of DS were recovered within 20 min. PS was again much less effective than polybrene for neutralizing DS. The bleeding effect of DS and its correction by polybrene was studied by using the rat tail transection model. Very large doses of DS (greater than 10 mg/kg) were required to get a modest prolongation of bleeding time. The injection of equivalent doses of polybrene in animals pretreated by DS induced a strong bleeding effect associated with a drop in platelet and leukocyte counts. Animal models are thus inappropriate for investigating the correction of DS-induced bleeding, because high doses of both DS and neutralizing agents are required in these models. Our results indicate that, provided the doses of neutralizing agents remain below their established levels of toxicity in man, DS could if necessary be neutralized completely by polybrene and partially by PS.     

Neutralization of heparin by cellular blood elements.

These studies were performed to evaluate heparin neutralization with a kinetic assay. Celite-induced clotting was followed using thrombelastography (TEG) on sequential samples of heparinized whole blood and plasma with and without platelets. Platelet-poor plasma and platelet-poor whole blood do not show the capability of neutralizing heparin activity with time in the TEG. When heparinized (1.15 U/ml) platelet plasma systems are incubated at room temperature for one hour, however, appreciable antiheparin activity is noted. The addition of erythrocytes to this same platelet concentration accelerates antiheparin activity, and the equivalent neutralization occurs at 15 minutes. Three different heparin concentrations were tried, and the platelet-plasma antiheparin activity was always slower to reach equilibrium than the matched platelet-erythrocyte-plasma combinations. No added antiheparin activity can be attributed to the presence of erythrocytes since both systems reach the same end points of clottability. Thus, platelets and erythrocytes interact in the neutralization of heparin, and their interaction does not provide more neutralization than a platelet-plasma system, only faster kinetics. This effect may be related to the red cell-platelet mixing in the thrombelastograph that results in the accelerated PF-4 release or other effects. These studies emphasize the importance of cellular blood elements in the neutralization of intravenous heparin in canines. We conclude that evaluation of platelet function, hematocrit, and platelet count in conjunction with a plasma assay (PTT) may provide more effective clinical heparin therapy with less chance of bleeding complications.     

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.     

Study on neutralization of low molecular weight heparin (LHG) by protamine sulfate and its neutralization characteristics.

The neutralizing effects of protamine sulfate (PS) on anticoagulant activities of low molecular weight heparin (LHG) and conventional sodium heparin (Heparin) were investigated. The in vitro anti-factor Xa and APTT-prolonging activities of Heparin were almost completely neutralized by PS, whereas the activities of LHG remained partially intact in the presence of PS. Crossed immunoelectrophoresis of antithrombin III (AT III) and affinity chromatography of LHG- and Heparin-cellulose showed that AT III was substantially less dissociated from its binding to LHG than to Heparin in the presence of PS. As in vitro, the in vivo anticoagulant activities of Heparin administered i.v. to rabbits were almost completely neutralized by PS, while the anti-factor Xa and APTT-prolonging activities of LHG remained partially intact in the presence of PS. The thrombin time-prolonging activity of LHG, however, was completely inhibited by PS. Since the bleeding effect of Heparin or LHG is considered mainly due to its anti-thrombin activity, PS may be used as an agent to neutralize LHG, as in the case of Heparin, when bleeding happens to occur during LHG treatment.     

Interaction of Cls̄ and Cl inactivator in the presence of heparin, dextran sulfate and protamine sulfate

When Cls̄ was mixed with Cl inactivator (ClINA) and the mixture was added with ATEe (acetyl tyrosine ethyl ester), the hydrolysis of ATEe decreased. The addition of heparin or dextran sulfate resulted in more hydrolysis of ATEe by the mixture of Cls̄ and ClINA, thus indicating the inhibition of ClINA activity. When the mixture of Cls̄ and ClINA was added to S-2238 (H-D-Phe-Pip-Arg-pNA), the hydrolysis of S-2238 by the mixture was the same as its hydrolysis by Cls̄ alone, thus ClINA being unable to inhibit Cls̄'s capacity to hydrolyze S-2238. The addition of heparin did not affect the extent of hydrolysis of S-2238 by the mixture of Cls̄ and ClINA. When protamine sulfate was added to Cls̄, Cls̄ activity was inhibited, but the addition of ClINA to the mixture of Cls̄ and protamine sulfate resulted in the same extent of inhibition of Cls̄ as the inhibition of Cls̄ by ClINA in the absence of protamine sulfate. It may be possible that Cls̄ has two sites, one interacting with both ClINA and protamine sulfate, the other being responsible for hydrolysis of S-2238 which is not inhibited by ClINA. As to influences of polycations and anions on hemolytic activities of the complement system, polyanions inhibited both the classical and alternative pathways to the same extent, but polycations primarily inhibited the classical pathway, and the extent of the inhibition of the alternative pathway by polycation was small.     

Interaction of C1s and C1 inactivator in the presence of heparin, dextran sulfate and protamine sulfate

When Cls? was mixed with Cl inactivator (ClINA) and the mixture was added with ATEe (acetyl tyrosine ethyl ester), the hydrolysis of ATEe decreased. The addition of heparin or dextran sulfate resulted in more hydrolysis of ATEe by the mixture of Cls? and ClINA, thus indicating the inhibition of ClINA activity. When the mixture of Cls? and ClINA was added to S-2238 (H-D-Phe-Pip-Arg-pNA), the hydrolysis of S-2238 by the mixture was the same as its hydrolysis by Cls? alone, thus ClINA being unable to inhibit Cls?'s capacity to hydrolyze S-2238. The addition of heparin did not affect the extent of hydrolysis of S-2238 by the mixture of Cls? and ClINA. When protamine sulfate was added to Cls?, Cls? activity was inhibited, but the addition of ClINA to the mixture of Cls? and protamine sulfate resulted in the same extent of inhibition of Cls? as the inhibition of Cls? by ClINA in the absence of protamine sulfate. It may be possible that Cls? has two sites, one interacting with both ClINA and protamine sulfate, the other being responsible for hydrolysis of S-2238 which is not inhibited by ClINA. As to influences of polycations and anions on hemolytic activities of the complement system, polyanions inhibited both the classical and alternative pathways to the same extent, but polycations primarily inhibited the classical pathway, and the extent of the inhibition of the alternative pathway by polycation was small.     

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

Measurement of the heparin neutralizing capacity of protamine.

Several methods of estimating the heparin neutralizing capacity of protamine were investigated for their reliability and practicability. The results from two chemical methods were compared with those from two in vitro biological assays, one of which was the method of the British Pharmacopoeia (1973). An in vivo method using mice was used to assess the accuracy of the in vitro test methods. Three standard heparin preparations were tested against the W.H.O. 1st International Reference Preparation for Protamine. Two of the heparin preparations were of mucosal origin, one of which was the 3rd International Standard, and the third heparin preparation was of lung origin (the 2nd International Standard for Heparin). The mean neutralization values (all methods) of heparin by protamine for a House Reference Preparation and the 3rd and 2nd International Standards for Heparin were 95.8, 109.8 and 89.9 units per mg of Protamine, respectively. All methods read the House Reference Preparation to have a lower value than the 3rd International Standard, which had a higher value than the 2nd International Standard for Heparin. There was a constant relationship between the results of any one method and those of another. The chemical and in vitro biological methods gave results of comparable precision although the latter required a greater degree of technical skill and time to perform.     

Effects of aprotinin on heparin activities and heparin neutralization with protamine.

In order to investigate the new situation in which aprotinin is proposed as a novel approach to reducing post operative bleeding, specially in cardiopulmonary bypass (CPB) surgery during which heparin and protamine are commonly used, preliminary in vitro and in vivo studies have been performed. Aprotinin increases the anticoagulant heparin effects in vitro, and the hemorrhage time in vivo. But in addition to protamine, there are no statistically significant differences with heparin-protamine situation, indicating aprotinin does not disturb the neutralizing activities of protamine on heparin.     

Neutralization of heparin by histone and its subfractions

The neutralization of heparin by histone and its subfractions has been systematically studied by measuring the effect of heparin on the esterolytic and proteolytic activity of thrombin. These results were compared with protamine sulfate, a most commonly used heparin-neutralizing agent. This study reveals that potencies of different fractions of histone are not similar. The antiheparin potency is in the order: lysine-rich histone greater than crude histone greater than arginine-rich histone. Histone binds strongly to heparin - Sepharose gel. The ability of histone to bind heparin can be utilized to fractionate heparin. By affinity chromatography on histone - Sepharose gel commercial heparin has been fractionated into components having a wide range of anticoagulant activities. The highest activity fraction, eluted around 1.0 NaCl, has 66% higher anticoagulant activity than the commercial heparin used.     

Neutralization of heparin by prothrombin activation products

The neutralization of heparin by active site blocked meizothrombin and thrombin, prothrombin fragment 1.2, fragment 1 and fragment 2 was probed by the heparin-dependent factor Xa inactivation by antithrombin III (AT III). Meizothrombin had no effect on the inactivation of factor Xa, whereas thrombin had an inhibitory effect (IC50 = 700 nM). After factor Xa catalyzed cleavage of meizothrombin, the resulting products, prothrombin fragment 1.2 plus thrombin, did not show any heparin neutralizing properties. However, after isolation of the reaction products, both thrombin and prothrombin fragment 1.2 exhibited heparin neutralizing properties in the factor Xa inactivation reaction. The IC50-values were 700 nM and 100 nM, respectively. Prothrombin fragment 1, when present at 125 nM, caused a 50% reduction of the heparin-dependent rate of inactivation of factor Xa and prothrombin fragment 2 had no effect at all. From this we conclude that, in addition to the thrombin part of the prothrombin molecule, the fragment 1 region also exhibits a rather high affinity for heparin.     

At high heparin concentrations, protamine concentrations which reverse heparin anticoagulant effects are insufficient to reverse heparin anti-platelet effects

Combined effects of heparin and protamine on plasma clot structure and platelet function were studied. Anticoagulant effects were monitored as changes in aPTT. Clot structure was defined in terms of fibrin fiber mass/length ratio (μ) and clot elastic modulus (EM). Platelet function was studied utilizing platelet aggregation and platelet force development (PFD) measurements. Heparin (1 U/ml) prolonged the aPTT from 30 to >300 seconds, reduced PFD from 5,100 to 0 dynes, decreased μ (in batroxobin-induced gels) from 1.36 to 1.08 × 10¹³ daltons/cm and decreased clot EM from 9,600 to 2000 dynes/cm². Varying amounts of protamine reversed these effects: 16 μg/ml normalized the aPTT, 20 μg/ml normalized PFD, 32 μg/ml corrected μ, and 20 μg/ml returned EM to baseline. At high heparin concentrations (4 U/ml), protamine concentrations which corrected anticoagulant effects were inadequate to reverse antiplatelet effects. A protamine concentration of 40 μg/ml normalized the aPTT and μ, but 140 μg/ml of protamine was required to reverse heparin suppression of force development and clot elastic modulus. Excess protamine inhibited clotting and platelet function. In plasma containing 1 u heparin/ml, 140 μg protamine/ml reduced PFD by 83%, prolonged the aPTT by 63%, and reduced clot EM by 75%. In heparin free plasma, >75 μg protamine/ml prolonged the aPTT. Thus, platelet function and clot structure are sensitive to protamine during heparin neutralization, and anti-platelet effects of heparin may persist when the aPTT is completely corrected. Excess protamine inhibits platelet function and compromises clot structure.     

Standard heparin enhances the antithrombotic activity of dermatan sulfate in the rabbit but CY 216 does not

Standard heparin (SH) and dermatan sulfate (DS) two glycosaminoglycans with different pharmacological targets are effective antithrombotic agents in the rabbit. We have investigated the antithrombotic activity of the association DS plus SH. It was found that doses as low as 25 micrograms/kg for DS and 10 micrograms/kg for SH were ineffective when injected separately but generated a high and significant antithrombotic activity when injected together. These results were confirmed when higher doses of each compound were delivered in association. Further experiments were performed to determine if the enhancement of the antithrombotic activity of DS by HS resulted from its anti-factor IIa or anti-factor Xa activity or from its moiety without affinity to AT III. A low molecular weight heparin (CY 216) with an anti-factor Xa/anti-factor IIa ratio of 5, the synthetic pentasaccharide bearing the minimum binding sequence to antithrombin III, and a low affinity fraction of SH to AT III did not increase the antithrombotic activity of DS; in contrast a high affinity fraction of SH to AT III had the same effect than SH. We conclude that the enhancement of the antithrombotic activity of DS by SH mainly results from its anti-factor IIa activity.     

Neutralisation of heparan sulphate and low molecular weight heparin by protamine

The neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.     

A comparison of the antithrombotic and haemorrhagic effects of a low molecular weight heparin (LHN-1) and conventional heparin

The antithrombotic effects after intravenous administration of a low molecular weight heparin (LHN-1) and conventional heparin were compared in a rabbit model of experimental thrombosis, where thrombus formation was induced by a combination of endothelial damage and stasis. Both compounds were able to prevent thrombosis completely. However, LHN-1 was significantly less potent than conventional heparin, the ratio between doses with the same antithrombotic effect being 2.4:1 on a weight basis. Bleeding times after administration of LHN-1 and conventional heparin were determined by tail transsection in anaesthetized rats and by template bleeding in the ear of conscious pigs. Given intravenously at a dose ratio of 2.4:1 (w/w), LHN-1 affected APTT less than conventional heparin, whereas the effects on haemostasis were not significantly different. In conclusion, it was found that after intravenous administration LHN-1 prevented experimental thrombosis as effectively as conventional heparin. However, the correlation between antithrombotic and haemorrhagic effects of LHN-1 was the same as that of conventional heparin. The corresponding relation in man remains to be determined.