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 the antithrombotic effects of heparin and Fraxiparin by protamine sulfate.

In general, the in vitro anti Xa activity of low molecular weight heparins is neutralized to a lesser degree than the anti Xa activity of unfractionated heparin. To determine whether these differences occur in vivo, a rabbit stasis thrombosis model and a rat laser-induced thrombosis model were utilized. In the laser model, a similar degree of neutralization of the antithrombotic activity of heparin and Fraxiparin was obtained. However, in the stasis thrombosis model, significant antithrombotic activity of Fraxiparin remained after equigravimetric protamine administration. Ex vivo APTT, thrombin time, Heptest, amidolytic anti Xa and anti IIa assays were performed. A coefficient (r = .806) was obtained for the correlation of Heptest activity to antithrombotic effect in the stasis thrombosis model, while the coefficients obtained for the other tests ranged from .152-.570. However, after neutralization by protamine, the thrombin time exhibited the highest correlation coefficient (r = .685) between ex vivo activity and residual antithrombotic effect. Since Fraxiparin retains antithrombotic activity after protamine administration, clinical benefit may be observed for this low molecular weight heparin as compared to unfractionated heparin after neutralization.     

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.     

A comparative study of precipitation and paracoagulation by protamine sulfate and ethanol gelation tests

A comparative experimental study was undertaken to determine the specificity and sensitivity of 3 methods commonly used for the detection of soluble fibrin monomer complexes and fibrin degradation products (fdp). Non-enzymatic fibrin formation (paracoagulation) induced by low concentrations of protamine sulfate (PS) occurred in the presence of fibrin monomer or early fdp and did not occur in the presence of fibrinogen alone. Ethanol gelation was found to lack this specificity. Though sensitive to fibrin monomer, ethanol gelation also occurred with fibrinogen itself in the presence of a low molecular weight protein isolated from the plasma of certain patients. Ethanol gelation was found to be insensitive to early fdp. The precipitation reaction induced by PS was sensitive to the major portion of total fibrinogen and cannot be used to identify the presence of fibrin monomer or fdp. It is concluded that only PS-induced paracoagulation should be considered specific for thrombin elaboration.     

Intracarotid infusions of protamine sulfate disrupt the blood-brain barrier of rabbits

This study examined the effect of intracarotid infusions of protamine sulfate on the blood-brain barrier of the rabbit. Evans blue, which binds tightly to serum albumin, served as the marker for blood-brain barrier disruption. Brains from 12 of 14 rabbits receiving 30-50 mg of protamine sulfate in an intracarotid infusion over 3 min demonstrated intense Evans blue-albumin staining in the distribution of the infused internal carotid artery. In contrast, brains from 5 animals receiving 0.9% saline and from 5 animals receiving 50 mg of protamine sulfate by the intravenous route showed no evidence of blood-brain barrier disruption. Premixing protamine sulfate with heparin prior to intracarotid administration greatly reduced or abolished Evans blue staining of the brain as did delayed administration of the marker dye. Thus, the interaction of the positively charged protamine molecule with the vascular endothelium of cerebral capillaries transiently alters the permeability of the blood-brain barrier in the rabbit.     

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.     

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.     

Comparison between a modified ethanol gelation test and protamine sulfate test. Experimental studies.

A comparative experimental study has been made to correlate the protamine sulfate test and a modified ethanol gelation test, based on clinical observations of the solubility of a gel formed at 20 degrees C (Godal and Abildgaard procedure) when it was transferred to a bath at 37 degrees C. Two different results were obtained: the gel remained insoluble at 37 degrees C or it became completely soluble, with intermediate degrees of partial solubility. Our studies indicate that this is due to the amount of fibrin monomers formed and the level of fibrinogen: the first are responsible for the insolubility of the gel and the second for its solubility. This furnishes us with useful information for diagnostic purposes. We found the protamine sulfate test more sensitive than the ethanol gelation test, and its sensitivity increased when fibrinogen level decreased. An insoluble gelation test is a sure indication of the presence of fibrin monomers, but a soluble gel calls for the protamine sulfate test to confirm this or the existence of high fibrinogen level.     

Albumin content in brain and CSF after intracarotid infusion of protamine sulfate: a longitudinal study

The endogenous serum albumin content was determined by immunoelectrophoresis in brain and cisternal CSF 1, 24, and 72 h after a transient opening of the blood-brain barrier. Protamine sulfate, 5 mg in 100 microliters 0.9% NaCl, was infused during 30 s into the internal carotid artery via a catheter in the external carotid artery in conscious rats. The albumin content in CSF was 0.08 +/- 0.03 g/liter before protamine infusion and 0.09 +/- 0.02 g/liter in rats infused with saline only. The levels were significantly increased one and 24 h after protamine infusion (0.37 +/- 0.19 and 0.23 +/- 0.09 g/liter, P less than 0.001) but not at 72 h (0.14 +/- 0.05 g/liter). The albumin content in the right (injected) hemisphere decreased with time but was significantly higher than that in the left hemisphere at all times (P less than 0.001 1 and 24 h after protamine; P less than 0.01 at 72 h). There was no correlation between the albumin contents in brain and CSF. Pretreatment with dixyrazine, a phenothiazine derivate, significantly reduced the protamine-induced leakage of endogenous serum albumin into brain and CSF.     

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.     

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.     

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.     

Inhibition of low molecular weight heparin by protamine chloride in vivo

To determine the antagonization of anticoagulant and lipolytic effects of a low molecular weight [LMW] heparin preparation protamine chloride was given intravenously after i.v. injection of LMW or normal heparin. The effects of normal heparin on factor Xa, thrombin, aPTT, lipoprotein [LPL] and hepatic triglyceride lipase [HTGL] activities were neutralized immediately by i.v. protamine. The inhibition of thrombin and aPTT by LMW heparin were also abolished, whereas the effects on LPL and HTGL were counteracted to 80% and on factor Xa only to 40% by i.v. protamine chloride. No rebound of the anticoagulant or lipolytic effect was detected. It is assumed that haemorrhagic complication during therapy can be antagonized by protamine chloride. The incomplete inhibitory effect of protamine chloride on LPL, HTGL and factor Xa activities of LMW heparin indicate that protamine chloride requires more than 14 saccharide units in the heparin molecule for interaction.     

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.     

Non-clottable, protamine sulfate precipitable protein of blood. Studies on its in vitro generation and incorporation into the fibrin clot

Protamine sulfate (PS) was shown to precipitate a plasma protein which is thrombin unclottable, contains only 2.2% of fibrinogen-related antigen, but becomes incorporated into the fibrin clot. Experiments with a PS precipitable protein fraction isolated from plasma showed that as much as 40% of the fibrin clot protein could be composed of the PS precipitable fraction rather than fibrin itself. Incubation at 37° C for 24 hours of blood from normal subjects resulted in a doubling of the volume of PS precipitable material as well as a slight increase in fibrin clot protein. The presence of intact red cells was found to be necessary for the observed in vitro generation phenomenon. Blood from patients with active arterial disease contained significantly higher baseline levels of PS precipitable protein than normal subjects. It is speculated that a portion of fibrin clot protein in certain diseases may represent PS precipitable protein rather than fibrinogen.