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.     

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.     

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.     

Interrelationship of protamine and platelet factor 4 in the neutralization of heparin

To determine the interaction of platelet factor 4 (PF4) and protamine sulfate in the neutralization of heparin in plasma in vitro studies were carried out using a tritium-labeled heparin and a PF4 tagged with 14C. Plasmas treated with various combinations of PF4, protamine and heparin were chromatographed on Sephadex G200 and the fractions were tested for both radioactivity and antithrombin activity. PF4 was comparable to protamine in its ability to neutralize heparin, but the complexes formed with heparin were different. In contrast to protamine, when heparinized plasma was treated with an excess of PF4, no large PF4-heparin complexes were formed and none of the PF4-heparin complexes which did form were able to activate antithrombin III (ATIII). Also, incubation of PF4-neutralized, heparinized plasma at 37 degrees C did not result in liberation of heparin and prolongation of the thrombin clotting time as was found with protamine-neutralized plasma. The action of protamine and PF4 is complimentary. When half the neutralizing dose of each was added together to heparinized plasma, no immediate antithrombin activity remained. When a neutralizing dose of protamine was added to PF4-neutralized, heparinized plasma, the protamine displaced the PF4 from its complexes with heparin. The large protamine-heparin complexes which formed also contained PF4 but could not activate fresh ATIII as has been demonstrated with protamine-heparin complexes without PF4. On incubation of the protamine-PF4-neutralized, heparinized plasmas for 5 hours at 37 degrees C, the large complexes were broken down but no active heparin appeared. The results of these experiments may have some bearing on the amount of protamine needed for the neutralization of heparin following extracorporeal bypass procedures, when large amounts of PF4 may have been released from activated or disrupted platelets.     

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.     

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.     

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.     

The effect of lysed platelets on neutralization of heparin in vitro with protamine as measured by the activated coagulation time (ACT)

The effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/l heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% +/- 0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to anti-heparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.     

Heparin-associated thrombocytopenia: the antibody is not heparin specific.

In this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo release of anti-Xa clotting activity by a heparin analogue

The parenteral injection of a semi-synthetic heparin analogue (SSHA) releases anti-Xa clotting activity, lipoprotein lipase activity and PF₄ antigen. The increased anti-Xa activity is not neutralized by PF₄ or protamine sulphate. A second injection of the drug after 90 minutes, or an increase in dose, does not increase the level of induced anti-Xa clotting activity. Possible mechanisms of action include the release by SSHA of endogenous glycosaminoglycans with anti-Xa activity, and interference by released lipoprotein lipase of a modulator of anti-Xa activity. It is concluded that a drug with weak anticoagulant activity in vitro may nevertheless have significant antithrombotic potential.     

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.     

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.     

In vivo butyrylcholinesterase activity is not increased in Alzheimer's disease synapses

OBJECTIVE: We tested the premise that cholinesterase inhibitor therapy should target butyrylcholinesterase (BuChE) in Alzheimer's disease (AD), not acetylcholinesterase (AChE) alone, because both enzymes hydrolyze acetylcholine, and BuChE is increased in AD cerebral cortex. METHODS: To examine this issue in vivo, we quantified human cerebral cortical BuChE activity using tracer kinetic estimates (k(3)) of 1-[(11)C]methyl-4-piperidinyl n-butyrate ([(11)C]BMP) hydrolysis determined by positron emission tomography. Validation of the putative positron emission tomography method included regional distribution, positive correlation with age, and attenuation by the nonselective cholinesterase inhibitor physostigmine, but no attenuation by the AChE-selective inhibitor donepezil. Positron emission tomography scans in AD patients (n = 15) and control subjects (n = 12) measured both BuChE (using [(11)C]BMP) and AChE activity (using N-[(11)C] methylpiperidin-4-yl propionate, an established method). RESULTS: As expected, AChE activity in AD cerebral cortex was decreased to 75 +/- 13% of normal (p = 0.00001). Contrary to prediction, accompanying BuChE activity also was decreased to 82 +/- 14% of normal (p = 0.001). INTERPRETATION: Failure to observe increased [(11)C]BMP hydrolysis in vivo makes it less likely that incremental BuChE contributes importantly to acetylcholine hydrolysis in AD. The findings do not support the premise that inhibitor therapy should target BuChE so as to prevent increased levels of BuChE from hydrolyzing acetylcholine in AD cerebral cortex.     

Increased fibrinolytic activity after surgery induced by low dose heparin

Forty women undergoing surgery under general anaesthesia for hyperplasia mammae were randomized to treatment with low dose heparin (5000 IU twice daily) or not. Preoperatively, and repeated on the 3. postoperative day, assays of euglobulin clot lysis time (ELT) after venous stasis, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were performed. Compared to the control group heparin was found to give a significant rise in t-PA antigen before (24.0 vs. 11.2 ng/ml, p = 0.02), and especially after venous stasis (104.8 vs. 47.3 ng/ml, P = 0.007). t-PA activity was also significantly more increased after venous stasis in the heparin group than among the controls (4.2 vs. 1.4 U/ml, p = 0.04). This was also reflected in the ELT after venous stasis which was significantly shorter in the heparin group (p = 0.01). No differences in PAI were found between the groups. The present results point to a heparin-induced increase of t-PA synthesis in the endothelium, also giving rise to an increased level of circulating t-PA as measured immunologically. This effect of small dose heparin may play an important role in the prophylaxis against thrombo-emboli, in addition to the anticoagulant effect.     

In vivo studies on the inhibition of coagulation by fractionated heparin and by a heparin analogue. II. Effects of a heparin analogue

SSHA, a semi-synthetic heparin analogue, and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six healthy volunteers over a period of three days in a cross-over trial. Before injection and 2, 4, 6, 8 hrs afterwards, the heparin-like activity was measured with the APTT, the anti-Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induced proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). Repeated injections of SSHA twice daily led to increasing effects on two tests for heparin-like activity, whereas two other tests remained unchanged. Both drugs were tolerated equally well, side effects were not detected. Clinical studies are required to demonstrate whether SSHA is similar or superior to low-dose heparin for use in thrombosis prophylaxis.     

Changes in blood viscosity by heparin and argatroban.

Anticoagulants are effective for preventing both venous and arterial thrombosis. Although antithrombotic agents have been reported to reduce thrombin formation, to our knowledge, the relation between blood viscosity (BV) and antithrombotic agents has not been examined. We examined the effects on whole BV of various dosages of the antithrombotic agents-heparin sodium and argatroban. Thirty microliters of either drug was added to 3-ml samples of blood obtained from healthy male volunteers. Whole BV was then immediately examined with an oscillation-type viscometer. When either agent was added, BV decreased and coagulation time increased dose dependently. BV was 4.5+/-0.3 mPa.s in untreated blood but decreased in a dose-dependent manner to a minimum of 2.5+/-0.3 mPa.s with heparin sodium and decreased dose dependently in a sigmoid manner with argatroban. Because thrombin generation is inhibited in all antithrombotic therapies, this inhibition might be reflected by changes in BV. Our results suggest that BV in accordance with blood coagulability are indexes of thrombotic tendency and that decreasing BV prevents life-threatening thromboembolic conditions.     

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.     

Appearance of heparin antithrombin-active chains in vivo after injection of commercial heparin and in anaphylaxis

Recently it has been demonstrated that only one-third of commercial heparin binds with antithrombin III to enhance antithrombin activity, and that heparin is rapidly taken up by the RES. Commercial heparin was given to rabbits, dogs and men by subcutaneous and intravenous injection. Plasma samples were deproteinized with phenol, urine samples precipitated with benzidine, and the separated heparin measured for both antithrombin and metachromatic (dye-binding) activity compared to a reference heparin. Urinary excretion as heparin and uroheparin accounted for approximately 5% of the injected heparin. The extraction of heparin added to blood $\text{in}$$\text{vitro}$ gave identical values in metachromatic and antithrombin assays, as did heparin from blood samples immediately after intravenous injection. However, later blood samples after injection gave much higher values in the test for antithrombic activity than in the metachromatic test. Subcutaneous injection of heparin subjected to acid inactivation in vitro, reducing its antithrombin activity, likewise resulted in heparin appearing $\text{in}$$\text{vivo}$ with higher antithrombin activity. Endogenous heparin released to the circulation in canine anaphylaxis also gave much higher values in anti-thrombin assays. It is concluded that the difference in values is due to the conversion of antithrombin-inactive heparin chains to antithrombin-active heparin chains.     

Ex vivo release of eicosanoids after aneurysmal subarachnoid hemorrhage: a preliminary experience in humans.

Authors addressed the question whether the byosynthesis and the release of specific eicosanoids may occur in human brain cortex, and if the qualitative pattern of arachidonic acid metabolism is similar to that observed in experimental SAH. Human brain samples from 18 patients operated on for anterior communicating artery aneurysm (5 unruptured aneurysms considered as control cases, 7 patients operated on between Days 1 and 4 after SAH and 6 patients operated on between Days 10 and 14) were studied for the ex vivo release of 4 selected eicosanoids (Prostaglandin D2, E2, 6-keto-PGF1a and Leukotriene C4). Levels of arachidonate metabolites were determined by radioimmunoassay technique. PGD2 release is significantly lower in cases operated on delayed phase if compared to both control cases (p less than 0.05) and patients operated on in the acute phase, while there is no significant difference between the release of PGD2 in control cases and patients operated on in the acute phase. Release of 6-keto-PGF1a is significantly higher in patients operated on in a delayed phase (p less than 0.03 vs patients operated on in the acute phase and p less than 0.05 versus control cases). The release of LTC4 is significantly enhanced (p less than 0.05) in cases operated on in the acute phase if compared with unruptured aneurysms. The release of PGE2 is significantly enhanced in patients operated on in the acute phase (p less than 0.05) if compared to patients with unruptured aneurysm.(ABSTRACT TRUNCATED AT 250 WORDS)