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Hemodynamic changes have been documented during protamine infusion into heparinized but not unheparinized pigs and suggest that a protamine-heparin interaction might be responsible. This hypothesis was tested in four groups of pigs by varying the dosage and order of administration of these two drugs: Group I (n = 9) received heparin (3 mg/kg) followed by protamine (3 mg/kg); Group II (n = 9) received protamine (3 mg/kg) followed by heparin (3 mg/kg); Group III (n = 9) received protamine (25 mg/kg) followed by heparin (3 mg/kg); and Group IV (n = 16) received protamine-heparin complex (protamine 3 mg/kg and heparin 3 mg/kg mixed immediately prior to injection). Systemic and pulmonary arterial pressures, systemic and pulmonary vascular resistances, left ventricular end-diastolic pressure, central venous pressure, cardiac output, and heart rate were measured before and at 1.0, 2.5, 5.0, and 15 minutes after protamine, heparin, or protamine-heparin complex infusions. Immediately following protamine infusion, Group I pigs exhibited transiently but significantly increased pulmonary artery pressure, pulmonary vascular resistance, systemic vascular resistance, and central venous pressure and decreased cardiac output with (Group Ib, n = 5) or without (Group Ia, n = 4) systemic hypotension. The fact that no hemodynamic changes occurred in Group II confirms that infusion of clinical doses of protamine produces no hemodynamic changes in unheparinized pigs. Protamine alone in high doses (Group III) produced hemodynamic changes similar to clinical-dose protamine reversal of heparin (Group I). This effect suggests that the presence of heparin in the circulation lowers the threshold for protamine-mediated hemodynamic responses. Infusion of heparin (3 mg/kg) into pigs 15 minutes after treatment with high (25 mg/kg) (Group III) but not clinical (3 mg/kg) (Group II) doses of protamine produced hemodynamic effects similar to clinical-dose protamine reversal of heparin (Group I), suggesting that a protamine-heparin interaction may be responsible. These results also suggest a rapid inactivation in vivo of clinical doses (3 mg/kg) (Group II) of infused protamine. Protamine-heparin complex formed in vitro (Group IV) also produced hemodynamic changes similar to clinical-dose protamine reversal of heparin (Group I), suggesting that formation of this complex in vivo may be the protamine-heparin interaction responsible. Protamine-heparin complex may well be a useful tool in further elucidating the full effects of protamine reversal of heparin.
The use of protamine sulfate in patients has been associated with circulatory collapse and is suspected to directly depress left ventricular function. However, the cellular basis for these changes that occur after protamine administration are unknown. Accordingly, the first objective of this study was to determine the direct effects of protamine on isolated myocyte contractile function. Myocytes were isolated from porcine hearts and contractile function was examined at baseline and then after the administration of protamine in concentrations of 20, 40, or 80 micrograms/ml. These concentrations were chosen because they reflect the serum concentrations of protamine commonly obtained in patients. The presence of protamine resulted in a dose-dependent decline in myocyte contractile function. For example, in the presence of a 20 microgram/ml concentration of protamine myocyte contractile function did not change significantly from baseline values, whereas an 80 microgram/ml protamine concentration caused myocyte percent and velocity of shortening to fall by more than 35% from baseline values. In light of the fact that protamine directly depressed myocyte contractile function, a second objective of this study was to examine potential cellular mechanisms responsible for this effect. Accordingly, in the next series of experiments, the effects of protamine on the myocyte sarcolemmal beta-adrenergic receptor system were examined by measuring myocyte contractile function with the beta-adrenergic agonist isoproterenol (25 nmol/L), as well as with the concomitant addition of protamine and isoproterenol. In the presence of protamine, myocyte beta-adrenergic responsiveness was significantly reduced. For example, in the presence of an 80 microgram/ml dose of protamine, both myocyte percent and velocity of shortening fell by greater than 50% when compared with isoproterenol alone values (p < 0.05). To determine the reversibility of these protamine effects, we performed additional experiments in the presence of heparin. Incubation with heparin before protamine addition prevented the negative effects of protamine on myocyte function. However, the addition of heparin after protamine incubation failed to reverse the negative effects of protamine on myocyte function. In a final set of experiments, the effects of protamine on isolated myocyte electrophysiologic properties were examined using microelectrode techniques at baseline and with either 40 or 80 micrograms/ml doses of protamine. Myocyte resting membrane potential changed from baseline with the addition of a 40 micrograms/ml dose of protamine (-79.2 +/- 0.5 versus -75.2 +/- 0.8 mV (p < 0.05), with no further change at an 80 micrograms/ml dose of protamine (-73.0 +/- 1.3 mV).(ABSTRACT TRUNCATED AT 400 WORDS)
Reactions to intravenous protamine include rash, urticaria, bronchospasm, hypotension, and/or pulmonary artery pressure elevation. We have previously shown that in diabetic patients receiving daily protamine-insulin injections, the presence of anti-protamine IgE or IgG antibodies are significant risk factors for acute, life-threatening reactions when protamine is given intravenously. To study protamine reactions further, we measured serum anti-protamine IgE and IgG antibody levels, in-vitro basophil histamine release and intracutaneous skin testing to protamine serially in an NPH-insulin dependent diabetic who had a severe, protracted anaphylactic reaction to protamine. At the time of his protamine reaction, his serum contained 8.5 ng/ml of anti-protamine IgE and 1.3 micrograms/ml of anti-protamine IgG antibody. One month following the reaction both anti-protamine IgE and IgG increased to 16 ng/ml (twofold rise) and 90.5 micrograms/ml (70-fold rise), respectively. With time, both anti-protamine IgE and IgG antibody declined. Serial intradermal skin tests using protamine sulphate did not discriminate between the protamine reactor and nine normal control subjects who had no prior exposure nor any demonstrable serum IgE antibody to protamine. In-vitro basophil histamine release to protamine sulphate was inconclusive in discriminating between the protamine reactor and normal control subjects. We postulate that protamine may be an incomplete or univalent antigen that must first combine with a tissue macromolecule or possibly heparin to become a complete multivalent antigen capable of eliciting IgE antibody-dependent mediator release.
BACKGROUND: We investigated the role of tumor necrosis factor alpha (TNF-alpha) in protamine-induced cardiotoxicity and the possibility of preventing or decreasing this effect by anti TNF-alpha antibodies and heparin. METHODS: Isolated rat hearts were perfused for 60 min with Krebs-Henseleit solution (KH). The control group was perfused with KH alone, the KH > protamine > KH group was treated from the 20th to the 40th minute with protamine, and the KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF group was treated the same as the KH > protamine > KH group but with anti-TNF-alpha antibodies added throughout perfusion. The KH + heparin > protamine + heparin > KH + heparin group was treated the same as the KH > protamine > KH group but with heparin added to KH throughout perfusion. The KH > protamine > KH + heparin was perfused the same as the KH> protamine > KH group but with heparin added to KH for the last 20 min. Left ventricular (LV) function and coronary flow were measured every 10 min. TNF-alpha was measured in the coronary sinus effluent. Left ventricular TNF messenger RNA was determined in the control and KH > protamine > KH groups at baseline and after the 40-min perfusion. RESULTS: Protamine caused a significant decrease of peak systolic pressure and dP/dt (to 25% of baseline). Significant amounts of TNF-alpha in the effluent in the KH > protamine > KH group (102.3 +/- 15.5 pg/min) and TNF messenger RNA expression in left ventricular samples were detected. TNF-alpha was below detectable concentrations in the control, KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF, and KH + heparin > protamine + heparin > KH + heparin groups. TNF-alpha concentrations correlated with depression of LV peak systolic pressure (r = 0.984; P = 0.01) and first derivate of the increase of LV pressure (r = 0.976; P = 0.001). Heparin improved LV recovery and decreased protamine-induced TNF-alpha release (KH > protamine > KH + heparin group). CONCLUSIONS: Anti-TNF-alpha antibodies and heparin prevent protamine-induced TNF-alpha release and depression of LV function. Heparin improves protamine-induced depression of cardiac function.
To reduce the risks of protamine reactions after cardiopulmonary bypass (CPB), a heparin removal device (HRD) with plasma separation and poly-L-lysine (PLL) affinity adsorption was developed. To compare the efficacy of HRD with that of protamine, blood coagulation variables were evaluated in a swine model of CPB. Female Yorkshire swine were randomly divided into the HRD group (n = 6, weight 79.7 +/- 7.0 kg) and the protamine group (n = 6, weight 79.3 +/- 6.8 kg), and subjected to 60 min of right atrium-to-aortic, hypothermic (28 degrees C) CPB. After weaning from CPB, the right atrium was recannulated with a two-stage, dual lumen cannula in the HRD group. Blood flow was drained from the inferior vena cava, through the plasma separation chamber of the HRD where heparin was bound to PLL, and re-infused into the right atrium. The HRD run time was determined by an established mathematical model of first-order exponential depletion targeted to 90% heparin removal. In the protamine group, protamine was given in a 100 U heparin to 1 mg protamine ratio after CPB in a slow intravenous infusion. Hemodynamics, activated clotting time (ACT), activated partial thromboplastin time (APTT), and heparin concentration were obtained before, every 5 min during, and after the use of the HRD or before and after protamine administration, and 1 and 3 hours after HRD or protamine. Heparin concentration immediately after CPB was 4.90 +/- 0.19 U/ml in the HRD group and 3.94 +/- 0.63 U/ml in the protamine group, respectively (p > 0.05 between groups). The ACT was 994 +/- 7 sec in the HRD group and 768 +/- 55 sec in the protamine group, and APTT was greater than 150 sec in both groups (p > 0.05 between groups). In the HRD group, the HRD run time was determined to be 31.5 +/- 2.4 min for the targeted 90% heparin removal, and the plasma heparin concentration followed first-order depletion kinetics. In the protamine group, the full dose of protamine was administered over 15 min. Immediately after the HRD run or protamine administration, plasma heparin concentration decreased to 0.48 +/- 0.09 U/ml in the HRD group and 0.13 +/- 0.02 U/ml in the protamine group (p < 0.01 between groups); likewise, ACT decreased to 188 +/- 25 sec in the HRD group and 101 +/- 5 in the protamine group (p < 0.01 between groups). The APTT was not significantly different between the groups at any time during the experiment. Plasma heparin concentration and ACT were not significantly different three hours after the HRD run or protamine administration. The authors conclude that the HRD is capable of predictable reversal of systemic heparinization after CPB, and is an alternative to achieve heparin clearance in subjects who may develop adverse reactions to protamine.
UNLABELLED: The administration of protamine to neutralize the circulating heparin is common practice in cardiovascular surgery. The use of this drug is sometimes associated with hemodynamic alterations of varying degree and intensity (systemic hypotension, pulmonary hypertension and even cardiogenic shock). An intrinsic action of protamine has been suggested to be the cause of these vascular reactions. This action is blocked when protamine forms a complex with heparin, although in other cases it appears that the heparin-protamine complex is the factor responsible for these hemodynamic alterations. The aim of this experimental study was to characterize the vasodilatory action of protamine on the systemic circulation, determining whether or not it is dose-dependent; to analyze the role of endothelium; and to evaluate whether this vasodilatory effect is modified by the presence of heparin. MATERIALS AND METHODS: The abdominal aorta was dissected from eight New Zealand rabbits and then sectioned into vascular rings for study in an organ chamber. Mechanical disruption of endothelium was performed on some rings (n = 14). Once submaximal contraction was reached (ClK 80 mM), protamine sulfate with a final concentration in the organ chamber of 80-400 micrograms/ml was added to one of the groups (n = 12). In the second group (n = 12), equal concentrations of protamine were tested in the presence of heparin at a final concentration of 100 U/ml. RESULTS: The mean vasodilatation reached in the group of rings exposed only to protamine was 95.4 +/- 1.5% with respect to the submaximal contraction induced with ClK. In the second study group, the rings were exposed to protamine at equally increasing concentrations (80-400 micrograms/ml) but with the presence of heparin in the organ chamber. The mean vasodilatation in this group was 90 +/- 1.5. No statistically significant differences in vasodilatation were found between this group and the protamine without heparin group. On the other hand, in the endothelium-denuded rings (n = 14) exposed to isolated protamine and to protamine-heparin, no vasodilatory response was observed. CONCLUSION: Our results show that the administration in vitro of protamine induces endothelium-dependent vasodilatation of the systemic circulation. Likewise, this relaxing effect mediated through endothelium is not blocked when protamine forms a complex with heparin in comparable concentrations of both drugs. Based on these preliminary findings, we believe that in high-risk patients the prevention of systemic vasodilatation and cardiovascular collapse produced by protamine should move towards the use of other substances that can neutralize the anticoagulant effect of heparin or towards pre-medication guidelines that prevent these secondary effects in the case of protamine administration.
Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.
Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.
Protamine sulphate is currently used for the reversal of heparin anticoagulation but is known to cause direct myocardial depression. The purpose of this study was to compare the effects of protamine sulphate on the isolated heart with and without cardioplegic ischaemia. Isolated rat hearts (Langendorff preparation) were electrically paced at 300 beats/min and perfused with Krebs-Henseleit solution. Five groups were tested: (1) control: no ischaemia, no protamine; (2) no ischaemia, protamine; (3) no ischaemia, protamine (time-matched control to groups 4 and 5); (4) control: ischaemia, no protamine; and (5) ischaemia, protamine. Protamine sulphate was infused for 15 min at 10 microg/ml. In groups 4 and 5, cardioplegic ischemia was maintained for 30 min at 30 degrees C before protamine exposure. Protamine decreased myocardial performance in a time- and dose-dependent manner. Protamine depressed mean (s.d.) myocardial left ventricular pressure in both non-ischaemic hearts (groups 2 and 3, to 49(4)% and 50(4)% from baseline, respectively) and post ischaemic hearts (group 5, to 28(8%). Mean (s.d.) left ventricular-developed pressure only partially recovered after protamine in post-ischaemic hearts (to 55(13)% of baseline) compared with full recovery of the non-ischaemic group. Protamine depressed coronary flow to 70(5)% and 74(8)% in non-ischaemic hearts (groups 2 and 3, respectively) and to 58(7)% in group 5. Coronary flow recovered completely at the end of the experiments in all protamine-treated groups. In conclusion, isolated rat hearts subjected to cardioplegic ischaemia are more vulnerable to protamine than are non-ischaemic hearts.
The effect of the rate of intravenous infusion of protamine on the acute hemodynamic and pulmonary effects of heparin neutralization was investigated in six adult sheep surgically instrumented for chronic studies. Bovine lung heparin at a dose of 200 IU/kg was injected intravenously over 10 sec, 5 min before the start of protamine administration. On separate experimental days, each sheep received protamine at the same dose of 2 mg/kg, but it was infused over four different time periods: 3 s, 30 s, 300 s, or 30 min. At an additional session, protamine was administered over 3 s without prior heparinization to assess the effect of protamine alone. The sequence of the sessions was randomized and performed blindly. Injecting protamine in unheparinized sheep produced no change in any of the measured variables. In contrast, when protamine was injected over 3 s in heparinized sheep, it induced a transient and significant (P less than 0.001) pulmonary hypertension (from 17.2 +/- 1.5 to 45.6 +/- 2.4 mmHg at 1 min) with an increased pulmonary (five-fold) and systemic (2.5-fold) vascular resistance; a decrease of cardiac output (from 3.85 +/- 0.43 to 1.93 +/- 0.29 l/min) without change in left atrial pressure (from 5.3 +/- 1.3 to 6.0 +/- 1.7 mmHg; P = NS); a significant (P less than 0.001) increase of plasma thromboxane B2 (TxB2) concentrations (from 349 +/- 131 to 974 +/- 218 pg/ml); leukopenia (76 +/- 4% of baseline white blood cell counts); and hypoxemia (PaO2 decreased from 81 +/- 3 to 63 +/- 4 mmHg at 2 min). Administering the same amount of protamine after heparin at a slower infusion rate significantly attenuated and delayed all components of the adverse response to protamine. This attenuation occurred in an infusion rate-dependent fashion, so that when protamine was infused over 30 min, no significant changes in any of the measured variables were noted. The time course of plasma heparin concentrations following protamine indicated that chemical heparin was completely neutralized over the time period of protamine infusion. These results demonstrate that the rate of generation of heparin-protamine complexes (as detected by changes of plasma concentrations of chemical heparin) during iv protamine infusion started 5 min after heparin administration is a factor involved in the generation of sufficient mediators required to initiate a characteristic physiologic response in sheep, including systemic and pulmonary vasoconstriction, TxB2 generation, and leukopenia. Infusing a neutralizing dose of protamine over 30 min avoids these adverse reactions in sheep.
The precise mechanism(s) of inhibitory action of protamine on endothelium-mediated vasorelaxation has not been fully elucidated. In addition, no information is available regarding the effects of a heparin-protamine complex on the endothelium-mediated relaxation. Employing isometric tension recording methods, we studied the effects of heparin, an anionic substance, on the protamine-induced inhibition of acetylcholine (ACh)-induced vasorelaxation in isolated rabbit small mesenteric artery. Protamine (> or = 50 micrograms/ml) inhibited ACh (0.03-10 microM)-induced relaxation under a norepinephrine (10 microM)-stimulated condition (P < 0.05). The ACh relaxation, even 20 min after washout of protamine (150 micrograms/ml), was still significantly inhibited as compared to the control (before protamine) ACh relaxation, and further, it was not significantly different from the ACh relaxation maximally inhibited in the presence of protamine. Preapplication of heparin (700 U/ml) almost abolished the protamine inhibition (50 & 150 micrograms/ml) of the ACh relaxation. However, heparin (700 U/ml), applied on washout of protamine (150 micrograms/ml), had no effect on the prolonged protamine inhibition. In conclusion, a heparin-protamine complex had no direct effect on the endothelium-mediated relaxation, and the inhibitory action of protamine on the endothelium-mediated relaxation might be due to its polycationic property. The prolongation of protamine inhibition and the lack of effects of heparin on the prolonged protamine inhibition may suggest a toxic effect of protamine on the endothelium.