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Intermediate and long-acting insulin preparations without protamine sulphate are complement activators in vitro.

Recently, we have documented an abnormal in vivo complement metabolism in Type 1 diabetic children treated with monocomponent porcine insulin Monotard MC and its correction after switch-over to human insulin Protaphane HM. This prompted us to investigate the ability of different kinds of insulin preparations to induce complement activation in vitro. Freshly collected serum samples from healthy blood donors were incubated with commercial rapid and intermediate or long-acting (by protamine sulphate (PS) or zinc) insulin preparations for 2 hours at 37 degrees C. The C3d content of the supernatants was measured by turbidimetry as a marker of C3 complement fraction consumption. Only long-acting preparations of insulins without protamine sulphate were associated with highly significant increased levels of C3d, whatever the source of insulin, animal or human. Moreover, addition of exogenous protamine sulphate was able to inhibit the C3 conversion. This effect was dose-dependent and peaked at the concentration of commercial NPH insulin preparations. The mechanism by which protamine sulphate inhibits complement activation in vitro could be related to its ability to interfere with the physical nature of the solid surfaces presented by the insulin crystals. Indeed, insulin crystals were rapidly cleared (< 5 min) in the incubated serum when small doses of protamine sulphate were added. The complement activating capacity of long-acting insulin without protamine was dose dependent, equivalent to the known complement activator Zymosan, and abolished in the presence of EDTA. In conclusion, the present study has documented the ability of some protracted insulin preparations to activate the complement system in vitro if they are devoided of protamine sulphate. On the other hand, short-acting and NPH insulins are not complement activators.

Animals↗

PubMed2003

Hand-held personal digital assistant program for the HEMOCHRON RxDx heparin and protamine dosing system.

The use of in vitro dosing assays for heparin and protamine during cardiac surgery has significantly improved overall postoperative patient outcome. The HEMOCHRON RxDx system (International Technidyne Corp, Edison, NJ) is widely used for anticoagulation management. Based on a series of consecutive in vitro tests, the RxDx system is used to quantify the patient's heparin requirement (heparin response test, HRT), measure the activated clotting time (ACT), calculate the blood heparin concentration and the required protamine dose (protamine response test, PRT), as well as determine the efficacy of heparin reversal (protamine dose assay, PDA-O). A hand-held personal digital assistant (PDA) program has been developed that performs the RxDx calculations used for anticoagulation management during cardiac surgery. The Palm m505 hand-held device (Palm, Inc., Santa Clara, CA) is used in concert with any standard Hemochron blood coagulation system. The Palm m505 device has been programmed to perform all the calculations required for the RxDx test system. Patient's body weight, height, and gender are entered into the program using the onscreen keypad and the template provided in the Hemochron program. The calculator automatically provides the patient's blood volume and the recommended heparin dose upon entering the baseline ACT and HRT values and a target ACT. At the end of the case, the optimal protamine dose is determined, and the total heparin level is calculated and displayed upon entry of ACT and PRT clotting times. Following protamine administration, the program calculates any additional protamine required to neutralize residual heparin using the data from a PDA-O test. The RxDx hand-held PDA is accurate, quick, simple, and easy to use, patient data are saved and can be retrieved. The inclusion of this rapid computing technology into the Hemochron RxDx system serves to expand the applications of the Hemochron RxDx system during cardiac interventions.

Alabama↗

PubMed1975

Protamine inhibition of the oxidative phosphorylation in intact, cytochrome c-depleted and restored mitochondria.

On the basis of polarographic data it is shown that protamine has a biphasic effect on the respiration of intact mitochondria. At lower protamine concentrations respiration is stimulated and this combined with a decrease of the respiratory control index; at higher ones respiration is inhibited and respiratory control is lost. In cytochrome c-depleted and restored mitochondria protamine effect on oxidative phosphorylation is only inhibitory. Increasing cytochrome c concentrations restore respiration in protamine-treated cytochrome c depleted mitochondria but not the respiratory control. Binding of cytochrome c to mitochondria is studied by determining from Scatchard plots the number of high affinity binding sites (n) and their stability constants (K). In absence of protamine in intact mitochondria n = 2.7 and K = 4.67-10(6) M-1; in cotochrome c depleted mitochondria n = 4.7 and K = 5.16-10(6) M-1. In both types of mitochondria protamine decreases significantly n as well as K. These data show that protamine may affect oxidative phosphorylation by causing desorption of cytochrome c from the inner mitochondrial membrane.

Adenosine Diphosphate↗

PubMed1991

[Complications caused by protamine. 1: Pharmacology and pathophysiology].

Protamine is a strongly alkaline polypeptide with a molecular weight of about 4500. Protamine solutions contain paraben compounds as antimicrobial agents. Rapid neutralization of heparin by protamine may cause an anaphylactoid reaction characterized by a non-immunogenic histamine release and by unknown mediators mechanisms. This response is associated with systemic peripheral vasodilation resulting in slight to moderate hypotension. Weak negative inotropic effects by mechanisms different from the reduction of ionized calcium concentrations may also contribute to systemic hypotension. Apart from these mostly slight reactions, severe reactions may occur with life-threatening systemic hypotension, bronchospasm and, in rare cases, death. They are caused by anaphylactic/anaphylactoid reactions resulting in catastrophic pulmonary vasoconstriction which induces right and eventually global ventricular failure. Sensitization to protamine (anaphylactic) and anaphylactoid reactions are the underlying mechanisms. The majority of anaphylactic/anaphylactoid reactions are associated with complement activation and the release of anaphylatoxins C3a and C5a. These activate the cyclo-oxygenase pathway of the arachidonic acid metabolism in yet unidentified cells, probably within the lung. As a result, thromboxane and prostaglandins are released. Thromboxane is the pivotal mediator responsible for the pulmonary vasoconstriction and, presumably, also for the bronchospasm during protamine reactions. The pronounced activation of polymorphonuclear leukocytes and the decrease in platelet counts may reflect a mere epiphenomenon. The degree of right ventricular afterload increase at which systemic hypotension requiring immediate therapy would occur depends mainly on the contractile state of the heart. Potential risk patients for severe protamine reactions are depot insulin-dependent diabetics and patients with prior exposure to protamine.

Anaphylaxis↗

PubMed1990

[Protamine precursors in human spermatozoa].

Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.

Amino Acid Sequence↗

PubMed1986

Prospective evaluation of risk of protamine reactions in patients with NPH insulin-dependent diabetes.

Patients with NPH (neutral protamine Hagedorn) insulin-dependent diabetes may have an increased risk for protamine reactions because of prior sensitization. During one year, we prospectively evaluated 50 at-risk cardiac surgery patients for clinical reactions and determined in vitro histamine release when protamine was added to a preoperative blood sample. We speculated that in vitro histamine release would predict a reaction to protamine given clinically for neutralization of heparin. Twenty-five patients randomly received prophylactic corticosteroid and/or antihistamine pretreatment for allergic reactions. The incidence of clinical reactions to protamine was 1/50 (2%) in NPH insulin-dependent diabetic patients (1/25 in pretreated patients vs 0/25 in patients not receiving pretreatment). One pretreated NPH diabetic patient released histamine in vitro but did not demonstrate clinical signs of a reaction following protamine administration. One other NPH diabetic patient pretreated with corticosteroids developed severe pulmonary hypertension despite the absence of in vitro histamine release. Therefore, in vitro histamine release does not predict protamine reactions.

Blood Pressure↗

PubMed1989

Anaphylactoid reactions to protamine: an often lethal complication in insulin-dependent diabetic patients undergoing vascular surgery.

Protamine is used routinely at our institution during arterial surgery to reverse the anticoagulant effect of heparin. Adverse fatal reactions to protamine are generally believed to be rare. However, major anaphylactoid reactions occurred in 11 of the last 1150 patients receiving this drug at our institution. Nine of these reactions occurred in 325 insulin-dependent diabetic patients (incidence, 3%), whereas only two occurred in the 825 patients not receiving insulin (incidence, 0.2%) (p less than 0.001). Ten of these reactions occurred within 10 minutes of protamine administration (15 to 35 mg), whereas one reaction occurred immediately after administration of a 5 mg test dose of protamine. Systolic blood pressure fell below 60 mm Hg in all of the 11 patients, and three patients had to be resuscitated with closed-chest massage. Initial treatment with epinephrine and steroids was successful in seven cases. Four patients required further resuscitative measures, including closed-chest massage. However, one of the patients died as a result of ventricular fibrillation resistant to treatment. Ten of the 11 patients, including the patient who died, had significant preexisting cardiac disease; six of the surviving 10 patients (60%) had perioperative myocardial infarctions and three died. Thus the total mortality rate was 36% (4/11). These data support the implication that neutral protamine Hagedorn (NPH) insulin produces an adverse reaction through immunologic presensitization of the patient. These data also show that, in the older vascular surgery population with a high incidence of significant cardiac disease, protamine reactions can be potentially lethal. Thus routine use of protamine should be avoided in diabetic patients receiving insulin.

Aged↗

PubMed1989

Decreased oxygen consumption as a toxic manifestation of protamine sulfate reversal of heparin anticoagulation.

Protamine sulfate has been observed to interfere with the control of isolated mitochondrial respiration in vitro. This study was designed to determine if oxygen consumption changes in intact animals occur in vivo during protamine administration. Oxygen consumption was assessed in seven dogs anticoagulated with heparin (150 IU/kg) and reversed 30 minutes later with protamine sulfate (1.5 mg/kg). Oxygen saturations measured included arterial (SaO2 arterial), mixed venous (SvO2 systemic), jugular (SvO2 jugular), portal (SvO2 portal), and coronary (SvO2 coronary). Cardiac output (CO) and carotid artery flow determinations allowed calculation of systemic oxygen consumption (VO2 systemic) and cerebral oxygen consumption VO2 cerebral. Hemodynamic measurements included arterial blood pressure (BP), pulmonary artery systolic and diastolic pressures (PAS, PAD), and heart rate (HR). Protamine sulfate administration resulted in hypotension (delta BP -64 mm Hg), pulmonary hypertension (delta PAS + 13 mm Hg, delta PAD + 11 mm Hg), and bradycardia (delta HR -30). Shortly after protamine administration, CO fell 54% and carotid artery flow fell more than 50%, yet declines in SvO2 systemic and SvO2 jugular were not observed. In fact these parameters increased 3% and 2%, respectively. VO2 systemic fell 55% and VO2 cerebral fell 57%. Similarly, SvO2 portal and SvO2 coronary increased 6% and 9%, respectively. Significant correlations existed between changes in VO2 systemic and BP (r = 0.05, p less than 0.001), HR (r = 0.3, p less than 0.01, PAD (r = -0.3, p less than 0.05, and CO (r = 0.8, p less than 0.001). Impaired oxygen utilization was most evident during the first 5 minutes after protamine administration. This investigation, for the first time, establishes that protamine sulfate decreases in vivo oxygen consumption, a finding that may account for certain of the drug's adverse side effects.

Animals↗

PubMed1989

Thromboxane mediation of pulmonary hemodynamic responses after neutralization of heparin by protamine in pigs.

Protamine neutralization of heparin is often associated with severe hemodynamic side-effects, including pulmonary hypertension and systemic hypotension. Because prostanoids may be involved, the authors studied the role of arachidonic acid metabolites, especially thromboxane A2, in this process. During anesthesia with enflurane and fentanyl, four groups of pigs were studied: Group 1 (n = 10) received heparin (250 IU/kg), followed by protamine (100 mg) after 15 minutes to neutralize the heparin. The same protocol was used in group 2 (n = 11), except that the thromboxane A2 receptor antagonist BM 13.177 (10 mg/kg) was infused 5 minutes before the protamine. The protocol for group 1 was also used for group 3 (n = 7) except that these animals were pretreated with indomethacin (10 mg/kg). Animals in group 4 (n = 10) were given protamine only (100 mg). Pulmonary artery pressure and pulmonary vascular resistance increased significantly in group 1 after protamine neutralization of heparin. This was accompanied by significant increases in plasma concentrations of the cyclooxygenase products thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha. Cyclooxygenase products increased to comparable degrees in group 2, but without hemodynamic effects. Leukocyte counts decreased comparably in both groups. Hemodynamic reactions, as well as changes in plasma prostanoid levels were absent in group 3, and group 4, but leukocyte counts were less affected in animals that received protamine alone. The results indicate that the hemodynamic side-effects of protamine are mediated by prostanoids and that thromboxane A2 release is the pivotal step, because side effects were effectively prevented by pretreatment with a thromboxane receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effect of RBCs on the activation of human complement by heparin-protamine complexes.

Complement activation on red cells by heparin-protamine complexes was studied by using whole human serum. C3 bound to red cells was measured by radiolabeled monoclonal antibody to C3, and fluid-phase C5a was determined by radioimmunoassay. Heparin and protamine in clinically relevant concentrations caused the binding of C3 to red cell membranes, and the measurement of C3 binding provided a sensitive indicator of complement activation produced by these complexes. Complement activation by these reagents occurred at concentration ratios of protamine and heparin at which protamine neutralized the anticoagulant effect of heparin. Heparin-protamine complexes appeared to bind to red cells and produce complement activation by the classic pathway. C5a generation with heparin-protamine complexes in serum was greatly enhanced in the presence of red cells and increased with increasing red cell concentration. This enhancement of complement activation in the presence of red cells was also seen as measured by depletion of available C3 hemolytic complement units in the fluid phase. Thus red cells seem to play an important role in activation of complement by heparin-protamine complexes.

Complement Activation↗

PubMed1987

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins.

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.

Acetylation↗

PubMed1985

Protamine-induced thrombocytopenia and leukopenia.

Protamine has been reported to cause thrombocytopenia and granulocytopenia. In this article, we report studies examining the relative contribution of protamine and heparin in the pathogenesis of this phenomenon, the dose-effect relationship, and the possible mechanism of cell loss. Protamine alone infused into experimental animals causes mild, transient granulocytopenia and thrombocytopenia. The sequential administration of heparin and protamine results in a more severe cytopenia lasting 30 to over 60 min. Organ scanning with 111In-labeled platelets shows a striking though transient accumulation of radioactivity in the lungs following heparin-protamine infusion. Platelet survival, however, is not shortened. Incubation of 125I-labeled protamine with blood cells in the presence of heparin results in tight binding of the drug to platelets and granulocytes. These observations suggest that protamine and heparin form a complex that binds to blood cells. The sequestration of coated cells in the lungs results in transient granulocytopenia and thrombocytopenia.

Animals↗

PubMed1985

[ELISA detection of protamine antibodies].

Protamine is frequently used as an adjuvant in insulin preparations. As alien protein protamine is immunogenic. We developed an ELISA for detection of protamine antibodies. As a strongly basic molecule protamine shows remarkable reactivity. Resulting methodical difficulties with regard to the assay are discussed. The course of antibody titres to protamine after immunization is shown in rabbits, goats, sheep and guinea pigs with positive results in all species. In humans protamine antibodies were detectable in 4% of 150 patients treated with protamine insulins.

Animals↗

PubMed1977

Antibodies reacting with salmon and human protamines in sera from infertile men and from vasectomized men and monkeys.

A micro-complement fixation test was modified for the detection of low-titred antibodies to protamines. Specific immunological reactions with salmon protamine were found with some sera obtained from infertile men and vasectomized men and monkeys. The possibilities that allergic reactions, observed in patients receiving salmon protamine, result from immunity to the fish protamine and from immunological cross-reaction between human and salmon protamines (in cases where auto-immunity to human protamine has developed prior to the administration of fish protamine) are discussed.

Animals↗

PubMed1982

Cardiovascular effects of protamine sulfate in man.

Systemic hypotension is commonly observed in association with the administration of protamine after cardiopulmonary bypass. Previous studies have not conclusively demonstrated whether protamine induces its effect by altering myocardial performance or by changing systemic vascular resistance (SVR) or both. To elucidate the hemodynamic effects of protamine sulfate administration (150 mg/m2 body surface area), we studied 22 patients following cardiopulmonary bypass. In Group I (N = 8) protamine was infused over 30 seconds and while in Group II (N = 8), over 60 seconds. Group III (N = 6) received calcium chloride (20 mg/kg) prior to protamine administration. The hemodynamic response was assessed by continuous recording of myocardial contractile element velocity (maximal value-Vpm), aortic blood flow, systemic and pulmonary arterial and right atrial pressures, and electrocardiogram. A significant (p less than 0.05) decrease in the systolic, diastolic and mean blood pressure was observed in all groups. A significant increase in cardiac index and a significant decrease in SVR was observed in all groups. A small depression in Vpm was detected in those patients who experienced a mean blood pressure fall greater than 10 mm Hh. Heart rate and left ventricular end-diastolic pressure (LVEDP) did not change significantly. The response to protamine sulfate among the three groups was similar. These results demonstrate that protamine-induced hypotension is primarily the result of peripheral vasodilatation only partically compensated by an increase in cardiac index. In some patients, these changes were associated with a small decline in myocardial contractile state. Hemodynamic changes were transient (less that 3 to 4 minutes), unrelated to the rate of administration, and not prevented by preinjection of calcium chloride.

Blood Pressure↗

PubMed1993

Anaphylaxis to protamine masquerading as an insulin allergy.

This is the case of a 62-year-old man referred for the evaluation of insulin allergy. This patient had reacted to the subcutaneous injection of Novolin 70/30 (Squibb, Princeton, N.J.) and Humulin NPH (Eli Lilly, Indianapolis, Ind.). These reactions were characterized by the immediate onset of diffuse pruritic urticaria and angioedema with progression to hypotension as well as a local reaction. Past history also included anaphylactic shock after intravenous administration of protamine sulfate used for heparin reversal during arterial bypass surgery. Immediate hypersensitivty skin testing to protamine containing (NPH) insulin and protamine sulfate USP were strongly positive, while Lente insulin (Eli Lilly, Indianapolis, Ind.) and controls were negative. RAST tests revealed the titers > 24 ng/ml of protamine specific IgE with 98 percent inhibition and 1163 ng/ml of protamine specific IgG with 29 percent inhibition, while levels of insulin specific antibodies were negligible. Subsequently, the patient was treated with non-protamine containing insulin preparation, Lente insulin, without further incident. This study confirms the diagnosis of Type I hypersensitivity to protamine sulfate masquerading as insulin allergy.

Anaphylaxis↗

PubMed1996

Regulation of protamine gene expression in an in vitro homologous system.

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

Animals↗

PubMed1996

Spermatogenesis of the lizard Lacerta vivipara: histological studies and amino acid sequence of a protamine lacertine 1.

The lizard Lacerta vivipara is a seasonal breeder with a well characterized reproductive cycle. An histological study of the lizard testis has been performed at different stages of spermatogenesis and the nuclear basic proteins content was assessed by electrophoretical analysis. Two protamines, lacertines 1 and 2, are present in spermatozoa in April and May. We have isolated lacertine1 and characterized a protamine with a mass of 4,963.7 Da. Amino acid sequence of this protamine (41 residues) was established from data provided by automated Edman degradation. It is characterized by a basic amino acid stretch in the N- and C-terminal regions and by a central part which only consists of 3 different intermingled amino acids. This protamine presents 62% homology with scylliorhinine Z3 from dog-fish Scylliorhinus caniculus and 58% homology with quail protamine. The reported lizard protamine sequence is the first reptilian protamine sequence available so far.

Amino Acid Sequence↗