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PubMedDate not supplied

Novel approach for optimizing the capacity and efficacy of a protamine filter for clinical extracorporeal heparin removal.

The authors previously reported the development of a blood filter device containing immobilized protamine (termed "protamine filter") that could be used at the conclusion of an extracorporeal blood procedure to prevent heparin and protamine induced complications. In vitro and in vivo experiments have fully demonstrated the feasibility and utility of the approach. The bottleneck limitations of this approach, however, lie in the lack of efficacy and capacity of the filter device. In this article, the authors describe a method to improve the efficacy in heparin adsorption, by incorporating a poly(ethylene glycol) spacer arm between the immobilized protamine and the fiber surface to enhance its freedom to dynamic motion. The authors also describe a method to increase the capacity of the filter, by using a poly-L-lysine based amplification method to augment protamine loading on the fiber, and to create multiple layers of immobilized protamine for heparin adsorption. Results show that with a poly(ethylene glycol) spacer arm of 3,400 Da, heparin adsorption on the protamine-poly(ethylene glycol) fibers was increased dramatically from a value of 9.1 mg heparin per gram of fibers in the control (i.e., without the poly[ethylene glycol] spacer) to 60 mg heparin/g fiber. The use of the amplification method with 110 kDa poly-L-lysine also yielded a threefold increase in protamine loading, and, consequently, an approximately fourfold enhancement in heparin adsorption (from 9.1 to 38.0 mg heparin/g fiber). A combination of these two methods would yield an optimized protamine filter that could meet all types of clinical needs in heparin removal. As assessed from the in vivo theoretical model reported previously for the protamine filter, a 95% heparin removal under cardiopulmonary bypass conditions could be achieved with a single optimized protamine filter with a size smaller than a hemodialyzer cartridge.

Hemofiltration↗

PubMed1994

Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc.

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA-Zn(II) column was observed for P1 protamines with one histidine in the N-terminal sequence (ram and boar protamines). The strong binding was found to be related to the presence of tyrosine, serine and threonine closely spaced to the histidyl side chain. In the case of human protamine P2, the strong retention on the IDA-Zn(II) column seems to result from the additive contribution of all the histidine residues of the molecule. Thus, strong retention of protamines in IMAC seems to depend on an additive contribution of amino-acid side chains: histidine, tyrosine, serine, threonine and perhaps arginine. The high affinity of protamines, more especially P2 protamines, for zinc suggests that this metal ion could play a role for their correct folding and binding to DNA.

Amino Acid Sequence↗

PubMed1994

The phospho-opsin phosphatase from bovine rod outer segments. An insight into the mechanism of stimulation of type-2A protein phosphatase activity by protamine.

The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of alpha subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 micrograms/ml; 50 microM). However, when phospho-peptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.

Amino Acid Sequence↗

PubMed1998

Protamine enhances the proliferative activity of hepatocyte growth factor in rats.

The effect of protamine on the proliferative activity of hepatocyte growth factor (HGF) was examined in alpha-naphthyl isothiocyanate-intoxicated rats. Protamine pre-injection increased the hepatocyte labeling index induced by HGF four- to fivefold. A similar effect was also observed in partially hepatectomized rats. Because a cell surface heparin-like substance can bind to HGF and protamine has an affinity for heparin, protamine may affect HGF pharmacokinetics. In fact, protamine injection caused a transient increase in plasma HGF concentrations after administration of HGF and, in vitro, protamine eluted HGF prebound to heparin-Sepharose. Protamine also reduced the plasma clearance of HGF and increased 2.5-fold the exposure of hepatocytes to HGF in vivo. The enhancing effect of protamine on the mitogenic response of hepatocytes to HGF was also observed in vitro (approximately 2-fold after protamine pretreatment compared with HGF alone), suggesting that the enhancing effect of protamine on HGF-induced liver regeneration results from dual effects exerted by protamine 1) lowering the overall elimination of HGF and 2) directly stimulating hepatocyte mitosis induced by HGF.

1-Naphthylisothiocyanate↗

PubMed1991

Effect of protamine on ion conductance of upper portion of descending limb of long-looped nephron from hamsters.

To estimate the contribution of paracellular shunt pathway to the cation-selective permeability in the upper portion of the descending limb of long-looped nephron (LDLu) of hamsters, we observed the effect of protamine on salt-diffusion voltage (delta VT) and transmural resistance (RT). delta VT generated on reduction of lumen NaCl concentration was decreased from 12.0 +/- 1.4 to 7.3 +/- 1.2 mV when 100 micrograms/ml protamine were added to the lumen. Although the effect of protamine persisted after removal of the agent from the lumen, addition of 30 U/ml heparin reversed the delta VT toward the control level. The effect of protamine was dose dependent in the range from 3 to 1,000 micrograms/ml. Protamine was without effect from the bath. Studies on single salt dilution voltage revealed that 100 and 300 micrograms/ml protamine inhibited relative Na+ to Cl- permeability from 4.03 +/- 0.38 to 2.14 +/- 0.21 and from 3.75 +/- 0.37 to 1.36 +/- 0.09, respectively. Protamine markedly decreased the apparent transference number for Na+ but slightly increased the value for Cl-. Protamine also inhibited permeabilities for K+, Rb+, and Li+ relative to Cl-, indicating that the inhibitory effect of protamine was not confined to Na+ but was generalized to cations. Transmural cable analysis showed that 100 micrograms/ml protamine increased RT from 14.0 +/- 1.1 to 19.3 +/- 1.2 omega.cm2, with the effect being reversed by 30 U/ml heparin. Because the effect of protamine on RT was unaffected by ouabain in the bath, changes in RT may mainly represent those of the paracellular shunt resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Increased risk of severe protamine reactions in NPH insulin-dependent diabetics undergoing cardiac catheterization.

Protamine is widely used for reversing systemic heparinization after cardiac catheterization. Although rare, major reactions to protamine that simulate anaphylaxis occasionally occur and have previously been associated only with an allergic reaction to fish. Because neutral protamine Hagedorn (NPH) insulin includes protamine, it might be anticipated that NPH insulin-dependent diabetic patients would develop sensitivity to protamine. Of 866 consecutive patients undergoing cardiac catheterization over a 20 month period, 651 received protamine for reversal of heparinization. Of these, 8.5% (56/651) were diabetics and 2.3% (15/651) were NPH insulin-dependent diabetics. During this period seven patients were observed immediately after administration of protamine to have major adverse reactions that required the administration of catecholamines. One death ensued. Of the seven major reactions, four occurred in NPH insulin-dependent diabetics and one occurred in a patient with an allergy to fish. The incidence of major protamine reactions was 27% (4/15) in the NPH insulin-dependent diabetics vs 0.5% (3/636) in those with no history of NPH insulin use (p less than .001). This represents a 50-fold increased risk of a major reaction to protamine if the patient was receiving NPH insulin. Accordingly, we recommend that diabetics on NPH insulin and patients with allergies to fish undergo cardiac catheterization without the use of protamine or, when necessary, that protamine be administered cautiously in anticipation of a major adverse reaction.

Anaphylaxis↗

PubMed2001

Low molecular weight protamine as nontoxic heparin/low molecular weight heparin antidote (III): preliminary in vivo evaluation of efficacy and toxicity using a canine model.

Heparin employed in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine employed in heparin reversal, however, can cause severe adverse reactions. In an attempt to address this clinical problem, we developed low molecular weight protamine (LMWP) as a potentially effective and less toxic heparin antagonist. A homogeneous 1880-d peptide fragment, termed LMWP-TDSP5 and containing the amino acid sequence of VSRRRRRRGGRRRR, was derived directly from protamine by enzymatic digestion of protamine with thermolysin. In vitro studies demonstrated that TDSP5 was capable of neutralizing various anticoagulant functions of both heparin and commercial low molecular weight heparin preparations. In addition, TDSP5 exhibited significantly reduced crossreactivity toward mouse sera containing antiprotamine antibodies. TDSP5 showed a decrease in its potential in activating the complement system. All of these findings suggested the possibility of markedly reduced protamine toxicity for TDSP5. In this article, we conducted preliminary in vivo studies to further demonstrate the feasibility and utility of using LMWP as a nontoxic clinical protamine substitute. Dogs were chosen as test animals because they were known to magnify the typical human response to protamine. By using a full spectra of biological and clinical assays for heparin, including the anti-IIa and anti-Xa chromogenic assays and the activated partial, thromboplastin time and TCT clotting assays, TDSP5 showed that it could completely neutralize all these different anticoagulant functions of heparin in dogs. Although administration of protamine in dogs produced a significant reduction in mean arterial blood pressure (-14.9 mm Hg) and elevation in pulmonary artery systolic pressure (+5.0 mm Hg), the use of TDSP5 in dogs did not elicit any statistically significant change in any of the variables measured. Furthermore, the use of LMWP also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 82.1% and 60.0% of baseline, respectively, in dogs given intravenous protamine compared to 97.8% and 88.6% of baseline in dogs receiving TDSP5. These preliminary findings indicated that LMWP could potentially provide an effective and safe means to control both heparin- and protamine-induced complications.

Animals↗

PubMed1998

Comparison of the effects of enoximone and isoproterenol on protamine cardiotoxicity in anesthetized dogs.

In this study we investigated the effects of isoproterenol and enoximone on protamine cardiotoxicity because administration of protamine for heparin reversal during open heart surgery depresses left ventricular function. Eighteen mongrel dogs were entered into this study. After induction of general anesthesia and a stabilization period, a thermodilution catheter was inserted via the jugular vein. Another 2 catheters were inserted into the left ventricle and femoral artery. Heparin and protamine were used in all animals. Heparin dosage was 300 U/kg, and protamine dosage was 4.5 mg/kg. The animals were divided into 3 groups. Six animals received enoximone (5 micrograms/kg per min), 6 animals received isoproterenol (0.05 microgram/kg per min), and 6 animals received no inotropic agent. Measurements were performed before treatment, 5 min after protamine administration, and at 15-min intervals for 1 h. Cardiac output (CO), mean arterial pressure, pulmonary capillary wedge pressure, first derivative of left ventricular pressure (1 +/-) left ventricular systolic pressure, and heart rate were measured. CO was 1582 +/- 34 ml/min in the isoproterenol group (I + P), 1684 +/- 61 ml/min in the enoximone group (E + P), and 1471 +/- 37 ml/min in the protamine group (P) (p < 0.05 E + P vs I + P and P) 60 min after protamine administration. The first derivative of left ventricular pressure (dP/dt) was 1995 +/- 61 mmHg/sec in the I + P group, 2320 +/- 85 mmHg/sec in the E + P group, and 1816 +/- 48 mmHg/sec in the P group (p < 0.05 E + P vs I + P and P). In our experimental study, the isoproterenol and protamine combination did not increase hemodynamic activity. However, isoproterenol alone significantly increased hemodynamic activity as determined by dP/dt values. Protamine administration impairs the effects of beta agonists on the myocardium. In the protamine group, CO and pressure-dependent values were significantly reduced. Isoproterenol administration did not reverse this deterioration because of the loss of the beta-receptor activity. Inotropic agents acting through the beta-adrenergic system have partial effects on myocardium. Enoximone, a phosphodiesterase inhibitor, reverses deterioration of cardiac function after protamine administration because it increases myocardial function via the phosphodiesterase system.

Animals↗

PubMed1988

Frequency and specificity of protamine antibodies in diabetic and control subjects.

Protamines are cationic fish chromosomal proteins that retard absorption of isophane (NPH) insulins. Protamines are also administered in large doses for heparin neutralization in cardiac procedures. This study used a rapid enzyme-linked immunosorbent assay to examine frequency of protamine antibodies in diabetic and control populations. Antigen specificity of the IgG binding to protamine-coated plates was verified by competitive inhibition with other protamines, histone, glucagon, thyroid-stimulating hormone, arginine, and lysine. All antibodies tested cross-reacted completely with all protamines. Only 4 of 18 had any cross-reactivity with histones. None cross-reacted with the other inhibitors. In population surveys, 122 (38%) of 319 NPH insulin-treated diabetic subjects, 3 (8%) of 39 diabetic subjects treated with protamine-free lente insulins, and 5 (2.5%) of 202 normal control subjects had protamine antibody. No correlation was found between insulin and protamine antibodies. Because more than one-third of insulin-treated diabetic subjects have circulating IgG specific for protamine, they are potentially at risk for acute immunologic or anaphylactoid reactions when protamine is administered for heparin neutralization.

Adolescent↗

PubMed1985

Evidence for complement activation by protamine-heparin interaction after cardiopulmonary bypass.

Complement activation by the alternate pathway has been implicated in the pathophysiology of cardiopulmonary bypass (CPB), and laboratory studies suggest that the complement cascade may be activated by the protamine-heparin complex. To determine if the administration of protamine to patients receiving heparin activates complement, we studied 100 patients undergoing CPB by assaying levels of C3a and C4a (classic pathway) at regular intervals before and after protamine administration. In group I (90 patients), protamine was given at the usual interval (median 5 minutes) after CPB. In group II (10 patients), protamine was withheld until skin closure (median 45 minutes) after CPB. Results demonstrated that C4a was not activated during CPB in either group. After CPB, the C4a level in group I was 459 ng/dl and increased to 1047 ng/dl 10 minutes after protamine administration (p less than 0.001). In group II, the C4a level was 484 ng/dl at the end of CPB and 354 ng/dl 15 minutes later, which corresponds to the value immediately after protamine administration in group I. The delayed administration of protamine in group II caused a significant increase in C4a at the time of skin closure (1090 ng/dl; p less than 0.001). Corresponding results from C3a analysis before and after protamine administration confirmed the activation of complement cascade. Our study provides the first clinical evidence that the protamine-heparin complex activates complement via the classic (C4a) pathway. The hemodynamic effects of protamine after CPB may be related to complement activation.

Aged↗

PubMed1993

Protamine neutralization of the release of tissue factor pathway inhibitor activity by heparins.

The present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa-activity were taken, in order to detect a possible relation between the remaining anti-factor Xa activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs. There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamine. It is assumed that protamine displaces heparins from the binding sites of TFPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor Xa Inhibitors↗

PubMedDate not supplied

Tissue distribution, circulating half-life, and excretion of intravenously administered protamine sulfate.

Intravenous protamine reversal of heparin anticoagulation may cause adverse hemodynamic side effects, but little is known about protamine's tissue distribution, circulating half-life (t/2), and excretion. The latter were assessed by examining 125I Bolton-Hunter (125I BH) radiolabeled protamine kinetics in a rat model. Three groups were studied: Group I controls (n = 5) received intravenous 125I BH label alone; Group II (n = 10) received intravenous 125I BH radiolabeled protamine (0.15 mg/100 g); and Group III (n = 10) received intravenous heparin (15 IU/100 g) followed by intravenous 125I BH radiolabeled protamine (0.15 mg/100 g). Five animals in each group were killed at 3 min, and tissue radioactivity was quantitated. An additional five animals each in Groups II and III were followed up for 60 min to determine protamine's circulating t/2 and its renal excretion. The lungs, heart, and kidneys, compared with other organs, retained the most 125I BH radiolabeled protamine per gram tissue at 3 min. Retention of 125I BH radiolabeled protamine (Groups II & III) was greater (p < 0.05, Kruskal-Wallis) than control 125I BH label alone (Group I). Higher tissue 125I activity was observed in Group II than in Group III rats, suggesting that tissue retention of protamine was greater in the absence of prior heparin administration. Circulating t/2 was shorter (18 vs. 24 min) and urinary protamine 125I excretion was higher (34 vs. 24%) in Group III than in Group II, respectively, suggesting more rapid renal clearance of protamine in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Inhibition by protamine of catecholamine secretion and ion influxes in bovine adrenal medullary cells in culture.

To elucidate the mechanism of protamine-induced hypotension, we examined the effects of protamine on catecholamine secretion in bovine adrenal medullary cells and on the serum norepinephrine in the rat. 1) in bovine adrenal medullary cells in culture, protamine at concentrations of 10 to 100 micrograms/ml inhibited catecholamine secretion stimulated by carbachol. The inhibitory effect of protamine was diminished by heparin at concentrations of 3.5 to 14 U/ml. Protamine suppressed carbachol-stimulated 22Na+ influx and 45Ca++2 influx at a concentration similar to that which inhibited catecholamine secretion. Protamine (10-100 micrograms/ml) also inhibited veratridine-induced 22Na+ influx and 45Ca++ influx and 56 mM K(+)-evoked 45Ca++ influx. The inhibition of these ion influxes by protamine was closely correlated with that of catecholamine secretion. 2) In rats, i.v. administration of protamine (10 mg/kg) attenuated the arterial blood pressure and the serum norepinephrine. There was a high correlation (r = 0.96) between the serum norepinephrine and the arterial blood pressure in protaminetreated rats. Furthermore, pretreatment with heparin (1000 U/kg) abolished the protamine-induced decreases in arterial blood pressure and serum norepinephrine. Because protamine seems to inhibit catecholamine secretion by interfering with Na+ influx and Ca++ influx to adrenal medullary cells, the protamine-induced hypotension may be, at least in part, due to inhibition of norepinephrine release and ion channel activities of sympathetic nerve terminals in rats.

Adrenal Medulla↗

PubMed2000

Hemostatic effects of low-dose protamine following cardiopulmonary bypass.

Twenty-eight patients undergoing cardiac surgery were prospectively studied and were assigned to two groups. The patients received 0.8- (Group L) or 2.0-fold (Group H) dose of protamine for the neutralization after cardiopulmonary bypass (CPB) which was determined by Hepcon HMS(R) assay system in which the reagent chamber containing the concentration of protamine that completely neutralized the heparin had the shortest clotting time. Mean dose of protamine was 1.60 +/- 0.50 mg kg(-1) in Group L, and 3.56 +/- 1.48 mg kg(-1), respectively. Activated clotting times (ACT) were comparable between the two groups through this study period. In Group H, platelet counts significantly decreased to 69% of that before protamine administration, and plasma platelet factor 4 level significantly increased to approximate 2-fold of that before protamine administration just after protamine administration, respectively. However, these phenomena were not observed in Group L. In addition, these hemostatic changes occurred transiently just after protamine administration. We conclude that the low-dose protamine may prevent transient platelet depletion following CPB. Low-dose protamine can neutralize anticoagulation effect of heparin sufficiently and may mitigate protamine-induced platelet dysfunction.

Aged↗

PubMed1996

DNA and total protamine masses in individual sperm from fertile mammalian subjects.

The total amount of phosphorus and sulfur inside the nuclei of individual bull, stallion, hamster, human, and mouse sperm from fertile subjects has been measured using Particle Induced X-ray Emission (PIXE). Using the sulfur masses, we determined the total protamine (protamine 1 plus protamine 2) mass within the sperm nuclei of each species. Using the phosphorus masses, we determined the DNA mass present within the sperm nuclei of each species. The results reveal that although the relative proportion of protamine 1 to protamine 2 varies among the species examined, the total protamine mass to DNA mass ratio is similar in bull, stallion, hamster, and mouse sperm nuclei. In contrast, mature human sperm nuclei were found to contain significantly less protamine. This observation is consistent with other studies, which suggest that as much as 15% of the DNA in human sperm remain packaged by histones. Using the data obtained for bull sperm, the length of DNA that could be covered by each protamine 1 molecule in bull sperm has been estimated. Making the assumption that the size of the protamine 1 binding site on DNA is similar in the sperm of these species, the length of DNA covered by a single protamine 2 molecule also has been estimated.

Animals↗

PubMed1989

Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells.

Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration-dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly-L-lysine decreased collagen production to a lesser degree than protamine. Poly-L-arginine was toxic to the cells. Poly-L-glutamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.

Ascorbic Acid↗

PubMed1987

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa.

The basic nuclear proteins of a fraction of elongating spermatids from human testes and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCl) while protamine 2 alone was eluted by 50% GuCl. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.

Alkaline Phosphatase↗

PubMed2006

Protamine sulfate causes endothelium-independent vasorelaxation via inducible nitric oxide synthase pathway.

PURPOSE: The precise mechanism of systemic hypotension frequently observed with the use of protamine is unclear. Although it has been reported that protamine stimulates the release of nitric oxide (NO) from endothelium NO synthase (eNOS), the association with inducible NOS (iNOS) remains unknown, despite the induction of iNOS by lipopolysaccharides (LPS) and/or inflammatory cytokines during cardiopulmonary bypass (CPB). The purpose of this study was to determine whether protamine stimulates the release of NO from iNOS induced by LPS. METHODS: We performed prospective and controlled functional examinations with isolated endothelium-denuded thoracic aortas from 21 male Wister rats. Aortic strips were mounted in Krebs solution and treated with LPS (1 microg x mL(-1)) for six hours to induce iNOS. Changes in tension caused by L-arginine (a substrate of NOS), protamine or a heparin-protamine complex (heparin: protamine = 1 unit: 10 microg) were measured in strips pre-contracted by phenylephrine. RESULTS: No drug relaxed the strips before LPS-treatment, but each drug relaxed the strips in a dose-dependent manner after LPS-treatment (P < 0.05). Aminoguanidine (an iNOS inhibitor) and methylene blue (a guanylyl cyclase inhibitor) inhibited the relaxations. CONCLUSION: These results indicate that protamine and the heparin-protamine complex stimulated the release of NO from iNOS. As iNOS is induced during CPB, protamine or a heparin-protamine complex might cause systemic hypotension, at least in part, by stimulating iNOS.

Animals↗