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PubMed2001

Individualized heparin and protamine management in infants and children undergoing cardiac operations.

BACKGROUND: Measurements of activated coagulation time do not correlate with plasma concentration of heparin. This study investigated the effects of a patient-specific method to manage anticoagulation and its reversal in pediatric patients undergoing cardiopulmonary bypass. METHODS: Infants and children were randomly assigned to receive either a standard dose of heparin (300 IU/kg; group C, n = 13) or an individualized dose, calculated by an in vitro heparin dose-response test (group HC, n = 13). Protamine dose was based on a 1 mg/l mg ratio of total administered heparin for patients in group C and of the residual heparin concentration in group HC. RESULTS: Administered heparin was significantly higher and total protamine dose was significantly reduced in the HC group (both p < or = 0.001). There was less thrombin generation (p = 0.02) and fibrinolysis (p = 0.05) in group HC. Blood loss and requirement for transfusion of blood and fresh frozen plasma were also lower in group HC (all p < or =0.05). CONCLUSIONS: An individualized management of anticoagulation and its reversal results in less activation of the coagulation cascade, less fibrinolysis, and reduced blood loss and need for transfusions. Further studies are warranted to better define the clinical impact of these findings.

Anticoagulants↗
PubMed1980

Noncardiogenic pulmonary edema and peripheral vascular collapse following cardiopulmonary bypass: rare protamine reaction?

Four instances of severe anaphylactoid reaction occurring subsequent to cardiopulmonary bypass are described. These catastrophic reactions, from which 2 patients died, took place approximately an hour following administration of protamine and were characterized by marked peripheral vasodilatation, loss of capillary membrane integrity, and fulminant noncardiogenic pulmonary edema. Primary cardiac depression was not evident. We hypothesize that protamine was the causative agent in these unusually severe reactions. Differential diagnosis from other causes of acute cardiorespiratory dysfunction depended on early assessment of pulmonary artery and left ventricular filling pressures, cardiac output, respiratory mechanics, and arterial blood gases. Therapy was difficult; success in 1 of the patients seemed to have been effected in part by prompt administration of high-dose corticosteroids and maintenance of peripheral vascular tone with an alpha-adrenergic agonist.

Aged↗
PubMed1997

Effects of the bile salt sodium deoxycholate, protamine, and inflammatory mediators on the potassium permeability of the frog nerve perineurium.

An electrophysiological method was used to measure the potassium permeability (PK) of the perineurium of the sciatic nerve of frogs Rana temporaria and R. pipiens. Isolated but intact nerves were mounted in a grease-gap chamber, and compound action potential and DC potential monitored. Change in the DC potential (delta DC) in response to challenge with 100 mM [K+] Ringer was used to assess the K+ permeability of the perineurium, since change in DC potential under these conditions reflected changes in the axonal resting potential. The permeability of the perineurium was calculated from the published calibration curve relating delta DC to bathing [K+] in desheathed nerves of Abbott et al. (1997). In the control condition, PK was < 1.1 x 10(-6) cm.s-1. The bile salt sodium deoxycholate (DOC, 1-4 mM) caused a dose-dependent increase in PK, which reached a maximum of 1.7 x 10(-5) cm.s-1 after 2-min exposure to 4 mM DOC, but access of K+ to the endoneurial compartment was more restricted after DOC than after desheathing. Protamine phosphate (1 mM) and protamine sulphate (0.1-5 mg/ml equals 0.125-6.25 mM) had no effect on PK. Neither histamine (0.4-40 mg/ml), bradykinin (0.1-5 mg/ml) nor serotonin (5-hydroxytryptamine, 0.1-5 mg/ml) affected PK. The frog nerve perineurium appears to be relatively insensitive to chemical agents and inflammatory mediators, in contrast to the endothelial cells forming the endoneurial blood-nerve barrier and the blood-brain barrier.

Action Potentials↗
PubMed1999

Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor.

A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target.

Amino Acids↗
PubMed1994

A new method for cytodestruction of bladder epithelium using protamine sulfate and urea.

Bladder epithelium relies primarily on the presence of a surface glycosaminoglycan (GAG) layer and the structural integrity of cell-cell contact to maintain impermeability to toxic urinary wastes. Previous clinical studies evaluating bladder permeability characteristics in interstitial cystitis patients had indicated that epithelial desquamation occurs after treatment with protamine sulfate (PS) followed by hypertonic urea. The following study was performed using rabbits to further investigate this finding. The urinary bladder was evaluated for optimal treatment conditions for epithelial removal. Protamine sulfate (1 to 10 mg./ml.) and urea (100 to 200 gm./ml.) were instilled into the bladder at volumes ranging from 5 to 60 ml. to that required for near maximum distention. After incubation at room temperature for 15 minutes, the bladders were fixed and evaluated histologically for epithelial removal. The maximum epithelial removal occurred when the bladders were distended, and when PS concentration was 5 to 10 mg./ml. and urea at 200 gm./l. There was greater epithelium removal after repeated treatments. Epithelial cells that were removed were not viable based on Trypan blue staining. There was no significant increase of C14 labeled urea in the plasma after 15 minutes. Rabbits that were followed for 6 weeks after treatment did not show any histological evidence of increased collagen deposition and/or fibrosis. This procedure may have important clinical value since it may remove sufficient bladder epithelium in patients with transitional cell carcinoma to have therapeutic benefit. This offers a realistic option for selective, nontoxic destruction of bladder epithelium.

Administration, Intravesical↗
PubMed1977

The measurement of androgen receptors in human prostatic tissue utilizing sucrose density centrifugation and a protamine precipitation assay.

Methods have been established for the measurement of androgen receptors in the cytosol and crude nuclear extract of human prostatic tissue using sucrose density gradient centrifugation and a protamine precipitation assay. These studies have demonstrated the presence of a high affinity, heat labile protein that binds preferentially to dihydrotestosterone and that is present only in androgen target tissues. In studies of prostatic tissue from 15 patients sucrose density gradient centrifugation appeared more sensitive than the protamine precipitation assay in the identification of androgen receptors. These findings appear promising and suggest that in the near future a reliable assay may be available for the measurement of androgen receptors in prostatic carcinoma.

Centrifugation, Density Gradient↗
PubMed1992

Studies on the function mechanism of a Formosan grey mullet protamine-mugiline beta M6: interaction of the M6 and M6 fragments with DNA.

The interaction of three peptide segments of one component of Formosan grey mullet protamine (mugiline beta M6), obtained by chemical and enzymatic cleavage, with DNA was studied by spectroscopic measurement, thermal denaturation and circular dichroism. The data obtained were then compared with those of whole M6 and other fish protamines such as salmine of salmon and clupeine of herring. M6-B-I, which lacks C-terminal 11 amino acids in M6, showed significantly different properties. It showed remarkably high DNA aggregating ability which was due to a conformational change of DNA from B to A form. The conformational change of DNA induced by the binding of M6-B-I was reproduced by the carboxypeptidase B digestion of DNA-M6 complex. From these results, the arginine-rich, C-terminal domain of the M6 molecule was estimated to be essential for natural DNA binding.

Amino Acid Sequence↗
PubMed2001

Characterization of complexes of an antisense oligonucleotide with protamine and poly-L-lysine salts.

The objectives of this work were to study the interaction of an antisense oligonucleotide (ISIS 2302) with poly-L-lysine (PLL) and protamine salts, to determine the physico-chemical characteristics of the resulting complex systems and to analyze the influence of permeation enhancers (Na-chenodeoxycholate and Na-caprate) on the dissociation of the complexes. Specific conductivity, zeta potential, particle size distribution and dialysis studies of the resulting complex systems were performed. Conductometric titration defined the molar ratios between the ionic species in the complex. Zeta potential data confirmed the conductometric equivalence points and explained the good physical stability of charged complexes when compared to neutral complexes (+/-40 mV for PLL-based complexes and +/-25 mV for protamine sulfate complexes). The particle size was less than 175 nm for most systems. The incorporation of Na-chenodeoxycholate promoted complex dissociation, while Na-caprate gave opposite results. An increase in the ionic strength of the environment had a destabilizing effect and promoted dissociation of the complexes.

Drug Delivery Systems↗
PubMed1998

Genesis of a novel human sequence from the protamine PRM1 gene.

The members of the male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1-->PRM2-->TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1-->PRM2-->TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Animals↗
PubMed2003

Dramatic post-cardiotomy outcome, due to severe anaphylactic reaction to protamine.

Immunologic reactions to protamine sulfate during cardiac surgery are very rare. The frequency and outcome of such adverse reactions is unclear. We report a case of lethal anaphylactic reaction to protamine that occurred in a non-diabetic patient following the uneventful replacement of the ascending aorta. We also briefly review the mechanisms of this adverse reaction and emit some considerations on the management of this situation.

Anaphylaxis↗
PubMed1988

Purification and characterization of mouse protamines P1 and P2. Amino acid sequence of P2.

Two mouse protamines, denoted as P1 and P2, have been purified directly from mature sperm nuclei and characterized as distinct polypeptide species. The complete primary structure of P2 was determined by peptide sequencing analyses. P1 and P2 were purified by a sequence of cation-exchange chromatography on Bio-Rex 70 and permeation chromatography on Bio-Gel P10, both in the presence of guanidine hydrochloride. Biochemical analyses demonstrate P1 has a molecular weight of 7400 and is characterized by the presence of arginine, cysteine, lysine, and tyrosine. By contrast, P2 is unusual in containing an abundance of arginine, histidine, lysine, and cysteine, but no tyrosine. The primary structure of P2 was determined from the sequencing of overlapping, high-pressure liquid chromatography purified peptides generated by thermolysin and endoproteinase Lys-C digestions and by chemical cleavage at each of four serine residues. Sequence analyses have demonstrated that P2, with a molecular weight of 8841, contains 62 amino acids, in the sequence NH2-Arg-Gly-His-His-His-His-Arg-His-Arg-Arg-Cys- Ser-Arg-Lys-Arg- Leu-His-Arg-Ile-His-Lys-Arg-Arg-Arg-Ser-Cys-Arg-Arg-Arg-Arg-Arg-His-Ser- Cys-Arg - His-Arg-Arg- Arg-His-Arg-Arg-Gly-Cys-Arg-Arg-Ser-Arg-Arg-Arg-Arg-Arg-Cys-Arg-Cys-Arg- Lys-Cys - Arg-Arg- His-His-COOH. Thus, the primary structure includes six clusters of arginine and histidine, distributed throughout the polypeptide, each ranging from five to eight amino acids in length. Sequence comparisons of mouse and human protamines by the Dayhoff program have revealed greater homology exists between human P2 and mouse P2 than within the P1 family from the two mammalian species.

Amino Acid Sequence↗
PubMed1976

Modification of histone binding in calf thymus chromatin and in the chromatin-protamine complex by acetic anhydride.

A relationship between side-chain modification of histones and their displaceability from DNA has been investigated using calf thymus chromatin which was chemically acetylated with acetic anhydride. When the chromatin is treated with increasingly higher concentrations of the reagent, histones become acetylated to an increasingly greater extent, attaining the modification at 23-24 sites for histone I, 5-6 for IIb1, 9-10 for IIb2, 5-6 for III and 3-4 for IV. As the chromatin becomes more acetylated, NaCl concentrations required for histone removal are lowered. Saturation binding of protamine does not bring about either an increase in the number of acetylation sites of histones in chromatin or a decrease of the NaCl requirement for dissociation of the acetylated chromatins. A comparison of the present results with the extents of histone acetylation known to occur enzymatically in vivo indicates that the complete removal of somatic histones during transformation of chromatin in spermiogenesis cannot be explained on the basis of decreased binding of the histone to DNA by acetylation or by a combination of acetylation and protamine binding, suggesting that the displacement process may require some additional processes.

Acetates↗
PubMed1995

Reversible adsorption of soluble hexameric insulin onto the surface of insulin crystals cocrystallized with protamine: an electrostatic interaction.

Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 10(4) M-1 to 10(7) M-1, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.

Adsorption↗
PubMed2001

Protamine augments stretch induced calcium increase in vascular endothelium.

1. Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured by fluorescence intensity ratiometry using fura-2. 2. In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca(2+) transient in a dose dependent way. The response was completely dependent on external Ca(2+) and inhibited by gadolinium (Gd(3+)), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC). 3. Interestingly, the stretch induced Ca(2+) transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca(2+) response was inhibited neither by Gd(3+) nor by the deprivation of external Ca(2+), indicating that the SACatC is not responsible for this phenomenon. 4. In contrast, this augmentation was inhibited by depletion of intracellular Ca(2+) stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide. 5. These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca(2+)](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.

Calcium↗
PubMed1997

In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.

A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 micrograms per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung. Increasing the DNA to 100 micrograms per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expression by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy.

Animals↗
PubMed1998

Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery.

A previous study has shown an efficient, systemic transgene expression in mice via intravenous administration of a LPD formulation composed of DOTAP liposomes, protamine sulfate and plasmid DNA. In this study, factors affecting the in vivo performance of this formulation were further evaluated. A protocol in which liposomes were mixed with protamine before the addition of plasmid DNA was shown to produce small condensed particles with a diameter of about 135 nm. These particles were stable over time and gave a high level of gene expression in all tissues examined including lung, heart, spleen, liver and kidney with the highest level of expression in the lung. Inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid significantly decreased the in vivo activity of LPD. In contrast, inclusion of cholesterol as a helper lipid increased the in vivo transfection efficiency of LPD and more importantly, decrease the amount of cationic lipid required for the maximal level of gene expression. Studies on the interaction between mouse serum and LPD showed that LPD became negatively charged after exposure to serum, and LPDs containing different helper lipids varied in the amount of associated serum proteins. LPD containing DOPE was more enriched in a protein corresponding to albumin in molecular weight. These results suggest that the mechanism of LPD-mediated intravenous gene delivery might be different from that of in vitro lipofection and that serum protein association might be a major factor limiting the in vivo transfection by LPD.

Animals↗
PubMed1999

Nephelometric determination of micro amounts of nucleic acids with protamine sulfate.

Nucleic acids can form large particle complexes with protamine sulfate by electrostatic forces, which results in strong light scattering. Based on this, a nephelometric method is described for sensitive and convenient determination of nucleic acids with protamine sulfate by using a common spectrofluorimeter. Maximum light scattering is produced in the range of pH 2.2-4.4 with the same excitation and emission wavelengths at 365 nm. Under optimal conditions, the calibration curves are linear in the range 0.05-60.0 micrograms cm-3 for nucleic acids. The corresponding detection limits are 12.5 ng cm-3 for calf thymus DNA, 9.0 ng cm-3 for fish sperm DNA, and 18.0 ng cm-3 for yeast RNA, respectively. Six synthetic samples are determined with satisfactory results. The relative standard deviation of five replicate measurements is 3.2% for 2.0 micrograms cm-3 calf thymus DNA.

Animals↗