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PubMed1980

A comparison of the quantitative action of protamine and heparin on blood coagulation. Significance in clinical and laboratory usage.

Protamine is a considerably weaker anticoagulant than heparin. At a concentration in plasma of 0.05 mg (5 units/ml), heparin rendered the plasma incoagulable, while protamine produced only a mild-to-moderate lengthening of activated partial thromboplastin time (APTT) and prothrombin time (PT). Protamine concentrations between 0.3 and 1.0 mg/ml of plasma produced marked prolongation of APTT and PT, but the thrombin time (TT) was shortened. Between protamine concentrations of 1 and 2 mg/ml of plasma, the fibrinogen was precipitated to a variable degree, the APTT and PT were more than 200 sec, and the TT was moderately prolonged. High doses of protamine prolonged APTT, simulating heparin effect. Although APTT is prolonged both by heparin and protamine, TT is either normal or shortened in protamine excess and prolonged in heparin excess. To differentiate between the protamine and heparin excess, a combination of APTT and TT, supplemented by an estimation of fibrinogen and fibrin-fibrinogen split products, is recommended. The significance of these observations in the interpretation of the tests on blood coagulation is discussed.

Blood Coagulation Tests↗
PubMed1998

In-situ competition between protamine and fluorochromes for sperm DNA.

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.

Animals↗
PubMed2000

Purification and characterization of a novel protamine kinase in HL60 cells.

A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.

Calcitriol↗
PubMed1995

Protamine-induced pulmonary venoconstriction in heparinized pigs.

Reversal of heparin anticoagulation with protamine may be associated with acute pulmonary vasoconstriction. The specific site of pulmonary vasoconstriction has not been determined. This study was designed to determine the site of protamine-induced pulmonary vasoconstriction and the role of nitric oxide (NO) after protamine injection. Pigs were anesthetized and instrumented with catheters for monitoring pulmonary arterial, systemic arterial, and central venous pressures. Pulmonary capillary pressure was estimated using the arterial occlusion concept, while left atrial pressure was estimated from the equilibrium wedge pressure. Hemodynamic measurements were made during baseline, before and after heparin (200 U/kg), at peak pressure response after protamine injection (2 mg/kg), and 10 and 30 min thereafter. In the control group, pulmonary vascular resistance (PVR) values during baseline and after heparin were identical (2.7 +/- 0.4 mm Hg.L-1.min-1). At peak protamine response (1-2 min) PVR increased to 8.0 +/- 1.6, but returned to baseline value after 10 min (2.8 +/- 0.3) and remained stable for 30 min (2.2 +/- 0.3). The increase in PVR after protamine was primarily due to an increase in venous resistance from 1.0 +/- 0.2 to 4.9 +/- 1.4 mm Hg.L-1.min-1, and a much smaller increase in arterial resistance from 1.7 +/- 0.3 to 3.4 +/- 0.6 mm Hg.L-1.min-1. A second group was treated with nitrow-L-arginine (LNA, 20 mg/kg) to inhibit NO release, and then heparin and protamine were administered as in the first group. Heparin had no effect on pressures, but protamine increased PVR by the same magnitude as in Group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Extracellular calcium modulates the effects of protamine on rat myocardium.

UNLABELLED: We studied the effects of protamine (10-300 microg. mL(-1)) as well as its interaction with heparin in rat left ventricular papillary muscles in vitro at calcium concentrations of 0.5 and 1 mM under low (isotony) and high (isometry) loads. Protamine induced a negative inotropic effect that was less pronounced at calcium 0.5 mM (active force at protamine 300 microg/mL, 84 +/- 20 vs 57 +/- 15% of baseline, P: < 0.05); whereas at calcium 1 mM there was a marked contracture of the muscle. For the smallest concentrations of protamine and at calcium 0.5 mM, we observed a moderate positive inotropic effect that was suppressed by nifedipine. Protamine induced a negative lusitropic effect under low load and decreased postrest potentiation, suggesting an impairment in the functions of the sarcoplasmic reticulum. Heparin was able to inhibit and reverse the negative inotropic effect of protamine. The negative inotropic effect of protamine is enhanced by an increase in extracellular calcium concentration. This negative inotropic effect is probably related to calcium overload and impairment in sarcoplasmic reticulum functions, and heparin can block these effects. IMPLICATIONS: The negative inotropic effect of protamine is enhanced by an increase in extracellular calcium concentration. This negative inotropic effect is probably related to calcium overload and impairment in sarcoplasmic reticulum functions, and heparin can block these effects.

Animals↗
PubMed1990

Protamine-induced histamine release in human skin mast cells.

Rapid intravenous (iv) infusion of protamine sulfate is associated with hypotension in humans. A possible mechanism for this hypotension is the release of inflammatory mediators, including histamine, from tissue mast cells lining the blood vessels. To determine whether protamine caused nonimmunologic release of histamine, histamine released from dispersed human skin mast cells exposed to protamine sulfate was measured. Skin from seven adult patients was washed, chopped into small tissue fragments, and incubated with collagenase, hyaluronidase, and DNAase. Dispersed mast cells were harvested after 12 h of short-term tissue culture, washed, and challenged with protamine sulfate. Histamine release was measured using an automated histamine analyzer and expressed as a per cent of total released histamine measured minus the spontaneous histamine release. Spontaneous histamine release averaged 6 +/- 1%. Protamine produced dose-related histamine release. At a concentration of 3 X 10(-3) M, protamine sulfate released 14 +/- 2% (P less than 0.05), which significantly differed from spontaneous release. This study demonstrates that protamine sulfate causes nonimmunologic histamine release in dispersed human skin mast cells. However, histamine release occurred only at concentrations much greater than those used in clinical practice. Thus, these data do not support the hypothesis that nonimmunologic histamine release is a likely mechanism for protamine-induced hypotension in vivo.

Adult↗
PubMed1997

The effects of heparinase 1 and protamine on platelet reactivity.

BACKGROUND: Protamine is currently the most widely used drug for the reversal of heparin anticoagulation. Heparinase 1 (heparinase) is being evaluated as a possible alternative to protamine for the reversal of heparin anticoagulation. The authors evaluated the effects of equivalent doses of heparinase and protamine on platelet reactivity by measuring agonist-induced P-selectin expression. METHODS: After Institutional Review Board (IRB) approval, informed consent was obtained from 12 healthy volunteers and 8 patients undergoing surgery requiring cardiopulmonary bypass (CPB). Twenty-four ml of blood was obtained from each volunteer; 10 ml of blood was obtained from each patient before the CPB, and another 10 ml was obtained after CPB. Heparin was neutralized using heparinase or protamine. Platelet reactivity was assessed by measuring the expression of P-selectin after stimulation of platelets with increasing concentrations of a thrombin receptor agonist peptide (TRAP). Data were analyzed using analysis of variance. P < 0.05 was considered significant. RESULTS: For the healthy volunteers, the activated coagulation times (ACTs) of the heparinized samples returned to baseline values with heparinase (12.5 U/ml) or protamine (32.5 microg/ml). For the 8 patients, the ACTs returned to baseline with heparinase (20 U/ml) or protamine (50 microg/ml). The authors found no difference in the expression of P-selectin in samples neutralized with heparinase, but samples neutralized with protamine showed a significant decrease in the expression of P-selectin when compared with heparinized samples. CONCLUSIONS: At dosages that reverse the anticoagulant effects of heparin, heparinase has minimal effects on platelets, whereas platelet reactivity was markedly inhibited by protamine.

Adult↗
PubMed1991

Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei.

Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.

Amino Acid Sequence↗
PubMed1982

Enhancement of pneumococcal transfection by protamine sulfate.

Protamine sulfate enhanced transfection of Streptococcus pneumoniae by DNA of omega 3 phage by factors as large as 10(5)-fold, provided it was present at the time the cells were added to the DNA. For DNA concentrations well below 1 microgram/ml, the optimum amount of protamine sulfate was near 1 microgram/ml of cells. Higher DNA concentrations required more protamine for maximum effect, and in all cases transfection fell when protamine was in excess. Transformation was not enhanced by low protamine levels and was inhibited by higher levels. A recipient strain with low but finite endonuclease activity and normal transformability showed higher transfection than did the wild type at low DNA concentrations but less than did the wild type at high DNA concentrations. Protamine sulfate enhanced its transfection at low, but not high, DNA concentrations. The behavior of this strain and the enhancement of transfection by protamine sulfate of wild-type cells were each consistent with less cutting of the donor DNA at the cell surface, which is part of the normal entry process in naturally competent gram-positive bacteria. Less cutting would lead to entry of fewer but longer strands that would be more efficient in reconstruction of the 33-megadalton phage replicon. We suggest that in this system protamine enhances transfection by inhibition of the surface nuclease action that is part of the normal entry process.

Bacteriophages↗
PubMed1979

Double-stranded protamine cDNA: synthesis and characterization.

Double-stranded protamine complementary DNA (cDNA) was synthesized from a protamine mRNA template via the single-stranded cDNA intermediate using avian myeloblastosis virus reverse transcriptase. Synthesis at 37 and 46 degrees C resulted in similar overall yields (greater than or equal to 18%), although the initial rate of synthesis was higher at 46 degrees C than at 37 degrees C. The DNA of the second strand of the double-stranded cDNA product was 84% resistant to prolonged digestion with excess S1 nuclease. The S1 nuclease resistant material ranged in size from 235 to 100 base pairs (bp) with an average length of 185 bp. Analysis of the products released from double-stranded protamine cDNA by depurination indicated that there were a number of cytosine-rich oligopyrimidine tracts in protamine mRNA, namely C4, C4U1, C5U1, C6U1, C6U4, and C7U1. On the basis of the amino acid sequences for rainbow trout protamines, C5U1, C6U1 and C7U1 must be located within the noncoding regions. Double-stranded protamine cDNA was cleaved at least once by the restriction endonucleases HaeIII and HhaI and in several places by HpaII. These restriction endonucleases cleave at sequences which have a high probability of occurring within the coding region of protamine mRNA, again based on the known amino acid sequences of the rainbow trout protamines.

DNA↗
PubMed1984

Transcription of a trout protamine gene in vitro: the effects of alteration of promoters.

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is alpha-amanitin sensitive (1 micrograms/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.

Animals↗
PubMed1991

Effect of protamine on ion conductance of ascending thin limb of Henle's loop from hamsters.

To evaluate the contribution of paracellular shunt pathway in ascending thin limb (ATL) of hamsters, we examined the effect of protamine, a selective blocker of paracellular conductance, on salt-diffusion voltage (dVT) and transmural resistance (RT) during in vitro microperfusion. Lumen-negative dVT generated on reduction of lumen NaCl concentration was increased further from -7.3 +/- 0.5 to -10.3 +/- 0.7 mV when 300 micrograms/ml protamine was added to the lumen, and calculated Na+/Cl- permeability ratio was decreased from 0.46 +/- 0.03 to 0.31 +/- 0.03. Although the effect of protamine persisted after removal of the agent from the lumen, addition of 30 U/ml heparin returned the dVT toward the control level. The effect of protamine was dose dependent from 30 to 300 micrograms/ml. Protamine also exerted its effect from the bath, and the effect was inhibited by heparin either from the lumen or from the bath. The inhibitory effect was almost the same when the orientation of imposed NaCl gradient was reversed. Inhibition of transcellular Cl- transport with 0.1 mM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) in the bath caused lumen-positive dVT. This voltage was decreased significantly by protamine. Protamine markedly decreased the apparent transference number for Na+ but slightly increased the value for Cl-. Transmural cable analysis showed that 300 micrograms/ml protamine added to the lumen increased RT from 0.59 +/- 0.10 to 1.20 +/- 0.20 omega.cm2, with the effect being reversed by 30 U/ml heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Protamine increases the permeability of cultured epithelial monolayers.

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of leukopenia on pulmonary hypertension after heparin-protamine in pigs.

Heparin neutralization by protamine after cardiac surgery and cardiopulmonary bypass may be associated with complement activation, transient leukopenia, thromboxane A2 release, and severe pulmonary hypertension. The role of leukocytes in the heparin-protamine reaction was studied in leukopenic pigs (n = 9) and a control group (n = 8). Leukopenia was induced by pretreatment with cyclophosphamide (30 mg.kg-1.day-1) for 6-7 days. During general anesthesia and after catheterization, baseline recordings of hemodynamics were performed and blood samples were withdrawn. Heparin (250 IU/kg) was injected and measurements were repeated after 10 min. Protamine sulfate (100 mg) was then infused over 2 min and measurements were performed after 2, 5, and 15 min. Prostanoid concentrations were measured by radioimmunoassays. In additional in vitro experiments, the release of thromboxane B2 from washed platelets and leukocytes after heparin-protamine stimulation was measured. Pretreatment with cyclophosphamide reduced leukocyte counts by 95.5% and the number of neutrophils by greater than 99.9%. Protamine infusion increased mean pulmonary arterial pressure by 74 and 46% and pulmonary vascular resistance by 185 and 384% in control and leukopenic animals, respectively. Thromboxane B2 concentrations increased in both groups. Stimulation by heparin, protamine, or heparin and protamine in sequence did not induce any thromboxane A2 release from washed blood cells. It is concluded that leukocytes do not contribute to pulmonary hypertension after heparin-protamine.

Animals↗
PubMed1998

Effect of protamine on cation-selective permeability in hamster medullary thick ascending limb of Henle's loop.

To estimate the contribution of the paracellular shunt pathway to cation-selective permeability in the hamster medullary thick ascending limb of Henle's loop, we observed the effect of protamine, a selective blocker of paracellular conductance, on salt-diffusion voltage (dVT) in the isolated nephron segment perfused in vitro. When 300 microg/ml protamine was added to the lumen, the lumen-positive dVT generated upon reduction of the lumen NaCl concentration was decreased from 5.1 +/- 0.9 to 0.8 +/- 0.8 mV and the calculated Na+/Cl- permeability ratio was decreased from 1.40 +/- 0.14 to 0.86 +/- 0.08. Although the effect of protamine persisted after removal of the agent from the lumen, addition of 30 U/ml heparin, which neutralizes protamine, returned the dVT toward the control level. This effect was almost the same when the orientation of the imposed NaCl gradient was reversed. Protamine exhibited a similar effect on dVT in the presence of ouabain added to the bath. Protamine was without effect from the bath. Protamine did not affect the basel VT perfused with the control solution. Increased VT by decreasing perfusion pressure was inhibited by adding protamine from the lumen. These observations suggest that the paracellular pathway contributes to the cation selectivity of the medullary thick ascending limb. The cation selectivity of the paracellular shunt pathway may mainly account for the changes in VT which are either dependent on the luminal flow rate or transmural NaCl concentration gradient, while it may not contribute to the basal level of VT.

Animals↗
PubMed1993

Protamine and left ventricular function: a transesophageal echocardiography study.

The effect of protamine sulfate on left ventricular function was evaluated in 23 patients undergoing heart surgery requiring cardiopulmonary bypass (CPB). Ventricular function was assessed by measuring cardiac index with a thermodilution pulmonary artery catheter and fractional shortening with transesophageal echocardiography (TEE). After CPB, a heparin-neutralizing dose of protamine was infused continuously for 5 min. Hemodynamic variables were obtained and fractional shortening was measured before protamine administration (time 0), during protamine infusion (2.5 and 5 min), and after the infusion (10 and 15 min after the start of protamine). Patients with a preoperative ejection fraction > or = 50% had no statistically significant changes in either cardiac index or fractional shortening with protamine administration, when compared to time 0. However, patients with a preoperative ejection fraction < 50% had a significant decrease in fractional shortening which occurred during and immediately after protamine administration when compared to time 0 (P < 0.01), with no associated change in hemodynamics. Our results suggest that protamine may have a negative inotropic effect that is apparent in patients with impaired ventricular function.

Adult↗
PubMed2003

Adverse events after protamine administration following cardiopulmonary bypass in infants and children.

UNLABELLED: We performed this study to determine the incidence of and risk factors for adverse events (AEs) in infants and children after the IV administration of protamine after cardiopulmonary bypass. In a retrospective cohort study, all relevant anesthesia records from a 3-yr period were examined to identify AEs after protamine. The AEs were then grouped into three categories by applying increasingly strict criteria. Among 1249 anesthesia records, there were no documented episodes of isolated or hypotension-associated right-sided cardiac failure or acute pulmonary dysfunction. The incidence of systemic hypotension after protamine was between 1.76% (95% confidence interval [CI], 1.11%-2.65%) and 2.88% (95% CI, 2.03%-3.97%), depending on the strictness of case definition. To identify risk factors, we performed a nested case-control study in which unmatched controls were randomly selected from the parent cohort at a 4:1 ratio to cases. Cases of hypotension after protamine were more likely during operations on girls (odds ratio [OR], 6.47; 95% CI, 1.66-32.8), after larger doses of protamine (OR, 1.88; 95% CI, 1.03-3.63), or after smaller doses of heparin (OR, 0.49; 95% CI, 0.17-0.67). IMPLICATIONS: Systemic hypotension after protamine administration occurred in 1.76%-2.88% of pediatric patients having cardiac surgery. Female sex, larger protamine dose, and smaller heparin dose were each associated with increased risk. The development of protamine alternatives or prophylactic therapies may be useful for reducing the frequency of these events.

Adolescent↗
PubMed1999

Noncardiogenic pulmonary edema immediately following rapid protamine administration.

OBJECTIVE: To report the case of a rare, potentially preventable, immediate noncardiogenic pulmonary edema reaction to the rapid administration of protamine during coronary artery bypass graft (CABG) surgery. CASE SUMMARY: A 74-year-old white man was administered a 250-mg bolus of protamine sulfate toward the end of CABG surgery to reverse the heparin anticoagulation. Immediately following the administration of protamine, oxygen saturation declined, pink frothy sputum was suctioned from the trachea, and 1500 mL of serous fluid was removed from the airway. The patient was stabilized, but the surgeons were unable to close his chest because of the profound edema. Chest closure occurred on hospital day 6, with discharge from the intensive care unit on hospital day 28. DISCUSSION: Noncardiogenic pulmonary edema is a rare adverse event that occurs in 0.2% of cardiopulmonary bypass patients, with mortality rates approaching 30%. Complement activation or direct pharmacologic release of histamine by high concentrations of protamine is the suspected cause. High concentrations of protamine in the lungs may directly release histamine, with significant vasodilating effects. CONCLUSIONS: Immediate reversal of heparin anticoagulation with protamine is necessary to control bleeding; however, rapid protamine injection can be associated with life-threatening pulmonary edema. Slower, cautious administration and accurate calculation of protamine doses may prevent such an event.

Aged↗