Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protamines”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMedDate not supplied

A protamine filter for extracorporeal blood heparin removal.

Heparin employed in extracorporeal circuits often leads to hemorrhagic complications. Protamine employed for heparin neutralization can cause adverse hemodynamic responses. To control both types of complications, the authors propose an approach that consists of placing a filter device containing immobilized protamine (defined as a protamine filter) at the termination of the extracorporeal blood circulation (ECBC) procedure. This protamine filter would remove heparin from the extracorporeal circuit before heparin is returned to the patient. Meanwhile, the filter would also permit external protamine treatment. Since protamine toxicity generally results from the interaction of protamine with certain cells present in the liver, lungs, and tissues, the use of external protamine would minimize its potential adverse effects. Protamine was immobilized on a hollow fiber bundle obtained from a conventional hemodialyzer. Preliminary studies show that the protamine bound bundle is capable of neutralizing the anticoagulant activity of heparin both in vitro and in vivo. In addition, the protamine filter has abolished the hypotensive response normally associated with protamine reversal of heparin, as indicated by the insignificant changes in blood pressure, pulse rate, pulmonary artery systolic/diastolic pressures, and cardiac output. Further in vivo studies involving the use of dogs, as well as investigation of the activation of the complement system by the protamine filter, are currently being conducted.

Animals↗
PubMed2001

The effects of heparin, protamine, and heparin/protamine reversal on platelet function under conditions of arterial shear stress.

UNLABELLED: Platelet dysfunction contributes to blood loss after cardiopulmonary bypass. This study examined the antiplatelet effects of heparin, protamine, and varying heparin/protamine ratios in an in vitro physiologic model and further elucidated the mechanism of the antiplatelet and anticoagulant effects of protamine. We used the Clot Signature Analyzer (CSA(TM)), a system that analyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under conditions simulating arterial flow: collagen-induced thrombus formation (CITF) under moderate shear and high shear platelet activation, platelet hemostasis time (PHT). In addition, platelet aggregometry, celite activated clotting time (Hepcon(TM) ACT), prothrombin time (PT), and partial thromboplastin time (PTT) were measured. Both PHT and the CITF were prolonged by heparin at 20 microg/mL, protamine at 20 and 40 microg/mL, and heparin/protamine ratios of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by heparin 20 microg/mL and protamine alone at 20 and 40 microg/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 microg/mL prolonged the PT and PTT. Dependency on thrombin, protein kinase C activation, and nonspecific charge effects were examined. The direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polylysine prolonged the CITF, but not the PHT. The effect of protamine on the PTT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and moderate shear collagen-dependent platelet activation; however, the latter is not mediated by its antithrombin activity. Protamine's antithrombin effect may explain its inhibition of platelet activation at high shear stress. Protamine's nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation. IMPLICATIONS: This study suggests that protamine reversal of heparin's antiplatelet effect occurs within a narrow window because of the direct antiplatelet effects of protamine. Antithrombin effects may explain the inhibition of shear activation of platelets by both heparin and protamine. Nonspecific charge effects of protamine may explain the inhibition of collagen platelet activation in the presence of medium shear.

Aged↗
PubMedDate not supplied

Allergy to protamine.

Recent advances in medicine, such as cardiac catheterization, phoresis, dialysis, and cardiopulmonary bypass technology, have increased the need for heparin anticoagulation. To antagonize heparin's effect and prevent hemorrhagic complications after the procedure, protamine has likewise been used more frequently. With its increased use have come increased reports of adverse protamine reactions consisting of rash, urticaria, elevation of pulmonary artery pressure, systemic hypotension, and, at times, death. The elevation of pulmonary artery pressure, which appears to be a rather common occurrence in animals, may be an isolated finding without clinical consequences in humans. However, this pulmonary vasoconstriction may, when severe, lead to acute right-sided heart failure and systemic hypotension. Other protamine reactions involve a decrease in systemic vascular resistance and systemic hypotension without changes in pulmonary artery pressure. Causes of acute protamine reactions may involve the generation of anaphyatoxins and prostanoids either from protamine-heparin complexes or complement-fixing antiprotamine IgG antibodies, from inhibition of plasma Carboxypeptidase N, from crosslinking of cell-surface antiprotamine IgE on mast cells and basophils with subsequent mediator release, or from potentiation of IgE-mediated release of histamine through a polycationin-recognition site. Although we have come a long way in understanding the mechanisms by which protamine can cause its ill effects in humans, more work is clearly needed to define, in prospective studies, the incidence of and risk factors for protamine reactions in various patient groups, and to delineate more clearly which mechanisms are involved in each clinical type of acute protamine reaction. Hopefully, this will lead to strategies and protamine alternatives that will prevent or diminish, in frequency or severity, adverse protamine reactions. Alternatively, a clearer picture of the risk factors important for protamine reactions and the predictive value of diagnostic tests (e.g., protamine IgE antibody) can also minimize the clinical impact of this increasingly common adverse event.

Acute Disease↗
PubMed1987

Interrelationship of protamine and platelet factor 4 in the neutralization of heparin.

To determine the interaction of platelet factor 4 (PF4) and protamine sulfate in the neutralization of heparin in plasma in vitro studies were carried out using a tritium-labeled heparin and a PF4 tagged with 14C. Plasmas treated with various combinations of PF4, protamine and heparin were chromatographed on Sephadex G200 and the fractions were tested for both radioactivity and antithrombin activity. PF4 was comparable to protamine in its ability to neutralize heparin, but the complexes formed with heparin were different. In contrast to protamine, when heparinized plasma was treated with an excess of PF4, no large PF4-heparin complexes were formed and none of the PF4-heparin complexes which did form were able to activate antithrombin III (ATIII). Also, incubation of PF4-neutralized, heparinized plasma at 37 degrees C did not result in liberation of heparin and prolongation of the thrombin clotting time as was found with protamine-neutralized plasma. The action of protamine and PF4 is complimentary. When half the neutralizing dose of each was added together to heparinized plasma, no immediate antithrombin activity remained. When a neutralizing dose of protamine was added to PF4-neutralized, heparinized plasma, the protamine displaced the PF4 from its complexes with heparin. The large protamine-heparin complexes which formed also contained PF4 but could not activate fresh ATIII as has been demonstrated with protamine-heparin complexes without PF4. On incubation of the protamine-PF4-neutralized, heparinized plasmas for 5 hours at 37 degrees C, the large complexes were broken down but no active heparin appeared. The results of these experiments may have some bearing on the amount of protamine needed for the neutralization of heparin following extracorporeal bypass procedures, when large amounts of PF4 may have been released from activated or disrupted platelets.

Chromatography↗
PubMed1991

The role of plasma proteins in formation of obstructive protamine complexes.

Formation of complexes between heparin and protamine (in saline), or heparin, plasma proteins, and protamine (in plasma) was assessed by measurements of light transmission through different test solutions. To examine the formation of these complexes, 125I-labeled protamine was used. Addition of 125I-protamine to plasma or blood resulted in the sedimentation of 125I-protamine in the form of insoluble complexes. This complex formation was not affected by the presence of heparin, suggesting that protamine-plasma protein interaction may be primarily responsible for precipitation of 125I-protamine. To assess the capability of these complexes to obstruct the pulmonary circulation, an in vitro experimental model was developed. Citrated serum, plasma, blood, or saline were allowed to flow through a glass bead column with the help of a peristaltic pump. A pressure transducer positioned before the column allowed pressure measurements at a constant flow rate during the experiment. Mixing of protamine with plasma or blood prior to their passage through the glass bead column resulted in a significant increase in pressure suggesting that the column was being clogged with insoluble complexes. The increase in pressure occurred both in the presence and absence of heparin in plasma or blood. Under identical experimental conditions, the increase in pressure was insignificant when protamine was added to saline or serum regardless of whether heparin was present or absent. This was further confirmed by the use of 125I-protamine. These observations suggest that protamine forms insoluble complexes with certain plasma proteins. Based on these observations, it is hypothesized that following intravenous administration, protamine immediately forms complexes in circulating blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1996

Nitric oxide inhibition attenuates systemic hypotension produced by protamine.

BACKGROUND: Protamine reversal of heparin anticoagulation often causes systemic hypotension, and in vitro studies suggest that this may be mediated by release of nitric oxide from the endothelium. The present investigations were designed to evaluate the direct myocardial effects of protamine and to determine in vivo whether nitric oxide inhibition can prevent hypotension during protamine infusion. METHODS/RESULTS: Protamine sulfate (50 microg/ml) was added to perfusate of eight isolated rabbit heart preparations; in six other preparations, a similar concentration of prolamine was added to heparinized (5 U/ml) Krebs perfusate. Left ventricular developed pressure, maximum rate of pressure rise, and heart rate declined significantly (p < 0.01) in hearts exposed to protamine only (65.0% +/- 6.6%, 55.5% +/- 6.0%, and 87.6% +/- 2.5% of baseline, respectively), whereas protamine added to heparinized perfusate caused little change in developed pressure, maximum rate of pressure rise, and heart rate (85.3% +/- 5.4%, 84.9% +/- 5.5%, and 98.8% +/- 1.6%). To study systemic effects of protamine, we measured hemodynamic parameters in 12 heparinized dogs (150 U/kg). During protamine infusion (1.5 mg/kg intravenously over 30 seconds), mean blood pressure decreased by 46% +/- 7% from baseline (P < 0.05), cardiac output decreased by 38% +/- 4% (p < 0.05), and systemic vascular resistance decreased bv by 14& +/- 9%. After hemodynamic stabilization, Ng-monomethyl-L-arginine (2 mg/kg), a competitive inhibitor of nitric oxide synthesis, was administered to six dogs, and methylene blue (2 mg/kg), an inhibitor of cyclic guanosine monophosphate synthesis, was administered to the remaining six dogs. After treatment with Ng-monomethyl-L-arginine and methylene blue, the second infusion of protamine sulfate caused no significant change in blood pressure or cardiac output. In an additional six dogs, Ng-monomethyl-L-arginine pretreatment (5 mg/kg) blocked the effects of the first dose of protamine. The effect of Ng-monomethyl-L-arginine could be reversed by the addition of (6 mg/kg) L-arginine but not D-arginine. CONCLUSIONS: Protamine-heparin complex does not cause direct myocardial depression but does lead to severe hypotension in vivo. The finding that hypotension can be blocked by inhibitors of the nitric oxide pathway confirms previous in vitro studies indicating that the effects of protamine are mediated, in part, by the vascular endothelium. Further, these studies suggest a novel approach to prevention of hemodynamic complications caused by heparin reversal after cardiopulmonary bypass.

Animals↗
PubMed1994

Mechanical and electrophysiological effects of protamine on isolated ventricular myocardium: evidence for calcium overload.

OBJECTIVE: The aim was to examine in vitro the cellular cardiac effects of protamine, the cationic polypeptide employed to reverse heparin anticoagulation, and to define its mechanisms of action. METHODS: Isometric contractile force and action potential characteristics after rest and at frequencies up to 3 Hz were recorded in guinea pig ventricular papillary muscle. The actions of protamine (10-300 micrograms.ml-1) were compared to those of heparin (10, 30 units.ml-1), and to heparin (10 units.ml-1) neutralised with equivalent (100 micrograms.ml-1) or excess (200 micrograms.ml-1) protamine. The effects of protamine were also examined using (1) muscle rapid cooling contractures (RCC) to assess intracellular Ca2+ stores and (2) the whole cell voltage clamp method to evaluate K+ and Ca2+ currents of isolated ventricular myocytes. RESULTS: Protamine (100-300 micrograms.ml-1) depressed contractions by 35-65% at 3 Hz, whereas contractions were enhanced by 150-500% at lower rates (resting state, 0.5 Hz), with a concomitant rise in resting force. Protamine caused a resting depolarisation from -90 to -76 mV and depressed action potential amplitude. In contrast, heparin altered contractile or action potential characteristics minimally. In 26 mM K+ solution with 0.1 microM isoprenaline, 30-300 micrograms.ml-1 protamine caused dose dependent depression of late peaking force development and slow action potential prolongation. After 15 min rest, when RCC were not normally elicited, rest RCC became prominent in 100-300 micrograms.ml-1 protamine. Effects of heparin with 100 micrograms.ml-1 excess protamine were similar to those of 100 micrograms.ml-1 protamine alone. Voltage clamp of isolated myocytes revealed that 10 micrograms.ml-1 protamine irreversibly decreased current through inwardly rectifying K+ channels (IK1), increased leakage current, and decreased inward Ca2+ current (ICa). CONCLUSIONS: The loss of the normal force-frequency relation, partial depolarisation, rise in resting tension, and appearance of rested state rapid cooling contractures suggest that unbound protamine can lead to excess intracellular Ca2+, mediated by an alteration in membrane ionic conductances.

Action Potentials↗
PubMed1987

Both P1 and P2 protamine genes are expressed in mouse, hamster, and rat.

To date, in mammals except for the mouse and human, only one protamine variant has been isolated from sperm. These mammalian protamines share amino acid sequence homology with mouse protamine 1 (mP1), the tyrosine-containing variant. Southern blot analysis of restriction enzyme digests of hamster and rat liver DNA reveals the presence of sequences homologous to mP1, and also to mouse protamine 2 (mP2) cDNAs. Northern blots of hamster and rat total testis RNA probed with mP2 cDNA confirm that the protamine 2 gene in these species is transcribed into two size classes of mRNA of approximately 830 and 700 nucleotides. However, the relative abundance of the rat and hamster protamine 2 mRNAs (rP2 and hP2) in total testis is approximately 50-fold lower and 2- to 5-fold lower, respectively, than the mouse protamine 2 mRNA. Northern blot analysis of hamster and rat testis polysome gradients demonstrates that although the amount of rP2 mRNA and hP2 mRNA is reduced, both are present on polysomes. The decreased expression of rat and hamster protamine 2 mRNA relative to their protamine 1 counterparts contrasts protamine expression in the mouse testis, where approximately equal amounts of mP1 and mP2 protamine mRNAs are present. These results suggest differential expression of the P1 and P2 protamine genes in three closely related mammals.

Animals↗
PubMed1993

In vitro phosphorylation sites of stallion and bull P1-protamines for cyclic adenosine 3',5'-monophosphate-dependent protein kinase and protein kinase C.

Fish and mammalian protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm P1-protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion P1-protamine, and most probably the homologous Thr-43 in bull P1-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian protamine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of P1-protamines.

Amino Acid Sequence↗
PubMed1998

Protamine reversal of heparin affects platelet aggregation and activated clotting time after cardiopulmonary bypass.

UNLABELLED: Bleeding after cardiopulmonary bypass (CPB) is related to multiple factors. Excess protamine weakens clot structure and decreases platelet function; therefore, an increased activated clotting time (ACT) after protamine reversal of heparin may be misinterpreted as residual heparin anticoagulation. We evaluated the effects of protamine, recombinant platelet factor 4 (rPF4), and hexadimethrine on ACT in blood obtained after CPB. In addition, we examined the effect of protamine on in vitro platelet aggregation. Incremental doses of protamine, rPF4, and hexadimethrine were added to heparinized blood from CPB, and ACTs were performed. Incremental concentrations of protamine were added to heparinized platelet-rich plasma, and aggregometry was induced by adenosine diphosphate (ADP) and collagen. The mean heparin concentration at the end of CPB was 3.3 U/mL. Protamine to heparin ratios >1.3:1 produced a significant prolongation of the ACT that was not seen with rPF4 and was observed only with 5:1 hexadimethrine to heparin ratios. ADP-induced platelet aggregation was reduced with protamine administration > or =1.3:1. Excessive protamine reversal of heparin prolongs ACT and alters ADP-induced platelet aggregation in a dose-dependent manner in vitro. Additional protamine administered to treat a prolonged ACT may further increase clotting time, reduce platelet aggregation, and potentially contribute to excess bleeding after CPB. IMPLICATIONS: We found that excess protamine prolonged the activated clotting time and altered platelet function after cardiopulmonary bypass, whereas heparin antagonists, such as recombinant platelet factor 4 and hexadimethrine, exhibited a wider therapeutic range without adversely affecting the activated clotting time. Approaches to avoid excess protamine or use of alternative heparin antagonists after cardiopulmonary bypass may be beneficial.

Adenosine Diphosphate↗
PubMed1989

Protamine inhibits plasma carboxypeptidase N, the inactivator of anaphylatoxins and kinins.

Protamine given to neutralize heparin after extracorporeal circulation can trigger a catastrophic reaction in some patients. While searching for a biochemical basis for this reaction, protamine was tested as an inhibitor of human plasma carboxypeptidase N (CPN) or kininase I, the inactivator of anaphylatoxins and kinins. Human plasma and CPN purified from human plasma, (Mr = 280 K) or its isolated active subunit (Mr = 48 K) were the sources of enzyme. The hydrolysis of furylacryloyl (FA)-Ala-Lys was measured in a UV spectrophotometer and that of bradykinin and the synthetic C-terminal octapeptide of anaphylatoxin C3a (C3a8) by high performance liquid chromatography. Protamine inhibited the hydrolysis of FA-Ala-Lys by CPN, (IC50 = 3.2 X 10(-7) M); added human serum albumin (30 mg/ml) increased the IC50 to 7 X 10(-6) M. When plasma was the source of CPN, the IC50 was 2 X 10(-6) M. Protamine more effectively inhibited the hydrolysis of bradykinin and C3a8. The IC50 for protamine was 5 X 10(-8) M with CPN and bradykinin, 7 X 10(-8) M with CPN and C3a8 and with the 48 K subunit and bradykinin it was 7 X 10(-8) M of protamine. Heparin competes with CPN for protamine, because in high concentration (18 U/ml) it reverses the inhibition by protamine. Protamine did not inhibit angiotensin I converting enzyme (kininase II) or the endopeptidase 24.11 (enkephalinase). Kinetic studies showed the mechanism of protamine inhibition to be partially competitive; about 10-20% of the hydrolysis of bradykinin by CPN was not inhibited by protamine. Thus, by blocking the inactivation of mediators released in shock, protamine inhibition of CPN may be partially responsible for the catastrophic reaction observed to occur in some patients.

Anaphylatoxins↗
PubMed1995

Protamine does not affect the formation of cGMP or cAMP in pig vascular smooth muscle cells in response to vasodilators.

OBJECTIVES: Protamine has recently been shown to have a direct vasodilator action in isolated vascular tissue. As one possible mechanism for this action, it has been hypothesized that protamine might increase the response of vascular smooth muscle to the endothelium-derived relaxing factor, nitric oxide. In this study, we tested this hypothesis and examined the effect of protamine on other guanosine 3'5'-cyclic monophosphate (cGMP)- and adenosine 3'5'-cyclic monophosphate (cAMP)-dependent processes. DESIGN: Prospective, repeated measures analysis of concentration-response curves. SETTING: Anesthesia research laboratory in an academic medical center. SUBJECTS: Cultured coronary artery smooth muscle cells from pig heart. INTERVENTIONS: Sodium nitroprusside was used to mimic the action of the endothelium-derived relaxing factor by stimulating the soluble guanylyl cyclase and increasing intracellular cGMP. Atrial natriuretic peptide was used to stimulate the particulate guanylyl cyclase. Isoproterenol and forskolin were used to increase intracellular cAMP. The responses to these agents were determined in the presence and absence of protamine. MEASUREMENTS AND MAIN RESULTS: In cultured vascular smooth muscle cells, sodium nitroprusside increased cGMP, the second messenger for endothelium-derived relaxing factor, in a concentration-dependent manner. In cells treated with protamine (32 to 250 micrograms/mL), we could detect no effect of protamine on basal intracellular levels of cGMP until a concentration of 250 micrograms/mL of protamine was used. At this concentration, protamine increased basal cGMP concentrations from 4.2 +/- 0.3 to 9.0 +/- 0.6 pmol/mg protein (p < .001). The response of intracellular cGMP to sodium nitroprusside in cells treated with 250 micrograms/mL or other concentrations of protamine was not different from control. Likewise, we could detect no effect of protamine on intracellular cGMP stimulated with the atrial natriuretic peptide or on cAMP stimulated with the beta-adrenergic receptor agonist, isoproterenol, or with forskolin. CONCLUSIONS: These experiments show that protamine does not alter the responses of the intracellular second messengers, cGMP and cAMP, to the vasodilators sodium nitroprusside, atrial natriuretic peptide, isoproterenol, and forskolin. These results do not support the hypothesis that protamine sensitizes vascular smooth muscle cells to the endothelium-derived relaxing factor, nitric oxide.

Analysis of Variance↗
PubMed1999

Assessing heparin neutralization following cardiac surgery: sensitivity of thrombin time-based assays versus protamine titration methods.

Adequate assessment of heparin neutralization following cardiac surgery is critical in reducing the patient's exposure to protamine. Both excessive protamine and residual heparin have been associated with postoperative bleeding and poor patient recovery. The activated clotting time (ACT) is the preferred intraoperative heparin monitor, while both protamine titration (i.e. a protamine-containing ACT) and thrombin time methods have been used to detect circulating residual heparin after protamine administration. Following initial protamine dosing using the protamine response test (PRT), postoperative monitoring was employed in the operating room prior to transport of the patient to intensive care. Two point-of-care assays, the thrombin time (TT) and the protamine dose assay (PDA), were evaluated to determine their relative heparin sensitivity and their usefulness to quantitate protamine dose. The PDA, which is based on the ACT, was shown in laboratory and clinical studies to detect residual heparin above 0.25 units/ml and to quantify additional minidoses of protamine (as low as 25 mg) required to obtain complete heparin neutralization. Differential evaluation of the TT and heparin neutralized thrombin time (HNTT) was shown in laboratory studies to be more sensitive to small amounts of residual heparin than the ACT. Clinical evaluations confirmed that additional protamine is required in approximately 12% of cardiac surgical cases managed using the PRT system. Both the PDA and TT/HNTT provided useful postoperative assessment of the adequacy of heparin neutralization. The TT/HNTT had slightly improved heparin sensitivity even in the presence of significant fibrinogen loss. These point-of-care assays provide the opportunity to optimize heparin and protamine management in the cardiac surgery patient.

Anticoagulants↗
PubMed2005

Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains.

Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.

Animals↗
PubMed1997

Reactions of protamine with the molecular chaperone DnaK.

Molecular chaperones of the 70 kDa family mediate protein-protein interactions by selectively binding to partially unfolded segments of other proteins in an ATP-dependent activity cycle. Previous investigations of chaperone substrate selectivity have shown that chaperones have a propensity to bind to partially unfolded segments of polypeptides that contain bulky hydrophobic residues. However, recent investigations have shown that 70 kDa chaperones such as DnaK, which is expressed by Escherichia coli, also bind short basic peptides and even polycations. We report here that DnaK specifically binds to the polycation protamine when [protamine]/[DnaK] is near unity, whereas protamine induces the aggregation of DnaK when [protamine]/[DnaK] > or = 20. Complexes between DnaK and protamine were detected using fluorescently labeled protamine (protamine*) in conjunction with high performance size exclusion chromatography. We found that: (i) an unlabeled peptide of known affinity for DnaK partially inhibited the formation of DnaK-protamine* complexes; (ii) Mg-ATP (and Mg-gamma-S-ATP) significantly reduced the affinity of protamine* for DnaK; and (iii) the rate of DnaK-protamine* complex dissociation is highly temperature-sensitive, with apparent activation enthalpies (delta H*) equal to 32 +/- 4 and 28 +/- 1 kcal mol-1 in the absence of added nucleotide and in the presence of ADP, respectively. The results are consistent with the specific binding of protamine* at the (poly)peptide binding site of DnaK. A model is proposed to account for the protamine-induced aggregation of DnaK.

Amino Acid Sequence↗
PubMed1990

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes.

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.

Animals↗
PubMedDate not supplied

A filter device for the prevention of both heparin- and protamine-induced complications associated with extracorporeal therapy.

When extracorporeal blood circulation (ECBC) is used, systemic heparinization is necessary to prevent clotting of the blood in the extracorporeal circuit. However, the high circulating heparin concentration needed often leads to bleeding complications. To avoid these, protamine, a heparin antagonist, is administered at the conclusion of the ECBC procedure to reverse the anticoagulant activity of heparin. Intravenous administration of protamine can cause hypotension and shock. To date, there has been no real alternative to control the bleeding risks associated with systemic use of heparin and the adverse effects resulting from heparin reversal with protamine. A novel approach that might control both the heparin- and the protamine-induced complications is suggested. It consists of placing a blood-compatible filter device containing immobilized protamine (a protamine filter) at the distal end of the ECBC apparatus. The filter removes heparin after heparin serves its anticoagulant purpose in the extracorporeal circuit and before blood is returned to the patient. The filter also allows for an external protamine treatment. Since protamine toxicity results from the direct contact of protamine with cells of the liver, lungs, and other organ tissues, the use of an external protamine treatment would minimize it. Protamine was covalently immobilized onto a cellulosic hollow fiber bundle obtained from a clinically used hemodialyzer. The bundle was accessed to the vascular system of a dog by femoral artery and vein cannulation. In in-vivo experiments the protamine-bound fiber bundle not only removed heparin from the extracorporeal circuit, but also caused no clinically significant hemodynamic change in the animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMedDate not supplied

A protamine filter for extracorporeal heparin removal. Development, testing, blood compatibility evaluation, and future direction.

The authors previously developed a filter device containing immobilized protamine (termed "protamine filter") that could be used to remove heparin during extracorporeal perfusion. In vivo studies involving dogs showed that the protamine filter removed more than 50% of heparin from the animals' blood circuit in less than 20 min. In addition, the use of the protamine filter did not elicit statistically significant protamine induced hemodynamic and thrombocytopenic responses. Biocompatibility of the protamine filter was also evaluated, with the focus on its effect on the coagulation cascade, the complement system, and the blood antithrombin III levels. Results showed that heparin adsorbed to the protamine coated surface retained 20% of its original activated partial thromboplastin time activity, rendering the coated surface antithrombotic. Activation of the coagulation system by the protamine coated membrane and the untreated cellulose membrane, as measured by the elevation of prothrombin fragment F1 + 2 levels, was statistically identical. The CH50 hemolytic assay showed that the protamine coated membrane produced a reduction of 1.2 +/- 0.8% of the total complement levels, as compared to 9.4 +/- 1.6% by the untreated membrane. In addition, the change in C3a des Arginine levels after 30 min of circulation was 1.5 +/- 0.2 mg/ml by the protamine filter, as compared to 2.1 +/- 0.1 mg/ml by the untreated membrane. Unlike native heparin that would bind with antithrombin, heparin adsorbed on the protamine coated surface was devoid of such activity, and produced no depletion of circulating antithrombin. Because of the limited capacity of the protamine filter, the future system is envisioned to consist of two filters; while one filter is removing heparin the other will be regenerated. With a recently developed heparin sensor, it should be possible to design a sensor directed, biofeedback, two filter heparin removal system.

Adsorption↗