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PubMed1987

Intracarotid infusions of protamine sulfate disrupt the blood-brain barrier of rabbits.

This study examined the effect of intracarotid infusions of protamine sulfate on the blood-brain barrier of the rabbit. Evans blue, which binds tightly to serum albumin, served as the marker for blood-brain barrier disruption. Brains from 12 of 14 rabbits receiving 30-50 mg of protamine sulfate in an intracarotid infusion over 3 min demonstrated intense Evans blue-albumin staining in the distribution of the infused internal carotid artery. In contrast, brains from 5 animals receiving 0.9% saline and from 5 animals receiving 50 mg of protamine sulfate by the intravenous route showed no evidence of blood-brain barrier disruption. Premixing protamine sulfate with heparin prior to intracarotid administration greatly reduced or abolished Evans blue staining of the brain as did delayed administration of the marker dye. Thus, the interaction of the positively charged protamine molecule with the vascular endothelium of cerebral capillaries transiently alters the permeability of the blood-brain barrier in the rabbit.

Animals↗

PubMed1990

Albumin content in brain and CSF after intracarotid infusion of protamine sulfate: a longitudinal study.

The endogenous serum albumin content was determined by immunoelectrophoresis in brain and cisternal CSF 1, 24, and 72 h after a transient opening of the blood-brain barrier. Protamine sulfate, 5 mg in 100 microliters 0.9% NaCl, was infused during 30 s into the internal carotid artery via a catheter in the external carotid artery in conscious rats. The albumin content in CSF was 0.08 +/- 0.03 g/liter before protamine infusion and 0.09 +/- 0.02 g/liter in rats infused with saline only. The levels were significantly increased one and 24 h after protamine infusion (0.37 +/- 0.19 and 0.23 +/- 0.09 g/liter, P less than 0.001) but not at 72 h (0.14 +/- 0.05 g/liter). The albumin content in the right (injected) hemisphere decreased with time but was significantly higher than that in the left hemisphere at all times (P less than 0.001 1 and 24 h after protamine; P less than 0.01 at 72 h). There was no correlation between the albumin contents in brain and CSF. Pretreatment with dixyrazine, a phenothiazine derivate, significantly reduced the protamine-induced leakage of endogenous serum albumin into brain and CSF.

Animals↗

PubMed1996

Mast cell chymase in complex with heparin proteoglycan is regulated by protamine.

Protamines are polycationic proteins that are widely used for neutralisation of the anticoagulant action of heparin. However, several reports have shown adverse, mast cell-dependent reactions to protamine. The exact mechanism by which protamine causes these adverse effects is not clear. In the present study, the possibility that protamine may influence mast cell chymase function was investigated. Mast cell chymase is in vivo recovered in a macromolecular complex with heparin proteoglycan, and this interaction is essential for expression of optimal enzymatic activity. Protamine was shown to strongly reduce the activity of mast cell chymase by a mechanism that involved displacement of the chymase from heparin proteoglycan.

Animals↗

PubMed1988

Interaction of glucocorticoid receptor from rat liver with protamine and arginine.

The nontransformed glucocorticoid receptor (GR) from rat liver was found to bind to protamine-Sepharose and could be recovered by a salt gradient without a change in molecular configuration. The nontransformed GR also bound to arginine-Sepharose, but the transformed GR did not bind to either resin. Ligand-free GR interacted with both resins and was eluted without loss of its steroid binding ability. The bindings of GR to protamine- and arginine-Sepharose were saturable. The apparent dissociation constants of GR on protamine-Sepharose varied from 0.34 nM (-molybdate) to 0.68 nM (+ 10 mM molybdate) and those on arginine-Sepharose were 1.99 nM (-molybdate) and 0.65 nM (+ 10mM molybdate), respectively. The maximum binding capacity was achieved by arginine-Sepharose in the absence of molybdate. Higher salt concentrations (0.5 M NaCl) were required to elute GR from protamine-Sepharose than from arginine-Sepharose (approx 0.03 M NaCl). However, the effectiveness of several salts for the elution of GR was consistent in both resins as follows; MgCl2 = CaCl2 = Na2WO4 greater than (NH4)2SO4 = Na2MoO4 greater than arginine-HCl greater than lysine-HCl greater than KCI = NaCl. These results suggest that GR interacts with arginine residues in protamine. Chromatography using these resins resulted in 7-10-fold purification of occupied and unoccupied nontransformed GRs.

Animals↗

PubMed1983

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice.

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.

Animals↗

PubMed1983

Comparative study of protamine chloride and sulphate in relation to the heparin rebound phenomenon.

The heparin rebound phenomenon is observed when protamine sulphate, but not protamine chloride, is employed for the neutralization of heparin. On investigating the stability of several protamine compounds towards the protaminolytic activity of human plasma, we found that while protamine chloride was stable, both the sulphate and the phosphate were degradeable. The free base showed intermediary stability which persisted upon its conversion to either chloride or sulphate. Likewise, conversion of the sulphate into the chloride by means of an ion exchange column, did not alter its sensitivity. Apparently, the stability of protamine derivatives is not influenced by the specific anion bound to them but is rather acquired in the course of the manufacturing procedures involved in their preparation.

Blood Coagulation↗

PubMed1988

Does protamine chloride neutralize low molecular weight heparin sufficiently?

The heparin neutralizing properties of protamine chloride on conventional heparin (porcine mucosa) and on low molecular weight heparin (Kabi 2165) were studied in vitro. Protamine chloride neutralized 99% of the delaying effect of conventional heparin on the activated partial thromboplastin time, whereas only 70% of the effect of low molecular weight heparin was neutralized. The neutralizing effect of protamine chloride on the inhibition of factor Xa (clot test) was 95% for conventional heparin and 55% for low molecular weight heparin, whereas the effect of both heparin preparations on the thrombin inhibition could be completely neutralized. We conclude that conventional heparin is neutralized more effectively in vitro by protamine chloride than is the low molecular weight heparin. The findings do not exclude that protamine chloride is able to suppress in vivo bleedings caused by low molecular weight heparin.

Blood Coagulation↗

PubMed1982

Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa.

1. Sperm nuclei were isolated and purified from boar semen by a procedure involving differential solubilization of sperm tail and acellular materials by brief exposure to reducing reagent in the presence of cationic detergent, and sedimentation through 60% sucrose. The weight ratio of DNA:RNA: total protein: protamine in this preparation was 1.00: 0.02: 1.05: 0.75, and the molar ratio of phosphorus to arginine was 1.12. 2. Boar protamine was extracted with cold acid from ethanol precipitate of reduced and carboxymethylated nuclei in 6 M guanidine hydrochloride and purified by ion-exchange chromatography on CM-cellulose. The molecular weight of the protamine was estimated to be 6600 by the gel filtration method. The protamine consisted of a single amino terminus alanine and either half-cystine or arginine as carboxy terminus, and was composed of Thr, Ser3, Pro2, Ala2, Val2, Ile, His, Half-cystine9-10 and Arg26 . 3. Chymotryptic digestion gave rise to a single amino-terminal peptide, Ala-Arg-Tyr, and two carboxy-terminal peptides, Thr-Val-Ile-Arg-Cys-Arg2-Cys and Thr-Val-Ile-Arg-Cys-Arg2, which confirmed the heterogeneity of the protamine at the carboxy-terminal end.

Amino Acid Sequence↗

PubMed1987

Protein kinase C phosphorylation of protamine is Ca2+ independent, but the addition of DNA renders it Ca2+ dependent.

Protamine is a unique substrate of protein kinase C for its Ca2+-independent phosphorylation. The interaction between protein kinase C and protamine and the effect of DNA on the interaction was studied. Protein kinase C was retained in a protamine-immobilized Sepharose 4B column, even in the absence of Ca2+ and was eluted with ammonium sulfate or L-arginine. The eluted enzyme was fully activated by phosphatidylserine alone, when protamine was used as substrate. When DNA was included in the assay system, the activity elicited by phosphatidylserine alone was inhibited. The DNA effect on the activity in the presence of both Ca2+ and phosphatidylserine was much lower than on the activity elicited by phosphatidylserine alone, thereby demonstrating the Ca2+ sensitivity of protamine phosphorylation.

Animals↗

PubMed1990

Genomic sequences of human protamines whose genes, PRM1 and PRM2, are clustered.

Protamines are small, arginine-rich proteins involved in the condensation of sperm chromatin. Using cDNA clones, we have isolated the genes for both human protamines, i.e., protamine 1 (PRM1) and protamine 2 (PRM2), from a human cosmid library. Each of these genes contains a single intron consisting of 91 and 163 bp, respectively. From the 5'-noncoding region of PRM1 664 bp and from the 5'-noncoding region of PRM2 902 bp were determined. Both genes contain typical TATAA and CAAT boxes at conventional distances from the transcription start points, which by using primer extension experiments could be assigned to nucleotides -91 and -110 for PRM1 and PRM2 genes, respectively. Comparison of the 5'-noncoding regions of PRM1 and PRM2 genes reveals 12 different motifs in common, 8 of which are clustered in both genes and could reflect regulatory elements for testis- and spermatid-specific gene expression. Both human genes have been found to be clustered at a distance of 4.8 kb. Comparison of the genomic organization of human and mouse protamine genes revealed greater similarities between the two in the 5'-noncoding region.

Amino Acid Sequence↗

PubMed1990

Phospholipid independent phosphorylation of protamine by protein kinase C: effects of polyanions.

The ability of purified protein kinase C (PKC) to phosphorylate protamine sulphate was found to be totally independent of phospholipid cofactors, whereas the phosphorylation of protamine free base was markedly increased by the presence of phosphatidylserine (PS). The hypothesis of an activation of PKC by the sulphate groups of protamine sulphate was confirmed by the high phosphorylation of protamine free base in the presence of non-peptide polyanionic compounds, such as glycoaminoglycans or polynucleotides. The catalytic fragment of PKC supported protamine base phosphorylation with the same polyanionic dependency. Light scattering intensity measurements showed that this phosphorylation correlated to the substrate/cofactor aggregation. These data support the view that apparent phospholipid-independent activation of PKC results from the formation of aggregates in the assay and this could result in the non-specific activation of this enzyme through its catalytic domain.

Animals↗

PubMed2026

Protamine gene expression is associated with sperm motility in rams: An integrative experimental and gene network analysis.

Protamine 1 (PRM1) and protamine 2 (PRM2) are essential regulators of sperm chromatin condensation and genome integrity, and their dysregulation has been associated with impaired male fertility. However, their role in rams remains insufficiently characterized. This study investigated the relationship between protamine gene expression and semen quality in rams and explored their potential upstream regulatory mechanisms using gene regulatory network (GRN) analysis. Fifteen ejaculates from five rams were analyzed. Based on total sperm motility using computer-assisted sperm analysis (CASA), ejaculates were classified into a high-motility group (n = 8) and a low-motility group (n = 7). PRM1 and PRM2 expression levels were quantified by RT-qPCR. Following normality confirmation (p > 0.05), parametric tests were applied using the ejaculate as the biological experimental unit. Samples with reduced motility showed significantly lower expression of both protamines (p < 0.01). Moreover, progressive sperm motility was strongly correlated with both PRM1 (r = 0.71, p = 0.019) and PRM2 (r = 0.69, p = 0.03) transcript levels. Cross-species GRN inference using scGeneRAI and a reference human spermatogenesis dataset identified several hypothesis-generating candidate transcription factors, including HMGB4, HMGB1, H2AFZ, NKX6-1, and SMC3, consistently supported across multiple bootstrap resampling runs. These findings demonstrate a strong association between protamine expression and sperm motility in rams. While the identified candidate regulators provide a valuable framework for future species-specific validation, they also highlight promising candidate molecular biomarkers of male fertility in livestock.

Gene regulatory networks↗

PubMed2005

Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines.

Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

Animals↗

PubMed1984

Complement (C3, C4) consumption in cardiopulmonary bypass, cardioplegia, and protamine administration.

Anaphylatoxins produced by complement activation have been postulated to be responsible for postperfusion syndrome and protamine hypotension in patients undergoing cardiac surgical procedures. The consumption of serum complement components C3 and C4, which reflects the classic and alternate pathway activations of the complement system, was studied in 22 patients undergoing cardiac operations. Prior to the onset of cardiopulmonary bypass, the complement levels were within normal range. Rapid reduction in both C3 and C4 within minutes of cardiopulmonary bypass indicated rapid complement activation. Such a reduction in complement levels could not be accounted for by either hemodilution or transfusion of complement-poor blood. Aortic cross-clamping and cold potassium cardioplegia followed by myocardial reperfusion did not lead to further consumption of C3 and C4. Slow intravenous infusion of protamine sulfate after cardiopulmonary bypass did not change C3 and C4 levels significantly in our patients, although protamine and heparin-protamine complex have been shown to activate complement components in vitro. In another group of 9 similar cardiac surgical patients, C3 and C4 were found to return to normal levels within 24 hours after operation. This study thus confirms the rapid activation of the complement system by cardiopulmonary bypass but fails to demonstrate further activation of the complement system by cardioplegia or protamine administration.

Adult↗

PubMed1997

Changes in protamine 1 distribution in human sperm nucleus during in vitro sperm-oocyte interaction: an immunoelectron microscopic study.

OBJECTIVE: To determine whether the process of sperm nuclear destabilization would begin before sperm-oocyte fusion in humans. DESIGN: Changes in the distribution of human protamine 1 were investigated in human spermatozoa from the ejaculate, in spermatozoa selected by swim-up or Percoll techniques, and in spermatozoa bound to zona pellucida (ZP) from oocytes that failed to fertilize in an IVF program. SETTING: Center for Infertility and Assisted Reproductive Technology, and university departments. PATIENT(S): Fifteen couples undergoing an IVF program. INTERVENTION(S): Women underwent a similar superovulation induction protocol that consisted of GnRH agonist associated with hMG. MAIN OUTCOME MEASURE(S): Comparative immunoelectron microscopic study of sperm nucleus labeling with an anti-human protamine 1 specific protamine monoclonal antibody. RESULT(S): After selection by swim-up or by Percoll, spermatozoa show a significantly lower nuclear labeling than in the ejaculate. After binding to the ZP, labeling increases, more in spermatozoa selected by swim-up than by Percoll, but, after Percoll selection, labeling in zona-bound spermatozoa is lower than in the ejaculate. CONCLUSION(S): In humans sperm binding to the ZP induces differences in the accessibility of the anti-human protamine 1 antibody, which are consistent with structural rearrangements of the DNA-nucleoproteins complex. These modifications must be a prelude to sperm decondensation, protamines replacement by histones, and subsequent reactivation of the sperm genome in the oocyte.

Cell Nucleus↗

PubMed1999

Temporal expression of the transgenic human protamine gene cluster.

OBJECTIVE: To ascertain the fidelity of expression of the genes from the transgenic human sperm-specific nuclear packaging protamine-1-->protamine-2-->transition protein-2 (PRM1-->PRM2-->TNP2) locus. DESIGN: Controlled human transgene study. SETTING: Basic science laboratory. ANIMAL(S): Age-matched transgenic and nontransgenic mice. INTERVENTION(S): Transgenic mice containing the human protamine locus were mated. One testis from each offspring was frozen at -80 degrees C and the other was preserved in formalin. MAIN OUTCOME MEASURE(S): The temporal expression of the human and mouse protamines was evaluated by Northern blot analysis. Orientation of the transgenic locus was determined by Southern blot analysis. Tissue morphology was assessed histologically. RESULT(S): Conservation of transgenic morphology was confirmed. Head-to-tail integration of the PRM1--> PRM2-->TNP2 locus was shown. Temporal expression of the mouse and human protamine genes was maintained in the transgenic state. CONCLUSION(S): These results show that the head-to-tail concatomer of the PRMI-->PRM2-->TNP2 locus contains all the necessary elements for appropriate temporal expression while maintaining testicular structure and function.

Aging↗

PubMed1995

Reversal of heparin anticoagulation by recombinant platelet factor 4 and protamine sulfate in baboons during cardiopulmonary bypass.

The ability of recombinant platelet factor 4 and protamine to neutralize heparin after cardiopulmonary bypass was compared in anesthestized baboons. Clotting titration curves of heparinized baboon blood demonstrate an anticoagulant effect of protamine that is not seen with recombinant platelet factor 4. Neither drug caused meaningful changes in central pressures or cardiac output within 30 minutes after injection. After 30 minutes of cardiopulmonary bypass, recombinant platelet factor 4 normalized thrombin times and activated partial thromboplastin times within minutes of injection, but protamine did not. Neither drug altered bleeding times. Recombinant platelet factor 4 caused a species-specific leukopenia in baboons and significantly increased activated complement protein 3 (C3a) more than protamine. However, the increase in plasma C3a was small and neither drug caused a significant increase in plasma neutrophil elastase-alpha 1 proteinase inhibitor complex. We conclude that recombinant platelet factor 4 is effective and safe in baboons, does not have an anticoagulant effect with excess concentration, and reverses in vivo heparin more rapidly than protamine. The data support progression to a clinical trial.

Animals↗

PubMed1996

Protamine-induced hypotension in heart operations: application of the concept of ventricular-arterial coupling.

Protamine sulfate often causes hypotension during heparin neutralization. The concept of ventricular-arterial coupling was applied to determine whether a negative inotropic effect or a vasodilating effect of protamine was the major contributing factor to this hypotension. Thirty-five patients who underwent cardiac operations were studied during operation by measuring instantaneous left ventricular pressure and aortic flow to examine the end-systolic pressure-volume relationship. We obtained end-systolic elastance and effective arterial elastance values in a beat-to-beat fashion with a single-beat estimation method. In 28 of the 35 patients (80%), mean arterial pressure decreased more than 10 mm Hg with protamine infusion. Parameters were compared at the following three points: before a decrease in mean arterial pressure (control), at maximally decreased mean arterial pressure (maximum), and at a middle point between control and maximum values (midpoint). At both midpoint and maximum, mean arterial pressure decreased significantly (control 79.6 +/- 12.6 mm Hg, midpoint 66.5 +/- 10.8 mm Hg, maximum 52.7 +/- 9.9 mm Hg; p < 0.01). Similar changes were observed in effective arterial elastance (control 2.00 +/- 0.75 mm Hg/ml, midpoint 1.60 +/- 0.53 mm Hg/ml, maximum 1.31 +/- 0.46 mm Hg/ml; p < 0.01). Although the decrease in end-systolic elastance at midpoint (control 3.08 +/- 1.61 mm Hg/ml, midpoint 2.92 +/- 1.68 mm Hg/ml) did not reach statistical significance, end-systolic elastance significantly decreased at maximum (2.63 +/- 1.46 mm Hg/ml; p < 0.01). Continuous measurements showed that the decreases in mean arterial pressure and effective arterial elastance always preceded the depression of end-systolic elastance and that afterload reduction by vasodilating effect of protamine was the mechanism most likely to have initiated the hypotension. Delayed decrease in contractility may be ascribed to reduced coronary perfusion pressure caused by vasodilation or to a direct effect of protamine.

Adult↗