Anti-protamine Antibodies Research Articles

Heparin-induced thrombocytopaenia diagnostic testing after adult cardiac surgery: a single-centre experience and development.

To assess the incidence and outcome of heparin-induced thrombocytopaenia (HIT) in patients after cardiac surgery on cardiopulmonary bypass (CPB) and to review the time course of platelet counts and the use of different immunological diagnostic tests. All patients ≥18 years of age who underwent CPB from 2006 to 2015 and who were postoperatively admitted to our cardiac intensive care unit (ICU) were included in this retrospective study. Screening for heparin/platelet factor-4 antibodies was performed using an antibody test, which was later replaced with a screening test specific for IgG antibodies without IgA/IgM cross-reactivity. The enzyme immunoassay (EIA) for the detection of antibodies of all immunoglobulin classes against heparin/PF4 complexes was replaced with an IgG-specific EIA. HIT was confirmed by a heparin-induced platelet aggregation test until 2014. Among 4978 patients admitted between 2006 and 2015, 539 (11%) patients were evaluated for HIT. Patients were excluded because of age <18 years (n = 9), non-cardiac surgery without CPB (n = 10) or incomplete data (n = 3). Of the remaining 517 patients, 43 (8.3%) patients were HIT-positive. HIT incidence was 0.86%. The proportion of HIT-positive patients was similar in men and women (8.4% and 8.2%, respectively). Men and women with suspected HIT also had similar in-hospital mortality (odds ratio ≈ 1; P = 0.926). The incidence of HIT was lower in our study than previously reported. Novel immunological tests have improved to specifically detect IgG antibodies. Furthermore, they are able to detect anti-protamine antibodies, which may be present in patients with high clinical probability of testing negative for HIT. Incidence and clinical relevance of heparin/protamine antibodies will be subjects of future investigation.

New monoclonal antibodies specific for mammalian protamines P1 and P2

ABSTRACTThe expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies.Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline

Serological features of antibodies to protamine inducing thrombocytopenia and thrombosis.

A significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events. We extensively investigated the serological features of protamine antibodies, which developed in six patients who were clinically suspected to have heparin-induced thrombocytopenia (HIT). Three patients had thrombotic events. Sera were tested by four different commercially available immunoassays, a heparin-platelet aggregation test, and for their binding properties to heparin, platelet factor 4 (PF4), complex heparin-PF4, protamine, and protamine complex with heparin. Sera from four patients were also tested for the capability to induce platelet activation and the formation of platelet-monocyte heterotypic aggregates. The ELISA assay Zymutest HIA was strongly positive in all cases, the HPIA Asserachrome was borderline, and the gel centrifugation test PaDGIA was positive in two tested patients. Platelet aggregation tests were negative. Using a variation of the Zymutest HIA we demonstrate that IgG antibodies bound only to protamine or protamine complex with heparin, but not to heparin or PF4 only. Sera-induced platelet P-selectin expression and the formation of platelet-monocyte aggregates. Blood samples from one patient proofed positive concomitantly with the thromboembolic event. However, serological characteristics did not differ between antibodies associated with thromboembolic events from those without. These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide.

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery.

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, Bandicota indica

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.

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

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

Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (II): in vitro evaluation of efficacy and toxicity.

Patients undergoing anticoagulation with heparin or low molecular weight heparin (LMWH) require a superior antidote that possesses more selective biological actions and a better safety profile than protamine. We had previously developed 2 low molecular weight protamine (LMWP) fractions (TDSP4 and TDSP5) from thermolysin-digested protamine as potential nontoxic, heparin-neutralizing agents. In this, the second article in this series, studies focused on in vitro evaluation of heparin/LMWH-neutralizing efficacy and putative toxicity. These LMWP fractions, particularly TDSP5, were effective and fully capable of neutralizing a broad spectrum of heparin-induced anticoagulant activities (ie, aPTT, anti-Xa, and anti-IIa activities). Additionally, these LMWP fractions could neutralize the activities of commercial LMWH. As assessed by the anti-Xa assay, TDSP5 was as effective as, although less potent than, protamine in reversing the activity of Mono-Embolex (molecular weight 5000-7000) and 2 other different sizes (molecular weight of 3000 and 5000 d) of LMWH preparations. Furthermore, compared with protamine, TDSP5 exhibited a much-reduced toxicity and thus an improved safety profile, as reflected by its reduced ability to activate the complement system and cross-react with the antiprotamine antibodies, which are 2 primary indices of protamine toxicity.

Reduced Reactivity Towards Anti-Protamine Antibodies of a Low Molecular Weight Protamine Analogue

Protamine sulfate has been used routinely following cardiovascular surgery to reverse the anticoagulant activity of heparin. This clinical use, however, at times, is associated with adverse effects ranging from mild hypotension to severe, idiosyncratic fatal reactions [1–4]. In general, protamine responses can be distinguished into either via a nonimmunologic pathway or via an immunoglobin-mediated pathway. Adverse reactions produced by the first mechanism are more common and can be largely aborted using conventional pressor agents and slow protamine administration. Anaphylactic-type responses produced by the second pathway (termed “Type I protamine hypersensitivity”), however, are unpredictable, life threatening, and not readily manageable by clinical intervention. More than 100 protamine-related deaths have been reported in the literature that is attributed to this mechanism [1–4]. Owing to this immunopathogenic function of protamine, a large population of diabetic patients is at high risk to acquire severe protamine response following heparin reversal, primarily due to the routine use of protamine zinc insulin (PZI) or neutral protamine Hagedorn (NPH) by such patients. Indeed, several cases of anaphylaxis to protamine masquerading as insulin allergy have been reported to occur in protamine-containing insulin-dependent diabetes [5,6].

Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine.

To avoid bleeding complications, protamine is routinely used after cardiovascular surgery to neutralize the anticoagulant function of heparin. However, its clinical use is associated with adverse and sometimes fatal reactions. Based on literature review of the mechanism of heparin neutralization and protamine induced immunologic toxicity, we propose the following hypothesis: If a chain shortened low molecular weight protamine (LMWP) containing the heparin neutralizing domain could be derived from native protamine, it could be a potent and yet nontoxic heparin antagonist. In this study, we present results to validate this hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weight of approximately 1,100 daltons were successfully prepared by enzymatic digestion of protamine with thermolysin. In vitro studies show that such LMWP fragments completely neutralized the anticoagulant functions of heparin and LMWH, based on the anti-Xa chromogenic and aPTT clotting time assays. In vivo results reveal that although injection of protamine to mice led to obvious production of anti-protamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, these LMWP fragments exhibited a markedly reduced antigenicity and cross-reactivity toward the mice anti-protamine antibodies.

Low Molecular Weight Protamine: A Potential Nontoxic Heparin Antagonist

Protamine sulfate is the universal clinical antagonist to heparin and is used routinely after cardiovascular surgery to neutralize the anticoagulant function of heparin. Its clinical use, however, is associated with adverse effects including idiosyncratic fatal reactions. An examination of the mechanism of heparin neutralization and protamine toxicity suggests that the reversal of heparin anticoagulation may only require a small arginine-rich fragment of protamine to electrostatically dissociate antithrombin III from its binding to a specific pentasaccharide sequence in heparin. A review of literature indicates that chain-shortened peptide fragments derived from their parent proteins are normally accompanied with significantly reduced antigenicity and immunogenicity, which are two primary contributing factors to protamine-induced life-threatening toxic effects via an immunoglobulin-mediated pathway. Based on these observations, we propose our general hypothesis: if a chain-shortened low molecular weight protamine fragment containing the heparin-neutralizing domain could be derived directly from a native protamine, it could be a potent and nontoxic heparin antagonist. In this article, we present our experimental results to support the above hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weigh of approximately 1.1 kDa were prepared successfully by enzymatic digestion of native protamine with thermolysin. In vitro studies demonstrated that such LMWP fragments completely neutralized the anticoagulant functions of heparin, based on the anti-Xa chromogenic assay and aPTT clotting time assay. Our in vivo results indicated that while administration of protamine to mice led to obvious production of antiprotamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, the LMWP fragments showed a significantly reduced antigenicity or, in other words, cross-reactivity towards the mice antiprotamine antibodies produced by the administration of protamine.

Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine.

An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualized. The most compact parts of the nuclei are not yet stained, when the less compact parts have already been destroyed. This gives rise to a sperm-specific pattern which corresponds to the enhancement of microheterogeneities present in all sperm nuclei and to the local depolymerisation of the nuclear material during hydrolysis. The depolymerised parts of nuclei sit over the sections, and they also bind uranyl acetate, ethidium bromide and anti-protamine antibodies. Therefore, in sperm nuclei, the Feulgen-like staining at the ultrastructural level does not reveal the true DNA distribution. However, the amount of staining can be quantified to evaluate sperm chromatin compaction.

Prior Vasectomy and Anaphylaxis Following Protamine: No Cause and Effect

Adourian et al. in a case report identify a patient with a previous vasectomy antiprotamine IgG antibodies and 2 hypotensive episodes after receiving protamine. In presenting the case the authors imply that the patient suffered 2 anaphylactic reactions to protamine that IgG antibodies to protamine were a response to prior vasectomy. This author however argues that the case report does not support these implications and that any association among these observations remains speculative. He takes issue with the authors contentions that both reactions to protamine were anaphylactic and that IgG antibodies to protamine produce severe reactions to protamine. Furthermore although the patient could have produced antiprotamine antibodies during his vasectomy and from his exposure to intravenous protamine during catheterization the authors do not state why the vasectomy is to blame. Current evidence including the Adourian case is simply inadequate to implicate prior vasectomy as a risk factor for severe reactions to protamine.

Effects of antibodies to sperm surface fertilization antigen-1 on human sperm-zona pellucida interaction

To investigate the effects of antibodies to well-defined sperm surface antigens (the fertilization antigen [FA-1] and germ-cell antigen [GA-1]) and nuclear antigen (protamine) on human sperm-zona interaction. Number of total and acrosome-reacted human sperm bound to the human zona pellucida and the sperm movement characteristics assessed by computer-aided sperm analysis were evaluated after incubation of sperm with the antibodies. Academic research environment approved by the Institute Review Board. Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. Human oocytes were stored in salt solution at -80 degrees C until used. Spermatozoa were treated with the antibodies to various sperm antigens. Total and acrosome-reacted sperm bound to zona pellucida and sperm movement characteristics were evaluated after 3 to 5 hours of incubation of the antibodies with human sperm. Anti-FA-1 antibodies significantly reduced human sperm fusion with zona-free hamster oocytes and sperm binding to the human zona pellucida but did not affect binding of acrosome-reacted sperm and sperm movement characteristics. Anti-GA-1 and antiprotamine antibodies did not affect sperm-oocyte interaction, acrosomal reaction, or sperm motility. Antibodies to FA-1 but not to GA-1 and protamine inhibit human sperm-zona interaction.

Safety of protamine sulfate administration in vasectomized men

The majority of vasectomized men develop antibodies against different sperm antigens, including protamine. Due to the fact that salmon protamine is used clinically for heparin reversal and that a cross-reactivity has been observed between human and salmon protamine, vasectomized men may be at risk for adverse reactions to protamine sulfate. In order to explore this possibility, serum samples were analyzed for the presence of anti-sperm and anti-protamine antibodies in 20 vasectomized and 20 non-vasectomized men (controls) about to undergo major heart surgery requiring heparin reversal. The patients were closely monitored for any possible reaction following the injection of protamine. Anti-sperm antibodies were detected in the serum of 15 of the vasectomized men whereas only two of the non-vasectomized subjects had this type of antibody. Twenty-five percent of the vasectomized men presented with anti-protamine antibodies; in the control group, none of the patients had developed this type of antibody. Following the administration of protamine sulfate, none of the patients in either group showed any adverse reaction to the drug. It is concluded that vasectomized men are not at increased risk towards adverse reactions following the injection of protamine sulfate.

Vasectomy: Consequences of Autoimmunity to Sperm Antigens

Data from studies examining the effects of vasectomy in a large number of nonhuman primates vasectomized for periods ranging up to 14 years are summarized, and these findings and speculations are used as a framework with which to review the subject of autoimmunity and vasectomy. Attention is directed to autoimmunity to sperm antigens following vasectomy (factors affecting antisperm antibody levels, characteristics of circulating antisperm antibodies, antisperm antibodies in seminal plasma, and cellular immunity following vasectomy), and immunopathology of antisperm autoimmunity (local effects on the male reproductive tract and systemic effects on the male reproductive tract). The 6 hypotheses that have been advanced to explain individual variations in dynamics and types of antisperm antibodies produced following vasectomy are reviewed. 3 tests are commonly used to detect free antisperm antibodies after vasectomy: 1) the spermagglutination test; 2) the sperm immobilization test; and 3) the immunofluorescence test. Spermagglutinating (SA) antibodies, the most common type of antibody produced after vasectomy, occur in approximately 2/3 of vasectomized men and in a majority of vasectomized rhesus monkeys. Sperm-immobilizing (SI) antibodies are also produced in a large percentage (40%) of vasectomized men and rhesus monkeys. About 30% of vasectomized men also have antiprotamine antibodies.