Isolation and Purification of Protamine from the Cultured Takifugu flavidus and Its Physicochemical Properties.

Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured Takifugu flavidus is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of Takifugu flavidus and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured T. flavidus were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly ß-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.

Protamine dosing and its impact in cardiac surgery transfusion practice-A retrospective bi-institutional analysis.

BACKGROUND: Bleeding after cardiac surgery is common and continues to require 10-20% of the national blood supply. Transfusion of allogeneic blood is associated with increased morbidity and mortality. Excessive protamine in the absence of circulating heparin after weaning off CPB can cause anticoagulation and precipitate bleeding. Hence, adequate dose calculation of protamine is crucial yet under evaluated. STUDY DESIGN: Retrospective cohort study. METHODS: We conducted a retrospective bi-institutional analysis of cardiac surgical patients who underwent cardiopulmonary bypass (CPB)-assisted cardiac surgery to assess the impact of protamine dosing in transfusion practice. Total 762 patients were identified from two institutions using electronic medical records and the Society of Thoracic Surgery (STS) database who underwent cardiac surgery using CPB. Patients were similar in demographics and other baseline characteristics. We divided patients into two groups based on mg of protamine administered to neutralize each 100 U of unfractionated heparin (UFH)-low-ratio group (Protamine: UFH ≤ 0.8) and high-ratio group (Protamine: UFH > 0.8). RESULTS: We observed a higher rate of blood transfusion required in high-ratio group (ratio >0.8) compared with low-ratio group (ratio ≤0.8) (p < .001). The increased requirement was consistently demonstrated for intraoperative transfusions of red blood cells, plasma, platelets, and cryoprecipitate. CONCLUSION: High protamine to heparin ratio may cause increased bleeding and transfusion in cardiac surgical patients. Protamine to heparin ratio of 0.8 or lower is sufficient to neutralize circulating heparin after weaning off cardiopulmonary bypass.

Evaluation of Activated Clotting Times With a Heparin Reversal Protocol Designed to Conserve Protamine During Drug Shortages: A Retrospective, Cohort Study.

OBJECTIVE: To retrospectively evaluate a protamine conservation approach to heparin reversal implemented during times of critical shortages. This approach was aimed at maintaining access to cardiac surgical services. SETTING: In-patient hospital setting. PARTICIPANTS: Eight hundred-one cardiac surgical patients>18 years old. INTERVENTIONS: Patients undergoing cardiac surgery who received >30,000 U of heparin were given a single fixed vial protamine dose of 250 mg or a standard 1 mg of protamine to 100 U of heparin ratio-based dose to reverse heparin. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was differences in post-reversal activated clotting times between the 2 groups. The secondary endpoint was differences in the number of protamine vials used between the 2 reversal strategies. The first activated clotting times values measured after initial protamine administration were not different between the Low Dose and Conventional Dose groups (122.3 s v 120.6 s, 1.47 s, 99% CI -1.47 to 4.94, p = 0.16). The total amount of protamine administered in the Low Dose group was less than that in the Conventional Dose group (-100.5 mg, 99% CI -110.0 to -91.0, p < 0.0001), as were the number of 250 mg vials used per case (-0.69, 99% CI -0.75 to -0.63, p < 0.0001). The mean initial protamine doses between groups were 250 mg and 352 mg, p < 0.0001. The mean protamine vials used were 1.33 v 2.02, p < 0.0001. When the calculations were based on 50 mg vials, the number of vials used per case in the Low Dose group was even less (-2.16, 99% CI -2.36 to -1.97, p < 0.0001).) CONCLUSIONS: Conservation measures regarding critical medications and supplies during times of shortages can maintain access to important services within a community.

What's fishy about protamine? Clinical use, adverse reactions, and potential alternatives.

Protamine, a highly basic protein isolated from salmon sperm, is the only clinically available agent to reverse the anticoagulation of unfractionated heparin. Following intravenous administration, protamine binds to heparin in a nonspecific electrostatic interaction to reverse its anticoagulant effects. In clinical use, protamine is routinely administered to reverse high-dose heparin anticoagulation in cardiovascular procedures, including cardiac surgery with cardiopulmonary bypass. Despite the lack of supportive evidence regarding protamine's effectiveness to reverse low-molecular-weight heparin, it is recommended in guidelines with low-quality evidence. Different dosing strategies have been reported for reversing heparin in cardiac surgical patients based on empiric dosing, pharmacokinetics, or point-of-care measurements of heparin levels. Protamine administration is associated with a spectrum of adverse reactions that range from vasodilation to life-threatening cardiopulmonary dysfunction and shock. The life-threatening responses appear to be hypersensitivity reactions due to immunoglobulin E and/or immunoglobulin G antibodies. However, protamine and heparin-protamine complexes can activate complement inflammatory pathways and inhibit other coagulation factors. Although alternative agents for reversing heparin are not currently available for clinical use, additional research continues evaluating novel therapeutic approaches.

Heparin Rebound: An In-Depth Review.

The common conception of "heparin rebound" invokes heparin returning to circulation in the postoperative period after apparently adequate intraoperative reversal with protamine. This is believed to portend increased postoperative bleeding and provides the rationale for administering additional empiric doses of protamine in response to prolonged coagulation tests and/or bleeding. However, the relevant literature of the last 60+ years provides only a weak level of evidence that "rebounded" heparin itself is a significant etiology of postoperative bleeding after cardiac surgery with cardiopulmonary bypass. Notably, many of the most frequently cited heparin rebound investigators ultimately concluded that although exceedingly low levels of heparin activity could be detected by anti-Xa assay in some (but not all) patients postoperatively, there was no correlation with actual bleeding. An understanding of the literature requires a careful reading of the details because the investigators lacked standardized definitions for "heparin rebound" and "adequate reversal" while studying the phenomenon with significantly different experimental methodologies and laboratory tests. This review was undertaken to provide a modern understanding of the "heparin rebound" phenomenon to encourage an evidence-based approach to postoperative bleeding. Literature searches were conducted via PubMed using the following MeSH terms: heparin rebound, heparin reversal, protamine, platelet factor 4, and polybrene. Relevant English language articles were reviewed, with subsequent references obtained from the internal citations. Perspective is provided for both those who use HepCon-guided management and those who do not, as are practical recommendations for the modern era based on the published data and conclusions of the various investigators.

In vitro comparison of spatiotemporal fibrin clot formation dynamics in plasma treated with different protamine-heparin ratios.

INTRODUCTION: Our study aim was to explore how different protamine-heparin ratios impacted enzymatic coagulation and acellular fibrin clot growth in plasma using an in vitro model. We hypothesized that a low protamine-heparin ratio would be associated with superior fibrin clot growth dynamics. METHODS: We performed an in vitro study using 15 plasma samples from a commercial supplier. Different protamine-heparin ratios were added to each donor plasma sample: low ratio (0.7-1), traditional ratio (1-1), and high ratio (1.3-1) and clot formation dynamics were evaluated using a Thrombodynamics analyzer. Study outcomes were initial clot growth velocity and clot size at 30 min. RESULTS: Plasma samples treated with a one-to-one protamine-heparin ratio had significantly lower mean initial clot growth velocity compared to samples treated with a low protamine-heparin ratio; mean difference -2.3 µm/min (95% CI = -4.0 to -0.7, p = .004). Plasma samples treated with a one-to-one protamine-heparin ratio also had significantly smaller mean clot size at 30 min compared to samples treated with a low protamine-heparin ratio; mean difference -54.0 µm (95% CI = -107.6 to -0.4, p = .048). There were no significant differences in mean initial clot growth velocity or clot size at 30 min between plasma samples treated with a high protamine-heparin ratio and those treated with a one-to-one or low protamine-heparin ratio (all p > .05). CONCLUSIONS: Plasma samples treated with a low protamine-heparin ratio had superior clot growth velocity and larger clot size at 30 min compared to a one-to-one ratio, supporting the notion that a low protamine-heparin ratio may optimize enzymatic coagulation after cardiopulmonary bypass.

Compromiso cardíaco en el síndrome inflamatorio multisistémico post SARS-CoV-2 en niños / Cardiac involvement in post-SARS-CoV-2 multisystem inflammatory syndrome in children / Envolvimento cardíaco na síndrome inflamatória multissistêmica pós-SARS-CoV-2 em crianças

Objetivo: describir las características de ocho pacientes pediátricos que se presentaron con síndrome inflamatorio multisistémico (MIS-C) asociado a SARS-CoV-2 y compromiso cardíaco. Material y métodos: estudio descriptivo, retrospectivo de ocho pacientes con edades entre 1 y 13 años, con diagnóstico de MIS-C y compromiso cardíaco, asistidos en el CHPR. Se analiza su historia clínica, evolución y tratamiento. Resultados: los pacientes presentaron fiebre en el 100%, exantema e hiperemia conjuntival en el 88%, síntomas digestivos en el 50%, insuficiencia respiratoria en el 25% y shock en el 50%. Todos requirieron ingreso a cuidados intensivos. La alteración de la contractilidad cardíaca estuvo presente en el 63% de los pacientes, fue leve y segmentaria en el 80%, el 60% requirió soporte inotrópico por 3 días, recuperando una función normal en 7 días. La insuficiencia mitral se presentó en el 25% y el derrame pericárdico en el 38%, ambos de grado leve. Un paciente presentó dilatación de arterias coronarias con Z score < 2. El 85% de los pacientes presentó alteraciones del ECG, en el 29% se trató de alteración en la repolarización, en el 29% intervalo QTc prolongado, en el 15% bloqueo atrioventricular de 1er grado y bloqueo incompleto de rama derecha. Un paciente tuvo fibrilación auricular por 3 días con remisión espontánea a ritmo sinusal. Las troponinas estuvieron altas en el 57% de los pacientes y el ProBNP elevado en el 100%. Todos recibieron inmunoglobulinas, metilprednisolona y aspirina. Conclusiones: se presentaron ocho pacientes pediátricos con MIS-C y compromiso cardíaco, el 50% se presentó en shock, todos requirieron ingreso a cuidados intensivos. El 85% presento alteraciones en el ECG. El 63% presentó compromiso de la contractilidad sectorial y leve, se normalizó en 7 días. El 60% requirió soporte inotrópico por una media de 3 días.

Objective: describe the characteristics of 8 children who presented Multisystem Inflammatory Syndrome associated with SARS-CoV2 infections (MIS-C) and cardiac involvement. Material and methods: descriptive, retrospective study of 8 patients of between 1 and 13 years of age, diagnosed with MIS-C and cardiac involvement, assisted at the Pereira Rossell Children Hospital, analysis of their medical records, evolution and treatment. Results: the patients showed: fever in 100% of the cases, rash and conjunctival hyperemia in 88%, digestive symptoms in 50%, respiratory failure in 25% and shock in 50%. All required admission to Intensive Care. Cardiac contractility alteration was present in 63% of patients, the affectation was mild and segmental in 80%, 60% required inotropic support for 3 days and recovered normal functions in 7 days. Mitral regurgitation was present in 25% of the cases and pericardial effusion in 38%, mild in both cases. One patient had dilated coronary arteries with a Z score <2. 85% of the patients presented ECG abnormalities, 29% present alteration of repolarization, 29% prolonged QTc, 15% 1st degree atrioventricular block and incomplete right bundle branch block. One patient had atrial fibrillation for 3 days with spontaneous remission to sinus rhythm. Troponins were increased in 57% of the patients and ProBNP elevated in 100%. All patients received Immunoglobulins, Methylprednisolone and Aspirin. Conclusions: we present eight pediatric patients with MIS-C and cardiac involvement, 50% suffered shock, all required admission to Intensive Care. ECG abnormalities were found in 85% of the patients. Mild and segmental contractility compromise was found in 63% of the patients and normalized in 7 days. 60% required inotropic support for a mean of 3 days.

Objetivo: descrever as características de 8 pacientes pediátricos que apresentaram Síndrome Inflamatória Multissistêmica (MIS-C) associada ao SARS-CoV-2 e comprometimento cardíaco. Material e métodos: estudo descritivo, retrospectivo, de oito pacientes com idade entre 1 e 13 anos, com diagnóstico de MIS-C e comprometimento cardíaco, assistidos pelo CHPR. Seu prontuário médico, evolução e tratamento são analisados. Resultados: os pacientes apresentaram febre em 100%, erupção cutânea e hiperemia conjuntival em 88%, sintomas digestivos em 50%, insuficiência respiratória em 25% e choque em 50%. Todos necessitaram de internação nos cuidados intensivos. A alteração da contratilidade cardíaca esteve presente em 63% dos pacientes, foi leve e segmentar em 80%, 60% necessitaram de suporte inotrópico por 3 dias, recuperando a função normal em 7 dias. A regurgitação mitral ocorreu em 25% dos pacientes e o derrame pericárdico em 38%, ambos de grau leve. Um paciente apresentou dilatação da artéria coronária com escore Z < 2. 85% dos pacientes apresentaram anormalidades no ECG, 29% foram alterações de repolarização, 29% intervalo QTc prolongado em bloqueio atrioventricular de 1º grau a 15% e bloqueio incompleto do ramo direito. Um paciente apresentou fibrilação atrial por 3 dias com remissão espontânea ao ritmo sinusal. As troponinas foram elevadas em 57% dos doentes e ProBNP elevado em 100%. Todos receberam imunoglobulinas, Metilprednisolona e aspirina. Conclusões: houve oito pacientes pediátricos com SMIM-C e comprometimento cardíaco, 50% em choque, todos necessitaram de internação em terapia intensiva. 85% apresentaram elevações no ECG. 63% apresentaram comprometimento setorial e de contratilidade leve, normalizados em 7 dias. 60% necessitaram de suporte inotrópico por uma média de 3 dias.

Platelet Dysfunction in Cardiac Surgery: When is the Best Time to Assess It? An Observational Single Center Study.

Purpose. Cardiac surgery is characterized by a high risk of complications related to perioperative bleeding. Guidelines suggest the use of local algorithms based on perioperative point-of-care tests to assess and manage potential coagulation abnormalities. We investigated whether heparin reversal administration affects the adenosine-5-diphosphate (ADP) test values, thus identifying the earliest time point following cardio-pulmonary bypass that permits the promptest detection and treatment of potential platelet dysfunctions. Methods. This was a retrospective, single-center, observational study enrolling cardiac surgery patients requiring cardiac bypass. ADP-tests at 4 different time-points during surgery (T0: baseline, T1: at aortic de-clamping, T2: 10 minutes after protamine administration, and T3: at the end of surgery) were performed. Results. 63 patients undergoing elective cardiac surgery were studied. Baseline ADP-test values were almost constantly greater than intraoperative values, and end of surgery values were often greater than previous intraoperative values. The only difference that proved to be not statistically significant was between T1 and T2, with a clinically insignificant mean difference of -.2 U (95%CI of difference: -6.9 - 6.5 U). There was no correlation between the variation in ADP-test values pre- and post-protamine administration and the protamine-to-heparin ratio. Conclusion. The results of the present study support the hypothesis that the ADP-test could be performed early, at aortic de-clamping before protamine administration. This approach allows for the promptest assessment of a potential impairment in platelet function, and its timely correction.

The modification of the thrombin generation assay for the clinical assessment of hypercoagulability in patients receiving heparin therapy.

BACKGROUND: Heparin diminishes thrombin generation (TG) because it decreases the survival time of thrombin in plasma. Under heparin therapy, the TG curve therefore does not reflect the true hemostatic status of the patient. AIM: We investigated how far the in vitro addition of a heparin antagonist can restore the underlying TG capacity. MATERIALS & METHODS: Five different heparin antagonists were tested: polybrene, protamine sulfate, heparinase type 1, heparinase HEP-TEM, and (Z-GGR)2 -rhodamine (P2Rho). RESULTS AND CONCLUSION: Polybrene, P2Rho, and heparinase HEP-TEM effectively neutralized heparin at prophylactic and therapeutical doses of both low molecular weight and unfractionated heparin. The advantages and limits of each molecule and the most favorable combinations of TG-trigger and antagonist are discussed.

Study of in vitro thrombin generation after neutralization of heparin.

INTRODUCTION: Thrombin generation (TG) documents hypercoagulability. TG in platelet-poor plasma is exquisitely sensitive to heparins, which thus must be neutralized before testing. Heparinase and hexadimethrine bromide (polybrene) have been used for that purpose, but their effects per se on TG have been poorly studied so far. METHODS: (i) TG was studied in commercial normal pooled plasma (NPP; CryoCheck® , Cryopep) in absence or presence of neutralizing agents. (ii) NPP was spiked with increasing concentrations of unfractionated heparin (UFH; up to 1.0 IU/mL) or low-molecular-weight heparin (LMWH; enoxaparin up to 1.2 IU/mL) and TG studied after incubation of heparinase (Hepzyme® ; 15 minutes) or polybrene (0.025 mg/mL; 10 minutes). RESULTS: (i) With ThromboScreen reagent to initiate TG, addition of heparinase was associated with increased peak, whereas polybrene caused lengthening of lag time and time to peak, compared with nonsupplemented NPP. (ii) With polybrene, TG was completely restored over the whole range of UFH and LMWH studied. By contrast, heparinase failed to fully restore TG in presence of UFH concentrations ≥0.8 IU/mL or LMWH concentrations ≥1.0 IU/mL. Those effects were matched with detectable tiny residual amounts of non-neutralized heparin (as assessed with an anti-Xa assay) and were less pronounced with a higher picomolar concentration of tissue factor (DrugScreen reagent). CONCLUSION: Polybrene fully restored TG of heparinized plasma at the expense of an alteration of TG, pointing to the need to use adapted reference ranges. Heparinase failed to do so in presence of high concentrations of both heparins.

Protamine-Induced Coronary Graft Thrombosis: A Review.

Perioperative myocardial infarction is a serious complication affecting a significant portion of patients undergoing coronary artery bypass graft surgery. This may arise due to coronary graft thrombosis, a rare but potentially fatal phenomenon associated with both congenital and acquired risk factors. Multiple case reports implicate the role of protamine in the development of such thromboses. The role of protamine in facilitating the regulation of hemostasis by reversing the anticoagulant effects of heparin in patients undergoing cardiopulmonary bypass is well-recognized. However, discussion of its potential contribution to coronary graft thrombosis and mechanisms by which this may occur is lacking. Furthermore, its narrow therapeutic index and side effect profile are such that its appropriateness as a universal reversal agent to heparin requires reconsideration. This article reviews the current body of evidence regarding the use of protamine in cardiac surgery and the limited case reports pertaining to its potential role in the pathophysiology of coronary graft thrombosis.

Can the Minimum Protamine Dose to Neutralize Heparin at the Completion of Cardiopulmonary Bypass be Significantly Lower than the Conventional Practice?

Systemic anticoagulation with heparin during cardiopulmonary bypass (CPB) should be neutralized by protamine administration to restore normal hemostasis. However, protamine has potentially serious side effects and excessive protamine can cause increased postoperative bleeding. Thus, our goal is to appropriately dose protamine at the completion of CPB to neutralize heparin so that neither residual heparin nor excessive protamine is present. We performed a retrospective study of 216 patients who underwent cardiac surgery to search for a safe minimum protamine dose (PD) when measuring heparin concentration (HC). In addition, we developed a formula to determine PD using total heparin dose (THD) and CPB time without measuring HC. When protamine-to-heparin ratio (P-to-H) is set at 1 mg protamine to 100 international unit (IU) heparin in HMS Plus Hemostasis Management System (HMS), we determined that 75% of the calculated total PD is a safe minimum PD to sufficiently neutralize circulating heparin after CPB. On average, this translates into either .37 mg protamine/100 IU heparin of THD or .54 mg/100 IU of the first heparin bolus. The formula we developed to calculate PD without measuring HC can provide a PD that strongly agrees with the safe minimum PD when measuring HC. The safe minimum PD to neutralize circulating heparin after CPB can be significantly lower than conventional dosing practices. Reduction of PD may decrease the risk of postoperative bleeding and protamine-related adverse events. Based on our data, we decreased P-to-H in HMS to examine whether it is possible to reduce PD further than the safe minimum PD determined in this study.

Reversal Activity and Toxicity of Heparin-Binding Copolymer after Subcutaneous Administration of Enoxaparin in Mice.

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.