Reversible fluorescence modulation of BSA stabilised copper nanoclusters for the selective detection of protamine and heparin.

Protamine and heparin are the most important polyionic drugs used during surgeries and extracorporeal therapies. In this article, a selective and sensitive fluorescence method for the detection of both protamine and heparin was developed by using bovine serum albumin stabilised copper nanoclusters. Blue emitting fluorescent copper nanoclusters were synthesized in aqueous solution using bovine serum albumin as a capping agent and a reducing agent. A one pot microwave assisted method was adopted to synthesize fluorescent copper nanoclusters showing emission at 410 nm upon excitation at 330 nm. The fluorescence of copper nanoclusters was found to be enhanced after the addition of protamine and the limit of detection obtained is 0.12 ng mL-1. The significant enhancement in fluorescence can be attributed to the electrostatic interactions between the copper nanocluster and protamine. In contrast, the enhanced fluorescence intensity of the copper nanocluster with protamine added was decreased after the addition of heparin, and the copper nanocluster regained its original fluorescence intensity. This can be attributed to the strong interaction of protamine with heparin and the limit of detection was calculated as 0.0406 ng mL-1. The selectivity and sensitivity of the sensor for both protamine and heparin were also determined in the presence of potentially co-existing biomolecules, cations, and anions and satisfactory results were obtained. Additionally the validity of the proposed protamine and heparin sensor was attested in real sample matrices such as human urine samples and human blood serum samples. The results exhibited that the recovery percentage of protamine and heparin reached 98-99% and 92-99% in urine samples and 97-99% in serum samples.

Impact of protamine I on colon cancer proliferation, invasion, migration, diagnosis and prognosis.

This paper investigates protamine I (PRM1) expression and its effects on proliferation, invasion and migration of colon cancer cells as well as its function in clinical diagnosis and prognosis. Gene chips were used to screen differentially expressed genes. PRM1 expression was detected by Western blotting and quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin and eosin (HE) staining and immunohistochemistry were utilized to compare the expression of PRM1 from multiple differentiation levels of colon cancer tissues. Cell viability, cell apoptosis and cell cycle were tested using the MTT assay and flow cytometry. Cell invasion and migration capability were tested using the Transwell assay and wound healing. In vivo effects of PRM1 on colon cancer were explored using a xenograft model. PRM1 expression in serum was detected by enzyme-linked immunosorbent assay (ELISA). The expression level of PRM1 was significantly higher in colon cancer tissues and the staining degree of PRM1 in poorly-differentiated was stronger. pcDNA3.1-PRM1 decreased cell apoptosis while it increased the proliferation, cell invasion and migration. The si-PRM1 group displayed an opposite tendency. The serum PRM1 level was significantly higher and could serve as a diagnostic biomarker for colon cancer.

Implementing a Statistical Model for Protamine Titration: Effects on Coagulation in Cardiac Surgical Patients.

OBJECTIVES: To implement a statistical model for protamine titration. DESIGN: Prospective randomized trial. SETTING: University hospital. PARTICIPANTS: Sixty (n = 30+30) patients scheduled for elective coronary artery bypass surgery were randomly assigned to 2 groups. INTERVENTIONS: Protamine dose calculated according to an algorithm established from a statistical model or to a fixed protamine-heparin dose ratio (1:1). MEASUREMENTS AND MAIN RESULTS: Both groups demonstrated comparable patient demographics and intraoperative data. Coagulation effects were evaluated using rotational thromboelastometry. Using the statistical model reduced (p<0.01) the protamine dose from 426±43 mg to 251±66 mg, followed by significantly (p<0.01) shorter intrinsic clotting time (208±29 seconds versus 244±52 seconds) and stronger clot firmness (p = 0.01), and effects on indices of extrinsic or fibrinogen coagulation pathways were insignificant. Test of residual heparin was negative in all patients after protamine administration, aligned with insignificant (p = 0.27) intergroup heparinase-verified clotting time differences. CONCLUSIONS: The statistical model for protamine titration is clinically feasible and protects the patient from exposure to excessive doses of protamine, with advantageous effects on coagulation as measured using rotational thromboelastometry. Significance regarding clinical outcome is yet to be defined.

combination effect of hypertonic disease with chronic pancreatitis on the processes maintain homeostasis.

OBJECTIVE: Introduction: Abnormalities comorbidity - a frequent phenomenon in medical practice. This determines the relevance of research processes maintaining homeostasis with a combination of various diseases. The aim of this study was to examine and compare the character of vegetative, antioxidant, kallikrein-kinin system and parameters of endogenous intoxication disorders in the patients with isolated essential hypertension and with combination of hypertonic disease and chronic pancreatitis. PATIENTS AND METHODS: Materials and Methods: Cardiointervalography was used in the research with definition of standard statistical and spectral heart rate variability. Determination of superoxide dismutase, glutathione, catalase, middle molecular peptides, total proteolytic activity of plasma by the hydrolysis of protamine sulfate, prekallikrein, kallikrein, α1 -proteinase inhibitor, α2 -macroglobulin and kininase II was conducted by laboratory methods. RESULTS: Results: Sympathicotonia with the moderate tension of adaptation processes, violation of antioxidant protection, kallikrein-kinin system and displays of endogenous intoxication were found in the patients with isolated hypertension. Reduction of sympathicotonia, reducing total power spectrum, increasing the share of humoral-metabolic effects on heart rate, tendency to asympathicotonia autonomic reactivity, lower levels of superoxide dismutase, glutathione, prekallikrein, α2 -macroglobulin, kininase II, higher levels of catalase, middle molecular peptides, total proteolytic activity of plasma kallikrein were observed upon accession the concomitant chronic pancreatitis. CONCLUSION: Conclusions: The signs of compensatory mechanisms disruption and increased autonomic nervous system imbalance with a decrease in ductility autonomous processes in the load were determined upon accession the concomitant chronic pancreatitis. The combination of pathologies also accompanied by more severe manifestations of endogenous intoxication, significant violations of antioxidant and kallikrein-kinin systems.

Flow Chronopotentiometry with Ion-Selective Membranes for Cation, Anion, and Polyion Detection.

We report here on the development of a chronopotentiometric readout for ion-selective electrodes that allows one to record transition times in continuous flow conditions without the necessity to stop the flow. A sample plug of 150 µL is injected into the carrier solution (0.5 mM NaCl) and subsequently transported to the detection cell (∼20 µL) at moderate flow rates (∼0.5 mL min(-1)), where a short current pulse (5s) is applied between the ionophore-based working electrode and a biocompatible and nonpolarizable Donnan exclusion anion-exchanger membrane reference/counter electrode. Flow conditions bear an influence on the thickness of the aqueous diffusion layer and result in a shift of the chronopotentiometric transition time with respect to stopped flow. Two models based on rotating disk electrodes and flow chronopotentiometry at metal-based electrodes were used to corroborate the data. The method was successfully applied to the determination of calcium, chloride, alkalinity, acidity, and protamine with a range of ion-selective membranes. Because of the limiting exposure time of ca. 20 s of the membranes with the sample, this approach is demonstrated to be useful for the detection of protamine in the therapeutic range of undiluted human blood.

Facile and sensitive detection of protamine by enhanced room-temperature phosphorescence of Mn-doped ZnS quantum dots.

Quantum dot (QD) nanohybrids provide an effective route to explore the new properties of materials and are increasingly used as highly valuable sensitive (bio) chemical probes. Interestingly, the room-temperature phosphorescence (RTP) of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS QDs could be remarkably enhanced by the addition of protamine. Based on the above finding, a simple, sensitive, and selective method for rapid detection of protamine was successfully designed. With this method, protamine as a cationic peptide interacts electrostatically with MPA-capped Mn-doped ZnS QDs to form MPA-capped Mn-doped ZnS QD/protamine complexes, which leads to the aggregation of QDs and enhances the RTP intensity. Under the optimized conditions, the RTP intensity change was linearly proportional to the concentration of protamine in the range 0.2-3.0 µg ml(-1), and the limit of detection was 0.14 µg ml(-1). The proposed method was successfully applied to detect protamine in protamine sulfate injection and human serum samples with satisfactory results, and the recovery ranged from 96.5 to 105.6%.

Serological features of antibodies to protamine inducing thrombocytopenia and thrombosis.

BACKGROUND: A significant proportion of patients undergoing cardiopulmonary bypass develop anti-protamine antibodies, with or without the association of thromboembolic events. METHODS: 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. RESULTS: 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. CONCLUSIONS: These data show that protamine-induced antibodies are specific and may induce platelet activation, which explains their association with thromboembolic events.

Reversal of heparin after cardiac surgery: protamine titration using a statistical model.

OBJECTIVE: To establish a statistical model for determination of protamine dose in conjunction with cardiopulmonary bypass. DESIGN: Prospective. SETTING: University hospital. PARTICIPANTS: Ninety consecutive cardiac surgical patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A series of clinically oriented variables were introduced into a statistical model for projection of the protamine dose after cardiopulmonary bypass. The following significant predictors were identified using multivariable regression analysis: The patient's body surface area, the administered dose of heparin, heparin clearance, and the preoperative platelet count. The statistical model projected the protamine dose within 3±23 mg of the point-of-care test used as reference. CONCLUSION: Protamine dosing based on statistical modeling represents an alternative to point-of-care tests.

Ionophore-based ion-selective optical nanosensors operating in exhaustive sensing mode.

Ion selective optical sensors are typically interrogated under conditions where the sample concentration is not altered during measurement. We describe here an alternative exhaustive detection mode for ion selective optical sensors. This exhaustive sensor concept is demonstrated with ionophore-based nanooptodes either selective for calcium or the polycationic heparin antidote protamine. In agreement with a theoretical treatment presented here, linear calibration curves were obtained in the exhaustive detection mode instead of the sigmoidal curves for equilibrium-based sensors. The response range can be tuned by adjusting the nanosensor loading. The nanosensors showed average diameters of below 100 nm and the sensor response was found to be dramatically faster than that for film-based optodes. Due to the strong binding affinity of the exhaustive nanosensors, total calcium concentration in human blood plasma was successfully determined. Optical determination of protamine in human blood plasma using the exhaustive nanosensors was attempted, but was found to be less successful.

Protamine requirements in cardiac surgery: effect of changes in the heparin reference standard.

OBJECTIVE: UFH (unfractionated heparin) and protamine are integral to cardiac surgery, and inappropriate dosing can predispose to coagulopathy and hemorrhage. The FDA (Food and Drug Administration) recently has instituted changes to UFH formulation and it is not known if this has influenced its susceptibility to neutralization by protamine. Hence, the authors sought to compare 2 commercial preparations of UFH (old and new) with regard to their neutralization by protamine in patients undergoing cardiopulmonary bypass (CPB). DESIGN: Prospective, observational, cohort study. SETTING: Tertiary care university hospital and associated research laboratory PARTICIPANTS: Twenty adult patients undergoing elective cardiac surgery with CPB. INTERVENTIONS: Blood samples were drawn preinduction, prior to, and 5 and 30 minutes following protamine, and 0 and 2 hours after ICU admission. Protamine titration assays were conducted in vitro on samples drawn prior to and following protamine administration. Anti-IIa and anti-Xa activity were assayed in all samples. RESULTS: Anti-IIa and anti-Xa activity were detected ubiquitously at all time points following CPB, and there were no differences in susceptibility to protamine neutralization between the 2 groups. In vitro protamine titration studies revealed that anti-IIa was more resistant to protamine neutralization compared to anti-Xa activity. CONCLUSIONS: The 'old' and 'new' formulations of UFH evaluated in this study were similar in their susceptibility to protamine neutralization. Circulating UFH is detected as early as 5 minutes after protamine administration and anti-IIa is more resistant to protamine neutralization as compared to anti-Xa activity. Further studies are required to quantify the precise dose of protamine following CPB.

Anticoagulation monitoring during extracorporeal circulation with the Hepcon/HMS device.

OBJECTIVE: The objective of our study was to compare the standard protocol of anticoagulation to the Hepcon/HMS. METHOD: This study included forty-four patients who underwent coronary bypass grafting surgery (CABG), or biological aortic valve replacement (AVR). Unfractionated heparin (UH) was used for patients who underwent operations in the control group (n = 22) (300U/Kg of UH with a goal of an ACT of 400s). The heparin was antagonized dose/dose by protamine. For the patients who underwent operations in the HMS group (n = 22), the heparin and protamine doses were assessed by the Hepcon/HMS device. RESULTS: The sex ratio amounted to 1.93 (29 men and 15 women) and the mean age was 70 ± 11 years. The patients in the HMS group had a chest closure time that was significantly shorter than patients in the control group. The times were, respectively, 42 ± 15 minutes and 68 ± 27 minutes (p = 0.001). The protamine/heparin ratio was significantly lower in the HMS group (0.62 ± 0.13 vs. 1 ± 0.11) (p = 0.0001). The postoperative bleeding amounted to 804 ± 729 ml in the HMS group versus 1416 ± 1103 in the control group (p = 0.016). In multivariate linear regression analysis, only two independent factors were significantly associated with bleeding: the Hepcon/HMS (OR = 0.1-p = 0.03) and the preoperative hemoglobin rate (OR = 1.4 - p = 0.05). Postoperatively, within 72 hours, the red blood cell transfusion was 1.04 ± 1.5 units for the HMS group and 2.1 ± 1.87 units for the control group (p = 0.05). CONCLUSION: During cardiac surgery under CPB, heparin and protamine titration with the Hepcon/HMS device could predict a lower protamine dose and lower postoperative bleeding without higher thromboembolic events, and lower perioperative red blood cell transfusion with a shorter chest closure time.

Reversible sensing of the anticoagulant heparin with protamine permselective membranes.

A permselective membrane electrode allows the rapid and operationally reversible detection of the polycationic polypeptide protamine in physiological samples. Anticoagulant levels of heparin can be measured in undiluted whole blood by adding a known excess of its antidote protamine to discrete blood samples.

Analysis of the complex formation of heparin with protamine by light scattering and analytical ultracentrifugation: implications for blood coagulation management.

Heparin, a linear glycosaminoglycan, is used in different forms in anticoagulation treatment. Protamine, a highly positive charged peptide containing about 32 amino acids, acts as an antagonist for heparin to restore normal blood coagulation. The complex formation of protamine with heparin was analyzed by a combination of analytical ultracentrifugation and light scattering. Titration of heparin with protamine in blood plasma preparations results in a drastic increase of turbidity, indicating the formation of nanoscale particles. A similar increase of turbidity was observed in physiological saline solution with or without human serum albumin (HSA). Particle size analysis by analytical ultracentrifugation revealed a particle radius of approximately 30 nm for unfractionated heparin and of approximately 60 nm for low molecular weight heparin upon complexation with excess protamine, in agreement with atomic force microscopy data. In the absence of HSA, larger and more heterogeneous particles were observed. The particles obtained were found to be stable for hours. The particle formation kinetics was analyzed by light scattering at different scattering angles and was found to be complete within several minutes. The time course of particle formation suggests a condensation reaction, with sigmoidal traces for low heparin concentrations and quasi-first-order reaction for high heparin concentrations. Under all conditions, the final scattering intensity reached after several minutes was found to be proportional to the amount of heparin in the blood plasma or buffer solution, provided that excess protamine was available and no multiple scattering occurred. On the basis of a direct relation between particle concentration and the heparin concentration present before protaminization, a light scattering assay was developed which permits the quantitative analysis of the heparin concentration in blood plasma and which could complement or even replace the activated clotting time test, which is currently the most commonly used method for blood coagulation management.

Novel interactions between UFH and TFPI in children.

The impact of age upon therapeutic response to unfractionated heparin (UFH) in children is proposed to reflect quantitative and potentially qualitative differences in coagulation proteins across childhood. This study explores the UFH-dependent tissue factor pathway inhibitor (TFPI) release in children compared to previously published data in adults. Children <16 years of age undergoing cardiac angiography formed the population for this prospective cohort study. TFPI release was measured prior to (baseline) and at 15, 30, 45 and 120 min post-UFH dose. This study demonstrated that, whilst the immediate release of TFPI post-UFH was similar in children compared to adults, TFPI release in children remained increased and consistent for a significantly longer period post-UFH administration compared to adults. Plasma TFPI levels in children did not demonstrate an UFH concentration -dependent reduction, as has been previously reported in adults. The prolonged TFPI-mediated anticoagulant levels observed in children administered UFH may contribute to the increased rate of major bleeding reported in children compared to adults. Furthermore, we postulate that this sustained UFH-dependent increase in TFPI levels in children may influence the binding of UFH to competitive plasma proteins, such as those involved in the immunological response to UFH associated with heparin-induced thrombocytopenia.

Postoperative bypass bleeding: a bypass-associated dilutional (BAD) coagulopathy?

A number of associations with post-bypass bleeding have been described in the accompanying paper. Herein we hypothesize that dilution is an underlying cause through a malign series of bypass-associated events. Heparinized blood behaves anomalously when diluted. Clotting times first shorten somewhat, then--as the dilution of whole blood approaches 50%--rapidly lengthen to unclottability. During cardiopulmonary bypass, low blood volume patients are at a significant risk of clotting factor dilution which will always be more severe than the level of whole blood dilution. If severe enough, this dilution may lower plasma clotting factors to a critical level and may result in excess protamine administration, secondary to overestimation of heparin. The presence of un-neutralized protamine combined with critically lowered clotting factors leads to marked coagulopathy.

Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

Humoral immune responses to testis antigens in sera from patients with prostate cancer.

Tumor vaccines represent one type of molecularly targeted therapy being investigated for the treatment of prostate cancer. Although many prostate-specific proteins are being tested as target antigens for prostate cancer vaccines, most are not natural targets of an immune response in patients with cancer. Using sera from cancer patients, several research groups have identified a large family of immunologically recognized proteins whose expression is normally confined to immune-privileged testis tissue but which may be expressed in cancers of different histological origins. These proteins, so-called cancer-testis (CT) antigens, are appealing targets for immune-based therapies because they are essentially tumor-restricted antigens and there is less risk of preexisting immune tolerance. In addition, specifically targeting these proteins by means of vaccines should reduce the risk of potential autoimmune reactions to normal tissues. In the current study, we hypothesize that prostate CT antigens can be identified using a SEREX screening method with sera from patients with prostate cancer and probing with a human testis cDNA expression library. We have identified several potential prostate cancer antigens with predominantly testis-specific expression in normal tissues, including MAD-CT-1 (protamine 2) and MAD-CT-2. Each was independently identified from different subjects with prostate cancer. Antigens identified by these studies can be investigated further as potential prostate cancer tumor antigens.

A simple and rapid label-free fluorimetric biosensor for protamine detection based on glutathione-capped CdTe quantum dots aggregation.

A novel fluorescent biosensor is developed, based on glutathione-capped CdTe quantum dots aggregation, for the determination of trace amount of an important drug, protamine. In this method with increasing the protamine concentration, the fluorescence of the quantum dots was quenched due to their aggregation. Different parameters affect the sensitivity, such as pH and the amount of the quantum dots, were optimized. Using the new optical biosensor, under the optimized conditions, protamine could be measured in the range of 2.0-200 ng mL(-1) with a detection limit of 1.0 ng mL(-)(1). The relative standard deviation for five replicates determination of 30.0 ng mL(-)(1) protamine was 1.26%. The influence of common interfering species on the protamine detection was studied. The results showed that the biosensor is highly selective and sensitive for the detection of protamine. The optical biosensor was successfully used for the determination of protamine in real samples.

Heparin-protamine complexes and C-reactive protein induce activation of the classical complement pathway: studies in patients undergoing cardiac surgery and in vitro.

The administration of protamine to patients undergoing cardiopulmonary bypass (CPB) to neutralize heparin and to reduce the risk of bleeding, induces activation of the classical complement pathway mainly by heparin-protamine complexes. We investigated whether C-reactive protein (CRP) contributes to protamine-induced complement activation. In 24 patients during myocardial revascularization, we measured complement, CRP, and complement-CRP complexes, reflecting CRP-mediated complement activation in vivo. We also incubated plasma from healthy volunteers and patients with heparin and protamine in vitro to study CRP-mediated complement activation. During CPB, CRP levels remained unchanged while C3 activation products increased. C4 activation occurred after protamine administration. CRP-complement complexes increased at the end of CPB and upon protamine administration. Incubation of plasma with heparin and protamine in vitro generated complement-CRP complexes, which was blocked by phosphorylcholine and stimulated by exogenous CRP. C4d-CRP complex formation after protamine administration correlated clinically with the incidence of postoperative arrhythmia. Protamine administration during cardiac surgery induces complement activation which in part is CRP-dependent, and correlates with postoperative arrhythmia.