Heparin Polyanions Research Articles

Coordination-driven self-assembly formation of Ru(II)-metallocycles based on 4-amino-1,8-naphthalimide Tröger's base supramolecular scaffold: Synthesis, characterization, and heparin-binding study

The synthesis, characterization, and heparin polyanion sensing properties of a series of [2 + 2] self-assembled Ru(II)-metallocycles based on a green emitting 4-amino-1,8-naphthalimide Tröger's base (TBNap) supramolecular scaffold are described here. TBNap was reacted in a 2:2 stoichiometric ratio with a series of half-sandwiched Ru2(II)-acceptors (M1 to M4) to isolate four new tetranuclear Ru(II)-metallocycles, TB-MC-1 to TB-MC-4. All the metallocycles were characterized using NMR (1H &13C), FT-IR, and ESI-MS. A positive solvatochromism was observed for the metallocycles in different solvents with varying polarity. The strong-fluorescence emission of metallocycles was drastically quenched upon the addition of increasing concentration of heparin polyanions and the Stern-Volmer quenching constants (KSV) were determined to be in the order of 105 M−1. All these results demonstrate the usefulness of metallocycles for sensing and quantifying the concentration of heparin polyanion.

Performance of the New Automated Latex Immunoturbidometric Assay in the Diagnosis of Heparin Induced Thrombocytopenia: A Single Institution Experience

Introduction: Heparin induced thrombocytopenia (HIT) is a severe prothrombotic condition, usually triggered by exposure to heparin products. It is characterized by platelet activation induced by the formation of antibodies to the platelet factor 4 (PF4)/ heparin polyanion complexes. Diagnostic algorithm includes clinical scoring (4T score) alongside serological test for detection of these antibodies (HIT-Ab), while serotonin release assay (SRA) remains the gold- standard for confirmation. The automated latex immunoturbidometric assay (LIA) has recently been FDA approved as a screening tool for HIT and is a potential alternative to the conventional particle immunofiltration assay (PIFA) for time-sensitive detection of HIT-Ab to guide treatment considerations. We recently introduced LIA in our institution. In this study, we present our experience with LIA in comparison to PIFA in the diagnosis of HIT. Methods: We retrospectively reviewed the charts of all the patients on whom a PIFA was ordered between March 2017 and March 2018 in our hospital. We collected information on the results of the PIFA and SRA (if available). We replaced PIFA with LIA for HIT screening. Then, we introduced a structured protocol for diagnosis of HIT in our institution by incorporating 4T scoring alongside LIA order in the electronic medical record (EMR), in December 2018. We reviewed the EMR of all the patients on whom HIT-Ab test (LIA) was ordered between January and June of 2019, and collected similar information as before. All the data were compiled in a single master excel sheet for calculation of performance characteristics (sensitivity, specificity, positive and negative predictive values) for both PIFA and LIA. A patient was considered to have the diagnosis of HIT if the result of SRA was available and positive. Results: In the first phase, a total of 31 orders for SRA was noted against 170 PIFA orders. Five patients had a positive SRA, of whom two were PIFA negative. Half the patients with a negative SRA result were positive for PIFA. Hence, the sensitivity and specificity of PIFA test for our study population were noted to be 60% and 50%, respectively. PIFA had a positive predictive value (PPV) of mere 18.75% for the diagnosis of HIT, whereas the negative predictive value (NPV) was found to be 86.66%. Introduction of structured protocol for HIT diagnosis substantially reduced the number of inappropriate SRA orders in the second phase. On review of data for six months with the new HIT-Ab test LIA, SRA was ordered in only eight patients, to go with 69 orders for the LIA. The result of LIA was positive in all three patients with a positive SRA, whereas it was false positive in four instances. Only one patient was negative for both LIA and SRA during this period. LIA was found to be 100% sensitive and 20% specific for the diagnosis of HIT in our sample. PPV and NPV for LIA were 42.85% and 100%, respectively. Conclusion: The sensitivity and specificity of LIA were found to be 100% and 20%, respectively, in our study population, which is different from the earlier report (Warkentin et al. 2017). The small sample size is a limitation of our study. Higher PPV and NPV for LIA, with its quick turnaround time, make it a useful alternative for the time-sensitive determination of post-test probability for HIT in patients. [HIT- Ab- Heparin Induced Thrombocytopenia Antibody, PIFA- Particle Immunofiltration Assay, LIA- Latex Immunoturbidometric Assay, SRA- Serotonin Release Assay, +ve- Positive, -ve - Negative, PPV- Positive Predictive Value, NPV- Negative Predictive Value] Disclosures No relevant conflicts of interest to declare.

Effective Implementation of a Structured Protocol for Facilitation of the Judicious Use of Antibody Test for Diagnosis of Heparin Induced Thrombocytopenia

Introduction: Heparin induced thrombocytopenia (HIT) is a serious prothrombotic condition, usually triggered by exposure to heparin products with formation of antibodies to platelet factor 4/ heparin polyanion complexes. Diagnostic algorithm for HIT combines clinical scoring (4T score) with time sensitive screening for HIT antibodies (HIT-ab), while serotonin release assay (SRA) is remains the gold standard for confirmatory diagnosis. The rate of utilization of 4T score was low in our institution, resulting in inappropriate orders for HIT-Ab test and subsequent administration of unnecessary alternative anticoagulation (AC) in patients with false positive results. In this project, we designed a structured HIT diagnostic workflow incorporating 4T score calculation in our electronic medical record (EMR) and replaced particle immunofiltration assay (PIFA) with latex immunoturbidometric assay (LIA) in our laboratory for HIT-Ab screening, with an aim to improve the rate of 4T utilization and accuracy of HIT diagnosis in a cost-efficient manner. Methods: In phase I, we performed a retrospective chart review of all patients with PIFA ordered between March 2017-March 2018. Two investigators independently calculated 4T, collected data on results of HIT-Ab, confirmatory SRA tests, and the duration of alternative AC from each record. Any variations in 4T score were resolved by a senior investigator. In phase II, we implemented a new workflow in the EMR incorporating mandatory calculation of 4T score with every order for HIT-Ab test. Our lab started using LIA in place of PIFA. Charts were reviewed on patients with HIT-Ab orders (LIA) from January-June of 2019. Results: On review of data from phase I, we noted that 4T score was documented in only 5 (0.02%) of 170 patients in whom a PIFA test was ordered. Per investigators assessment, 113 (66.4%) patients had low probability (4T ≤ 3), 47 (27.6%) had intermediate probability (4T 4 or 5), and 10 (5.8%) had a high probability (4T ≥ 6) for a diagnosis of HIT. SRA was ordered in 32 patients, although 17 of them had low probability per investigator assessment. PIFA test came back positive in 26 patients, of whom 16 had corresponding SRA results, and three samples were positive for SRA. PIFA was negative in two patients with confirmed HIT (SRA positive). A total of 19 patients received alternate AC in the first phase, 7 of them had low 4T score per our assessment. In phase II, 69 records were found with available LIA results, showing a relative decrease in HIT-Ab orders compared to earlier phase at the six months mark. Documentation of 4T score has been 100% by ordering physicians, a certain improvement from phase I. Investigator calculated 4T score showed low probability in 33 (47.8%) patients, intermediate probability in 31 (44.9%) patients, high probability in 5 (0.07%) patients. LIA was positive in 7 of the 69 ordered tests, 6 of whom scored high/intermediate in the 4T score. HIT diagnosis was confirmed in 3 of these 7 patients with a positive SRA result. During this period, all the 7 of the eight patients who received alternate AC had a high or intermediate probability for HIT as per 4T. Conclusion: Our study demonstrated that the successful implementation of a structured protocol for HIT diagnosis ensured 100% adherence to the calculation and documentation of 4T score by clinicians, and significantly reduced the number of inappropriate HIT-Ab test orders in our institution. Use of alternate AC was also more consistent with the level of probability for HIT. Table Disclosures No relevant conflicts of interest to declare.

Stoichiometry of Zn(II)–heparin–glycine complex, determined using data from elemental and thermal analysis

Ternary polymer Zn(II)–heparin–glycine complex with the composition {Na3[ZnHepGly]·H2O} n , where Hep4− is the monomer chain of a heparin polyanion and Gly– is the chain of a glycine anion, is isolated in a solid state from a water solution, and is characterized via elemental and thermal analysis.

Investigation of Luminescence Characteristics of Osmium(II) Complexes in the Presence of Heparin Polyanions.

The luminescence characteristics of six osmium carbonyl complexes with phenanthroline (phen) or bipyridine (bpy) and pyridine (py), 4-phenylpyridine (4-phpy), or triphenylphosphine (PPh3) complexes in the presence of polyanion heparin were studied in both ethanol and aqueous solutions. The influence of heparin on the luminescence of the complexes is heavily dependent on the type of ligands in the complexes and the solvent used. In the ethanol solutions, the heparin solution enhanced the luminescence of the five osmium complexes, with the strongest enhancement to the 4-phenylpyridine complexes; linear curves were obtained in the luminescence enhancement ratio (F/F 0) versus the heparin concentration range of 1–40 μg/mL. In aqueous solutions, heparin quenching of the complexes was more significant; a linear quenching curve was obtained with [Os(phen)2CO(PPh3)](PF6)2 in the lower concentration range of 1–12 μg/mL. The interaction of these complexes with heparin in the solutions is discussed. The complexes are shown to be successful in the fast and sensitive detection of heparin in commercial injectable samples.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Physiochemical Properties of Low and High Molecular Weight Poly(ethylene glycol)-Grafted Poly(ethylene imine) Copolymers and Their Complexes with Oligonucleotides

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.

Binding of magnesium and lanthanum ions to heparin in the presence of sodium ions. A relationship between 13C chemical shift displacements and counterion condensation theory

Interactions between beef lung heparin and Mg2+ and La3+ counterions, in the presence of sodium ions, have been investigated by 13C-nmr spectroscopy. The chemical shift displacements observed for the carbons of the main disaccharide unit of heparin, when analysed on the basis of counterion condensation theory, indicate that the association between the heparin polyanion and Mg2+ is a delocalized process. This is analogous to the Ca2+/Na+/heparin system, despite the fact that the binding affinity of heparin for Ca2+ is stronger than that for Mg2+. By contrast, the interaction with La3+ is in excess of the theoretical predicted, which suggests that there is at least a partial site-binding of this counterion. In Ca2+/M+/heparin systems in which M+ = Na+ or K+, the selective binding preference of heparin for the univalent cations may be related to the order of their crystallographic ionic radii. A critical assessment is offered of the validity of this nmr spectroscopic approach to the study of counterion binding to heparin.

Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides.

Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10(5) and greater than or equal to 3 X 10(6) USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase (EC 2.3.1.60) and kanamycin 6'-acetyltransferase (EC 2.3.1.55) enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10(5) and greater than or equal to 10(4) USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. We describe how heparin interferes with these two assays and demonstrate interference at heparin concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes.

Counter-ion dependence in salt-free, aqueous solutions of heparin

Chiroptical properties of heparin for various degrees of neutralization of the sulfate and carboxylic groups and for different counter-ions in salt-free aqueous solutions were investigated. Variations of optical rotation and ellipticity values at given wavelengths are compared to simultaneous pH and viscosity changes observed during the neutralization of heparin by sodium and calcium hydroxide. For Na +, variations of ellipticity at 210 nm are related to acid—base properties of uronic carboxylic groups. C.d. characteristics found for alkaline-earth counter-ions (Mg 2+, Ca 2+ and Ba 2+), as compared to Na +, are assigned to effects of divalent ions on the ionization behavior of carboxylic groups. Among the divalent counter-ions considered, Ca 2+ gave the strongest interaction with the heparin polyanion, but no specific complex formation was observed. O.r.d. and c.d. data are discussed on the basis of a randomly coiled structure for macromolecules composed of rigid, heterocyclic repeating-units that are independent of each other in so far as electronic transitions of chromophore groups contributing to optical activity are concerned.

Thermodynamics of mucopolysaccharide–dye binding. III. Thermodynamic and cooperativity parameters of acridine orange–heparin system

AbstractBinding isotherms for acridine orange (AO)–heparin systems can be evaluated solely on the basis of quantitative fluorescence spectroscopic measurements. The evaluation of thermodynamic parameters indicates that the interactions of AO with heparins from several animal sources are similar to each other in magnitude. Binding is highly exothermic (ΔH = −6 kcal mol−1) and is stabilized by dye–polymer and dye–dye (coopertive) interactions, as well as by entropic factors (ΔS = +7 e.u.). The predominant stabilizing factor appears to be the electrostatic attraction between the AO cation and the heparin polyanion, although the other factors are important as well. At 24°C the value of the cooperative binding constants for the various heparins range from 8.8 to 11.3 × 105M−1, corresponding to a free energy of −8 kcal mol−1. The degree of cooperativity, which is a direct measure of dye–dye interaction, varies with polymer:dye ratio; the theoretical basis for this variation remains to be elucidated. Electrophoretic data indicate that each heparin sample consists of a mixture of species, each with its own charge density. This precludes definitive interpretation of observed small differences in the values of the thermodynamic parameters among the various samples until each sample can be resolved into its components.

Action of the heparin polyanion on chromatin preparations obtained in solutions of low ionic strength

DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b.

Chalone-like effect abrogated by dextran sulphate and heparin polyanion pretreatment of target cells.

Chalone prepared from primary BALB/c mouse embryo fibroblasts caused a 33% reduction in incorporation of tritiated thymidine in the cultures of the second in vitro passage of BALB/c embryo fibroblasts, whereas chalone prepared from thymus, skin and spleen was without effect. Pretreatment of BALB/c secondary fibroblasts with the polyanions dextran sulphate and heparin abrogated the chalone effect. The polycations DEAE-dextran and polybrene were without effect. The effect of incubation with the dextran sulphate polyanion was reversed when followed by incubation with DEAE-dextran polycation.