Range Of Heparin Concentrations Research Articles

Heparin Binding to Resting and Activated Platelets

AbstractHeparin inhibits platelet function and can cause thrombocytopenia. In an effort to understand these phenomena, we have measured the binding of (3H)-heparin to resting and stimulated platelets. In platelet-rich plasma, a single class of saturable heparin binding sites was observed (apparent dissociation constant [kd] ~ 0.55μg/mL, R ~ 0.059 μg/109 cells). In gel-filtered platelets, a similar class of sites was present but with a greater binding capacity (apparent kd1 ~ 0.56 μg/mL, R ~ 0.44 μg/109 cells). Gel-filtered platelets that had been stimulated with thrombin displayed two classes of binding sites: a high-affinity class (apparent kd1 ~1.1 μg/mL, R1 ~ 0.39 μg/109 cells) corresponding to that of the unstimulated cells, and a low-affinity class (apparent kd2 ~13 μg/mL, R2 ~ 2.2 μg/109 cells). Heparin binding was also increased in platelet-rich plasma when the cells had been stimulated by adenosine diphosphate (ADP) to release, but not when ADP caused primary aggregation without release. Binding was not dependent on extracellular calcium, nor was it reduced by monoclonal antibodies to platelet membrane glycoproteins la/Ila, lb, llb/llla, or IV. Because the apparent dissociation constant of the high-affinity sites (~0.55 μg/mL) falls in the range of heparin concentrations achieved clinically, these binding sites may be involved in the platelet dysfunction and immune-mediated thrombocytopenia associated with therapeutic heparin. The low-affinity, high-capacity class of sites, which appears after cell stimulation, may participate in the process of platelet adhesion.

Ultrasensitive detection of heparin by exploiting the silver nanoparticle-enhanced fluorescence of graphitic carbon nitride (g-C3N4) quantum dots.

A composite (Ag-g-CNQDs) was prepared from graphitic carbon nitride quantum dots and silver nanoparticles by water phase synthesis. Aided by metal-enhanced fluorescence, the composite exhibits excitation-dependent red emission with a peak at 600nm with a quantum yield of 21%. If the composite is coated with polyethylenimine (PEI) to form the Ag-g-CNQD/PEI complexe, fluorescence is strongly reduced. Upon addition of heparin, the fluorescence of the system is enhanced because PEI has a higher affinity for heparin than Ag-g-CNQDs. The effect was used to design a fluorometric assay for heparin.The emission at 600nm increases linearly in the 0.025 to 2.5μM heparin concentration range, with a 8.2nM limit of detection. Graphical abstract Schematic illustration for fabricating a composite consisting of silver nanoparticles and graphitic carbon nitride quantum dots (Ag-g-CNQDs). Its redfluorescence is weak in presence of polyethyleneimine but restored on addition of heparin. This forms the basis for anew method for heparin detection.

Effects of Plasma Exchange and Heparin Concentration on the Serotonin Release Assay in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a hypercoagulable state caused by a transient antibody to heparin-bound platelet factor 4 (PF4). Treatment involves discontinuing heparin and administering a nonheparin anticoagulant. Procedures requiring heparin, such as cardiopulmonary bypass, are preferably delayed until the offending antibody is no longer detectable. For patients with a high-titer anti-PF4-heparin antibody and who require exposure to heparin, therapeutic plasma exchange (TPE) has been used to remove the antibody. Recent work indicates that a functional assay for detecting platelet-activating antibodies in HIT patients, the serotonin release assay (SRA), is preferable to ELISAs for anti-PF4-heparin antibodies for following the effectiveness of plasma exchange. Two cases of acute heparin-induced thrombocytopenia managed with plasma exchange before emergent cardiac surgery were evaluated with SRAs using a range of heparin concentrations that included those used in cardiopulmonary bypass. We observed that a single round of plasma exchange led to greater reduction in platelet reactivity at heparin concentrations between 1 and 3 U/mL than at lower concentrations, consistent with the impression that heparin-PF4-antibody complexes form optimally within a limited heparin concentration range. The findings suggest there may be a range of heparin concentration in which cardiac surgery may be safely performed in HIT patients, and that a single TPE in an emergent setting may lower antibody concentration sufficiently to lower platelet reactivity in the presence of heparin.

Significant heparin effect on bovine embryo development during sexed in vitro fertilization.

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

Selective, switchable fluorescent probe for heparin based on aggregation-induced emission

A positively charged tetraphenylethene (TPE) derivative, TPE-4MN, was synthesized as a probe for heparin based on aggregation induced emission. On the addition of 5.0 μg/mL of heparin, TPE-4MN showed an enhanced emission of about 10-fold. The change in fluorescence at 475 nm was linear over a range of heparin concentrations of 0–1.0 μg/mL with an R = 0.99988 and the limit of detection (LOD) was calculated to be 0.75 μg/mL. The mechanism of the detection was proven to be through an ion pairing interaction. TPE-4MN showed good selectivity for heparin over other types of polysaccharides and could easily distinguish heparin from heparan sulfate, a glycosaminoglycan having a similar structure to that of heparin.

Electrochemistry of heparin binding to tau protein on Au surfaces

ABSTRACT The tau protein is a neurodegenerative disease biomarker. The in vitro aggregation of tau is triggered by electrostatic charge imbalance induced by an anionic inducing agent, such as heparin. The binding of the tau-heparin complex is based on electrostatic interactions, but the exact binding mode of heparin to the tau protein has not been fully identified. In this work, the effects of the tau protein orientation on gold (Au) electrode to heparin were explored by the cyclic voltammetry and electrochemical impedance spectroscopy. To modulate the accessibility of N-terminal of the tau to heparin, the tau films on Au surfaces were fabricated in two ways: immobilization of tau via the N-terminal of tau protein (N-tau-Au) or by the Cys291/Cys322 residues, located in the R-repeat domains of the tau protein (Cys-tau-Au). The sulfur-Au bonding was characterized by X-ray photoelectron spectroscopy. The charge transfer resistance was measured for N-tau-Au and Cys-tau-Au as a function of heparin concentration. The heparin concentration range was varied from 0.2 pM to 216 μM with the optimal binding concentration at 21 nM (the highest charge transfer resistance value). The heparin binding to tau films was investigated in the presence of [Fe(CN) 6 ] 3−/4− or benzoquinone redox probes. The tau-heparin binding was greater for the Cys-tau-Au surface over N-tau-Au, indicating specific tau domains may be required for optimal heparin binding.

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.

Heparin Modifies the Immunogenicity of Positively-Charged Proteins.

AbstractAbstract 1435Platelet Factor 4 (PF4)/heparin (H) multimolecular complexes initiate an immune response that can ultimately lead to complications of Heparin-Induced Thrombocytopenia (HIT), a life-threatening prothrombotic disorder. We have previously shown that PF4:H multimolecular complexes assemble through non-specific electrostatic interactions and that other unrelated positively-charged proteins such as protamine (PRT) and lysozyme (Lys) exhibit similar biophysical interactions with heparin (ASH 2009; abstract # 1316). In these earlier studies, we showed that PRT/H and Lys/H, like PF4/H, show heparin-dependent binding over a range of heparin concentrations and that formation of multimolecular complexes occurs at distinct stoichiometric ratios (PRT/H at 3:1 and Lys/H at 5:1 molar ratios). We now extend these observations in vivo to show relevance to human disease. Using a murine immunization model, we show that mice injected with PRT/H and Lys/H multimolecular complexes, but not PRT alone, Lys alone or buffer, develop antigen-specific immune responses. In additional studies, we show that the immune response to PRT/H or Lys/H shares important biologic similarities with the humoral response to murine (m) PF4/H multimolecular complexes. Specifically, we demonstrate that antibody formation to PRT/H and Lys/H is heparin-dependent (occurs optimally at certain stoichiometric ratios) dose-dependent (requires threshold amounts of multimolecular complexes) and shows serologic transience. To demonstrate the clinical relevance of our findings, we examined patients undergoing cardiopulmonary bypass (CPB) for development of PRT/H antibodies. For these studies, we assayed the plasma from healthy subjects (n=45) and patients undergoing CPB (n=15) at three time points {baseline (BL), 5 days (5D) and 30 days (30D) after CPB} for the presence of PRT/H antibodies. As shown Figure 1A, plasma from normal subjects and patients undergoing CPB patients at BL and D5 displayed minimal reactivity in the PRT/H ELISA. However, by 30D, we observed that 4/15 patients (27%) developed significantly elevated levels of antibodies to PRT/H as compared to normals, or their respective samples obtained at baseline or 5D after surgery. Seropositive patients (filled symbols, n=4) as compared to seronegative patients (open symbols, n=3) recognized PRT/H and to some extent, PRT alone, but did not cross-react with other antigens including PRT/H, BSA, Lys, Lys/H or human PF4/H, Figure 1B; p<0.001). To identify the mechanism by which protein/heparin multimolecular complexes triggered immune activation, we incubated murine dendritic cells from non-immunized C57Bl/6 mice with heparin or buffer, protein (mPF4, PRT or Lys), or protein/H complexes and measured IL-12, a marker of dendritic cell activation. As shown in Figure 1C, we demonstrated that IL-12 levels were significantly increased in wells containing protein/H complexes as compared to wells containing uncomplexed protein, buffer or heparin. Taken together, these studies indicate that heparin significantly alters the biophysical and biological properties of positively-charged compounds through formation of macromolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo. [Display omitted] Disclosures:Arepally:Glaxo Smith Kline: Speakers Bureau; Paringenix: Research Funding; University Of New Mexico: Patents & Royalties; Amgen: Speakers Bureau.

Heparin Forms Macromolecular Complexes with Protamine and Lysozyme.

Abstract 1316Poster Board I-340The immune response in Heparin-Induced Thrombocytopenia (HIT) is directed to and initiated by large multimolecular complexes of Platelet Factor 4 (PF4) and heparin. We, and others, have previously shown that PF4 and heparin multimolecular assembly occurs through colloidal interactions, wherein heparin, a negatively charged polymeric compound, facilitates macromolecular assembly by binding and neutralizing PF4's positive charge (Suvarna, Blood 2007). In these same studies, we also demonstrated that changes in the molar ratios of the two reactants result in PF4/heparin (PF4:H) complexes with markedly altered biophysical properties and immunogenicity. Because PF4:H electrostatic interactions are non-specific, we hypothesized that other positively charged proteins would exhibit similar colloidal interactions with heparin. To test this hypothesis, we selected two positively charged proteins (protamine and lysozyme) and studied heparin-dependent complex formation by spectrophotometry (A280nm), and zeta potential (Zeta Sizer, Malvern, UK). Protamine sulfate (250, 125, 62.5, 31.2, 15.6 and 7.8 mcg/mL; Mw 5.1kDa) and lysozyme (1000, 500, 250 and 125 mcg/mL; Mw 14.3kDa) were mixed with various heparin concentrations (0-160 U/mL; activity 140U/mg; Mw 12kDa) and biophysical properties characterized by both instruments. Both protamine and lysozyme showed heparin-dependent complex formation, with peak particle formation occurring over a range of heparin concentrations (2-25 U/mL ) for both compounds. For protamine, particle formation was maximal at protamine:heparin (Pr:H) molar ratios of ∼2.5-3:1, whereas lysozyme formed peak particles at lysozyme;heparin (Ly:H) molar ratios of ∼5:1 (See figure). As with PF4:H complexes, size of complexes was dependent on mass amounts of protamine or lysozyme, with particle size increasing or decreasing in proportion to the amounts of protamine or lysozyme available for complex formation. These findings indicate that heparin is capable of forming macromolecular complexes with other proteins through charge dependent interactions. Additional in vitro and in vivo studies are underway to determine if Pr:H or Lys:H complexes exhibit cross-reactivity with PF4/heparin antibodies and if complex formation is associated with immunological consequences. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bevacizumab Immune Complexes Induce Thrombocytopenia and Thrombosis in Mice Transgenic for Human IgG Receptor FcYIIa

Background: The anti-VEGF drug, bevacizumab (Bev), has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Prevailing opinion on the molecular mechanism behind Bev-associated bleeding and thrombosis is that tissue factor driven coagulation, secondary to vascular endothelial cell dysfunction, may cause thrombosis due to VEGF suppression by Bev. Bev forms immune complexes (IC) with VEGF (vascular endothelial growth factor), a heparin-binding protein. In our previous in vitro studies we showed that, in the presence of heparin, Bev+VEGF immune complexes activate platelets via the IgG receptor FcγRIIa —a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia (HIT).Objectives: First, we investigated whether Bev-associated thrombosis might be replicated in mice. Because mouse platelets do not carry FcγRIIa, we used mice transgenic for this human IgG receptor (hFcR mice) in order to enable the signaling pathway identified above. Second, using human platelets in vitro, we studied the functional roles of heparin and platelet surface localization of IC in Bev-induced FcγRIIa activation.Methods: Bev+VEGF IC were preformed using VEGF165 or VEGF121 (similar to VEGF165 but lacking the heparin-binding domain). Platelet dense granule release and aggregation were measured by the serotonin release assay (SRA) and Chrono- Log aggregometers, respectively. Platelet surface localization was assessed by flow cytometry (50,000 events/test condition) and fluorescence microscopy using Alexa488- labeled Bev (Bev488). For in vivo studies, Bev+VEGF+Heparin IC (60–500 nM) or control reagents were injected intravenously into wild-type (WT) or hFcR mice. Platelet counts were measured 10–60 minutes following IC injection after obtaining blood (0.45 ml) by cardiac puncture. Immediately afterward, lungs were processed for hematoxylin and eosin staining and analyzed microscopically for evidence of thrombosis.Results: IC consisting of Bev+VEGF165+Heparin (0.2U/ml) caused thrombotic thrombocytopenia in hFcR but not WT mice, showing a requirement for FcγRIIa. Injection of Bev+VEGF121+Heparin (0.2U/ml) into hFcR mice did not cause thrombocytopenia, suggesting a requirement for the VEGF165 heparin binding domain. Bev+VEGF165 was without effect in the absence of heparin or in the presence of excess (200 U/ml) heparin demonstrating that a limited range of heparin concentrations enable Bev-induced thrombocytopenia and thrombosis. This mechanism is similar to that observed in HIT and our in vivo results were consistent with SRA and aggregation in vitro studies. By flow cytometry, maximal Fab-dependent Bev488 platelet surface binding occured only with VEGF165+0.2U/ml heparin. Saturating IV.3 (anti-FcγRIIa antibody) concentrations, present in all samples, excluded Bev-Fc binding to FcγRIIa. Furthermore, binding of Bev488+VEGF121+0.2 U/ml heparin was not detected, suggesting the VEGF heparin binding domain is required for heparin-enhanced surface binding.Conclusions: In the presence of heparin, Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of FcγRIIa receptor. This mechanism may be relevant to the thromboembolic complications observed in patients receiving Bev therapy.

Potentiation and inhibition of bFGF binding by heparin: a model for regulation of cellular response.

Basic fibroblast growth factor (bFGF) binds to cell surface tyrosine kinase receptor proteins and to heparan sulfate proteoglycans. The interaction of bFGF with heparan sulfate on the cell surface has been demonstrated to impact receptor binding and biological activity. bFGF receptor binding affinity is reduced on cells that do not express heparan sulfate. The addition of soluble heparin or heparan sulfate has been demonstrated to rescue the bFGF receptor binding affinity on heparan sulfate deficient cells yet has also been shown to inhibit binding under some conditions. While the chemical requirements of the heparin-bFGF-receptor interactions have been studied in detail, the possibility that heparin enhances bFGF binding in part by physically associating with the cell surface has not been fully evaluated. In the study presented here, we have investigated the possibility that heparin binding to the cell surface might play a role in modulating bFGF receptor binding and activity. Balb/c3T3 cells were treated with various concentrations of sodium chlorate, so as to express a range of endogenous heparan sulfate sites, and [(125)I]bFGF binding was assessed in the presence of a range of heparin concentrations. Low concentrations of heparin (0.1-30 nM) enhanced bFGF receptor binding to an extent that was inversely proportional to the amount of endogenous heparan sulfate sites present. At high concentrations (10 microM), heparin inhibited bFGF receptor binding in cells under all conditions. The ability of heparin to stimulate and inhibit bFGF-receptor binding correlated with altered bFGF-stimulated tyrosine kinase activity and cell proliferation. Under control and chlorate-treated conditions, [(125) I]heparin was observed to bind with a high affinity to a large number of binding sites on the cells (K(d) = 57 and 50 nM with 3.5 x 10(6) and 3.6 x 10(6) sites/cell for control and chlorate-treated cells, respectively). A mathematical model of this process revealed that the dual functions of heparin in bFGF binding were accurately represented by heparin cell binding-mediated stimulation and soluble heparin-mediated inhibition of bFGF receptor binding.

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Biphasic response of complement to heparin: fluid-phase generation of neoantigens in human serum and in a reconstituted alternative pathway amplification cycle.

We describe a study of the effects of heparin on complement activation through the use of assays for fragment C4d, fragment Bb, and the S-C5b-9 complex (S-MAC). In sera from healthy volunteers, virtually no change was observed in C4d either as a function of time or of heparin concentration, whereas changes in Bb and S-MAC were biphasic. This observation was explored in greater detail in the heparin concentration range 0.001-5.0 u/ml (5 x 10(-3) to 25 micrograms/ml). For both Bb and S-MAC, a significant (P < 0.05) increase in production was noted in the heparin concentration range, 0.01-0.5 u/ml (5 x 10(-2) to 2.5 micrograms/ml). At higher heparin concentrations, Bb and S-MAC production decreased markedly (P < 0.05). We reconstituted the alternative pathway amplification cycle (C3, factor B, and factor D) and studied Bb generation. With reactants at concentrations one tenth those of normal serum, we observed a maximum generation of 13.2 micrograms/ml Bb. Control and heparin at 5 x 10(-4) micrograms/ml generated Bb concentrations of 6.8 and 6.1 micrograms/ml, respectively, for a 2-min incubation; at 5 x 10(-3) micrograms/ml heparin, Bb was increased to 9.8 micrograms/ml. Using isoelectric focusing to study anionic pI shifts in heparin-bound factors B and D, it was found that factor B bound heparin only at the highest heparin concentration studied, i.e., 50 micrograms/ml; factor D, however, bound heparin at a much lower concentration (0.05 micrograms/ml). We conclude that, at low concentrations, heparin activates complement due to potentiation of the alternative pathway amplification cycle in the fluid phase.

Effect of heparin on whole blood activated partial thromboplastin time using a portable, whole blood coagulation monitor

To evaluate the responsiveness of whole blood activated partial thromboplastin time (aPTT) to varying heparin doses in vitro and to examine the ex vivo relationship of whole blood aPTT to plasma heparin concentration. Prospective, controlled laboratory study. Surgical suites and laboratory at a tertiary center. Surgical patients and volunteers at a tertiary center were eligible for inclusion in this study. In vitro evaluation was performed using specimens obtained from each of five, healthy volunteers. Ex vivo evaluation was performed using specimens obtained from 30 cardiac surgical patients before and after systemic administration of heparin for extracorporeal circulation. Blood specimens were obtained from volunteers and added to syringes containing varying amounts of unfractionated porcine heparin for in vitro evaluation. For ex vivo evaluation, blood specimens were obtained from patients before and after systemic administration of 20 U/kg of heparin. For the in vitro evaluation, specimens were divided into two aliquots after mixing with varying amounts of unfractionated porcine heparin. One aliquot was used to measure whole blood aPTT using a whole blood coagulation monitor immediately after blood collection and 3 mins later, and a second aliquot was used to determine plasma aPTT with a conventional, laboratory-based assay. Linear regression analysis demonstrated a high correlation (r = .94; r2 = .88) between aPTT assay systems and bias analysis demonstrated a mean aPTT measurement difference of 1.6 secs with +/- 2 SD limits of -15 to +18.2 secs. As indicated by comparable regression slopes, the in vitro aPTT responsiveness to increasing heparin concentration was similar with the two assay systems among individual subjects. Whole blood aPTT measurements after 3 mins of blood specimen storage were similar to immediate measurements. For ex vivo evaluation, blood specimens obtained from patients before and after systemic administration of heparin were divided into two aliquots. One aliquot was used to measure whole blood aPTT in duplicate and a second aliquot was used to measure plasma heparin concentration with an antifactor X active chromogenic assay. A high correlation (r = .89; r2 = .79) between whole blood aPTT and plasma heparin concentration was observed. Heparin responsiveness of whole blood aPTT, measured with a portable whole blood coagulation monitor, is similar to that of conventional laboratory aPTT over a clinically relevant range of heparin concentrations in vitro and ex vivo. On-site whole blood aPTT measurements should be useful in clinical situations requiring rapid aPTT results.

Heparin-Induced Increase in the International Normalized Ratio:Responses of 10 Commercial Thromboplastin Reagents

A clinician's concern about an erratic response to oral anticoagulation in a patient treated concurrently with heparin and warfarin led the authors to investigate the effect of heparin on INR values obtained with various commercial thromboplastin reagents. Studies conducted with pooled normal plasma and with pooled plasma from patients treated long-term with oral anticoagulants demonstrated a wide range of sensitivities to heparin of these reagents as characterized by prolongation of INR values. Innovin was unaffected by concentrations of heparin as high as 1 U/mL. In contrast, Ortho thromboplastin showed the greatest increase in INR values over the range of heparin concentrations studied. Three other thromboplastins including Neoplastine CI, Dade thromboplastin, and Simplastin A demonstrated only limited sensitivity to heparin. Prolongation of the INR by heparin was reversed by protamine in a dose-related manner and also by preincubation of the plasma with heparinase. Some patients treated with warfarin while on the authors' institutional protocol for heparin had plasma concentrations greater than 0.8 U/mL. When thromboplastin reagents sensitive to heparin were used with such specimens, the INR values obtained were falsely elevated. The authors suggest that reagents insensitive to heparin be employed to avoid this difficulty.

Inhibition by heparin of the human blood coagulation intrinsic pathway factor X activator.

The effect of heparin and other glycosaminoglycans on the activation of factor X by the phospholipid membrane-bound human factor IXa-factor VIIIa complex (intrinsic fXase) was studied. Standard heparin inhibited purified intrinsic fXase by 50% at approximately 0.08 unit/ml (0.4 microgram/ml), which is below the normal range of heparin concentrations achieved during antithrombotic therapy (0.2-0.7 unit/ml). Kinetic and binding experiments revealed that heparin behaves as a partial noncompetitive inhibitor. The inhibition constant of heparin with low affinity for antithrombin was indistinguishable from heparin with high affinity for antithrombin (Ki = 20 nM). Additionally, "low molecular weight" heparin, which also is used as an antithrombotic drug, was a potent inhibitor of intrinsic fXase (Ki = 60 nM). Dermatan sulfate inhibited intrinsic fXase much more weakly than standard heparin (IC50 = 80 micrograms/ml). The IC50 of the other mammalian glycosaminoglycans, chondroitin sulfate, keratan sulfate, and hyaluronic acid, were greater than 100 micrograms/ml. Purified prothrombinase and extrinsic fXase were not inhibited by heparin. We propose that part of the antithrombotic action of heparin and low molecular weight heparin is due to anti-thrombin-independent inhibition of intrinsic fXase and that heparin with low affinity for antithrombin may be useful as an antithrombotic agent.

Inhibition by heparin of thrombin-catalyzed activation of the factor VIII-von Willebrand factor complex.

The activation of factor VIII (fVIII) by thrombin is associated with heavy chain cleavages at Arg372 and Arg740 and light chain cleavage at Arg1689. In a defined, plasma-free assay of fVIII activation and at physiological ionic strength and pH, heparin inhibited the rate of activation of either human or porcine fVIII by thrombin in either the presence or absence of von Willebrand factor (vWf). The inhibitory effect of heparin was associated with inhibition of all three thrombin-catalyzed bond cleavages. At plasma concentrations of fVIII (approximately 1 nM) and vWf (approximately 35 nM), the rate of fVIII activation was inhibited by 50% at approximately 0.1 unit/ml heparin, which is below the normal range of heparin concentrations in plasma during therapeutic anticoagulation (0.2-0.7 unit/ml). We propose that, in addition to catalyzing the inhibition of thrombin and other intrinsic pathway coagulation proteases by antithrombin, heparin functions as an anticoagulant by direct inhibition of the activation of the fVIII-vWf complex by thrombin.

Predictive Performance of Three Methods of Activated Clotting Time Measurement in Neonatal ECMO Patients

Previously reported activated clotting time (ACT) data in adults demonstrated higher values with the HemoTec LRACT (HT) and TriMed ACTivator (TM) techniques than with the Hemochron System P214/215 (HC) technique throughout a range of heparin concentrations. This study sought to determine if a difference exists in ACT values of neonatal patients receiving ECMO. ACTs were performed in nine neonatal ECMO patients using the HC, HT, and TM techniques. Techniques were compared for positive or negative direction of any prediction difference (bias), and the typical value of a difference (precision). Simultaneous, duplicate, morning, and afternoon ACT comparisons were obtained using all three techniques. Forty-six comparisons of HC values in the 180-240 sec range were analyzed. All techniques produced results different from the same sample. The HT and TM techniques were upwardly biased by 51 and 148 sec, respectively, when evaluated against HC. HT was negatively biased by 123 sec when evaluated against TM. Because ACT values vary among techniques, ACT target ranges should be technique specific. Future references to ACT data should identify the equipment and procedures employed.

Heparin-stimulated inhibition of factor IXa generation and factor IXa neutralization in plasma

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.

Heparin-Stimulated Inhibition of Factor IXa Generation and Factor IXa Neutralization in Plasma

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.