Increasing Concentrations Of Heparin Research Articles

Heparin-mediated PCR interference in SARS-CoV-2 assays and subsequent reversal with heparinase I

Heparin is postulated to block the interaction of SARS-CoV-2 with highly glycosylated proteins which are critical for binding the angiotensin-converting enzyme 2 (ACE2), an essential mechanism for host-cell entry and viral replication. Intranasal heparin is under investigation for use as a SARS-CoV-2 preventative in the IntraNasal Heparin Trial (INHERIT, NCT05204550). Heparin directly interferes with real-time quantitative polymerase chain reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection. This study aimed to investigate the magnitude of heparin interference across various clinical laboratory testing platforms, and the reversal of any interference by degradation of heparin using the heparinase I enzyme in nasopharyngeal swab (NP) samples for SARS-CoV-2 analysis by RT-qPCR. Heparin-mediated PCR interference was evident at heparin concentrations as low as 10 IU/mL across all platforms tested, with the exclusion of the Hologic Panther Aptima SARS-CoV-2 assay. Rates of false negative or invalid results increased with increasing heparin concentrations on all platforms, except the Hologic Panther Aptima and Roche Cobas LIAT. Heparinase I reversed heparin-mediated PCR inhibition across in all samples tested, except those with initial Ct values >35. Our study shows that the use of heparin-containing nasal sprays interferes with the detection of SARS-CoV-2 in NP swab samples by RT-qPCR, a phenomenon that is not well recognised in the literature. Furthermore, this study has also demonstrated that heparin-mediated PCR inhibition can be prevented through heparinase I treatment, demonstrating restoration of clinically significant results with Ct values <35.

A pilot study evaluating the Calibrated Automated Thrombogram assay and application of plasma-thromboelastography for detection of hemostatic aberrations in horses with gastrointestinal disease

BackgroundCritically ill horses, such as horses with gastrointestinal (GI) disease, often suffer from hemostatic aberrations. Global hemostatic tests examining the initiation of coagulation, clot strength and fibrinolysis, such as the Calibrated Automated Thrombogram (CAT) and plasma-thromboelastography (TEG) have not been evaluated in horses. This study aimed to evaluate CAT and apply plasma-TEG in horses.Test performance of CAT was evaluated on equine platelet poor plasma with intra- and inter-assay variability (CV) and a heparin dilution curve. To examine clinical performance of both tests, group comparisons were assessed comparing healthy horses, horses with mild and severe GI disease with both CAT and plasma-TEG.ResultsFor CAT, intra- and inter-assay CVs were established for lag-time (1.7, 4.7%), endogenous thrombin potential (1.6, 4.6%), peak (2.6, 3.9%) and time to peak (ttPeak) (1.9, 3.4%). Increasing heparin concentrations led to the expected decrease in thrombin generation. In the group comparison analysis, CAT showed significant higher peak (p = 0.04) and ttPeak (p = 0.008) in the severe GI disease group compared to horses with mild GI disease and healthy horses, respectively. Plasma-TEG showed an increased angle (p = 0.032), maximum amplitude (p = 0.017) and shear elastic force (G) (p = 0.017) in the severe GI disease group compared to healthy horses.ConclusionsCAT performed well in horses. Both CAT and plasma-TEG identified hemostatic aberrations in horses with severe GI disease compared to healthy horses. Further studies including more horses, are needed to fully appreciate the use of CAT and plasma-TEG in this species.

Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy.

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0μm) over a volumetric field-of-view (165 × 4000 × 36 μm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p=0.03) despite comparable fibrin time (baseline=6.0 ± 1.3, protamine=6.4 ± 1.6min, p=0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline=0.11 ± 0.02 A.U, protamine=0.08 ± 0.01 A.U, p=0.008) with straighter fibers (baseline=0.918 ± 0.003A.U, protamine=0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients.

Sensitivity improved with Parylene-C passivized on Lamb wave sensor for aPTT measurement through monitoring whole blood reaction

Blood clotting time monitoring is of great importance in predicting the risk of haemorrhage and cardiovascular diseases. In this study, a new P-Lamb wave sensor was developed to measure activated partial thromboplastic time (aPTT) with enhanced sensitivity. The sensor utilizes a piezoelectric and hydrophobic material, Parylene-C, as a waveguide for the P-Lamb wave sensor in order to monitor the aPTT in whole blood samples. With a smooth surface, the P-Lamb wave sensor reduced energy loss and prevented leaky modes, with a decrease of 33% ± 1.25% in the surface roughness and an increase of 2.75% ± 0.15% in the signal. The experimental results demonstrated that, compared to a quartz crystal microbalance (QCM), a P-Lamb sensor with high sensitivity (the frequency shift of a P-Lamb sensor is approximately 200 times greater than that of a QCM) improved stability and enhanced repeatability. Parylene-C worked as a phonon coupling layer, which improved the stability and sensitivity of the Lamb sensor. The relative standards deviation (RSD) of the aPTT measurement was 2.11%, which was 1.22 times lower than reported values. The P-Lamb wave sensor was also tested for its sensitivity to different concentrations of heparin and was found to exhibit an increase in coagulation time with an increasing heparin concentration. Compared to the SYSMEX CS 5100 haematology analyser, the clinical coefficient index (R2) was 0.99467. Considering its compact size, low cost, and mass production of the chip unit, the developed P-Lamb wave sensor is a promising device for point-of-care diagnosis of haemostasis and personal health monitoring.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host’s invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold.

Extracellular matrix (ECM) maintains a reservoir of bioactive growth factors and matricellular proteins that provide bioinductive effects on local cells that influence phenotype and behaviors. Bioactive acellular ECM scaffolds can be used therapeutically to stimulate adaptive tissue repair. Fibroblast growth factor-2 (FGF-2) attenuates transforming growth factor-β1 (TGF-β1)-mediated cardiac fibrosis. Heparin glycosaminoglycan can influence FGF-2 bioactivity and could be leveraged to enhance tissue engineering strategies. We explored the effects of heparin on FGF-2 enhancement of bioactive ECM scaffold biomaterials for its antifibrotic effect on attenuating human cardiac myofibroblast activation. Increasing heparin concentration at a fixed concentration of FGF-2 markedly increased the amount of FGF-2 retained and eluted by ECM scaffolds. To explore synergistic bioinductive effects of heparin and FGF-2, collagen gel contraction assay using human cardiac myofibroblasts was performed in vitro. Myofibroblast activation was induced by profibrotic cytokine, TGF-β1. FGF-2 and heparin in combination reduced human cardiac myofibroblast-mediated collagen gel contraction to a greater extent than FGF-2 alone. These observations were confirmed for both human atrial and human ventricular cardiac fibroblasts. Cell death was not different between groups. In summary, heparin is an effective adjuvant to enhance FGF-2 loading and elution of acellular ECM scaffold biomaterials. Heparin increases the bioactive effects of FGF-2 in attenuating human cardiac myofibroblast activation in response to profibrotic TGF-β1. These data may inform tissue engineering strategies for myocardial repair to prevent fibrosis.

A simple and sensitive label-free fluorescence sensing of heparin based on Cdte quantum dots.

A rapid, simple and sensitive label-free fluorescence method was developed for the determination of trace amounts of an important drug, heparin. This new method was based on water-soluble glutathione-capped CdTe quantum dots (CdTe QDs) as the luminescent probe. CdTe QDs were prepared according to the published protocol and the sizes of these nanoparticles were verified through transmission electron microscopy (TEM), X-ray diffraction (XRD) and dynamic light scattering (DLS) with an average particle size of about 7 nm. The fluorescence intensity of glutathione-capped CdTe QDs increased with increasing heparin concentration. These changes were followed as the analytical signal. Effective variables such as pH, QD concentration and incubation time were optimized. At the optimum conditions, with this optical method, heparin could be measured within the range 10.0-200.0 ng mL(-1) with a low limit of detection, 2.0 ng mL(-1) . The constructed fluorescence sensor was also applied successfully for the determination of heparin in human serum. Copyright © 2015 John Wiley & Sons, Ltd.

Heparin increases the osteogenic effect of recombinant human bone morphogenetic protein-2 in the rabbit bone defect model

In order to perform successful dental implantations and solve other problems such as bone deficiencies, it is important to find suitable biomaterials to enhance osteoinduction. Heparin has been known to enhance bone morphogenetic protein 2 (BMP-2) induced-bone formation. We histomorphometrically investigated the enhancing effects and roles of heparin to recombinant human bone morphogenetic proteins-2 (rhBMP-2). For morphologic study, stem cells obtained from rabbit adipose tissue were divided into four groups based on heparin concentrations, with a constant rhBMP-2 concentration. They were cultured for 2, 4, and 8 days. Alkaline phosphatase and calcium content were measured as time schedules. In addition, a mixture of rhBMP-2 and heparin was blotted into multiporous anorganic bovine bone and was inserted into calvarial defects in rabbits. The harvested tissues were stained using the Hematoxylin & Eosin (H&E) and Masson's trichrome (MT) stain. And the areas of newly formed bone in the grafted material were analyzed. In morphologic results, the degree of osteoblastic differentiation was significantly increased with increasing heparin concentrations, but the cellular degeneration was accelerated at a higher concentration of heparin as time passed. In histological results, the more higher the concentration of heparin, larger newly formed bone in grafted materials was also observed in the initial period. However, the increased amount of the newly formed bone in grafted materials was progressively decreased at higher concentrations of heparin as time passed. In conclusion, heparin might have an influence on the osteoinductive effects of rhBMP-2 during the initial stage of bone formation.

Microfluidic device and system for point-of-care blood coagulation measurement based on electrical impedance sensing

Blood coagulation tests are an integral part of diagnosis and treatment of patients with cardiac disorders. The activated partial thromboplastin time test (aPTT or APTT) is performed on a large scale, in laboratories, to monitor the functioning of the intrinsic and common pathways of the blood coagulation cascade and the concentration of anticoagulants such as heparin among patients. A polydimethylsiloxane (PDMS) based microfluidic device has been fabricated and tested to perform APTT as a point-of-care device using whole blood samples by detecting the change in electrical impedance of blood during coagulation. A portable setup consisting of the device and a lock-in amplifier circuit was built and tested for repeatability and stand alone usage. A frequency sweep test was performed on the device and the optimal frequency of operation for maximum output sensitivity was determined. The devices were also tested for their sensitivity to different concentrations of heparin and found to exhibit an expected increase in coagulation time for increasing heparin concentration.

Encapsulation and Controlled Release of Heparin from Electrospun Poly(L-Lactide-co-ε-Caprolactone) Nanofibers

Poly(L-lactide-co-ε-caprolactone) nanofibers with heparin incorporated were successfully fabricated by coaxial electrospinning. The morphologies of electrospun nanofibers were studied by scanning electron microscopy (SEM), and a significant decrease in fiber diameter was observed with increasing heparin concentration. The transmission electron microscopy (TEM) images indicated that coaxial electrospinning could generate core–shell structure nanofibers which have the potential to encapsulate drugs (heparin in this study) into the core part of nanofibers. Approximately 80% of the encapsulated heparin was sustainedly and stably released from the fibrous composite in 14 days by a diffusion/erosion coupled mechanism. The release behavior of heparin from blend electrospun nanofibers was also studied and showed an obvious burst release in the initial stage. An in vitro proliferation test was conducted to study the effect of heparin released from nanofibers, and the results suggest that the heparin maintains its bioactivity after encapsulating with and delivery through coaxially electrospun fibers.

Interlaboratory variation in heparin monitoring: Lessons from the Quality Management Program of Ontario coagulation surveys

Unfractionated heparin (UFH) monitoring is subject to substantial interlaboratory variation. We analysed results of annual coagulation surveys administered by the Quality Management Program - Laboratory Services (Toronto, ON, Canada) from 2003 to 2007 to evaluate variation in UFH monitoring across Ontario. Participating laboratories performed an activated partial thromboplastin time (APTT) utilising their local methodology on lyophilised human plasma spiked with UFH. In the 2006 and 2007 surveys, laboratories licensed to perform anti-Xa assays also reported anti-Xa activity results. The APTT differed significantly between heparin-sensitive and heparin-insensitive methods (p<0.0005). Within-method variation was observed and increased with increasing heparin concentration. Among laboratories performing an APTT and anti-Xa, the coefficient of variation was greater in the anti-Xa than in the APTT for both the 2006 (64.0% vs. 10.5%) and 2007 (15.0% vs. 11.6%) surveys. Substantial interlaboratory variation in UFH monitoring, both between and within APTT methods, was observed and was not reduced by use of an anti-Xa assay.

A Multicolor Cationic Conjugated Polymer for Naked-Eye Detection and Quantification of Heparin

A new cationic polyfluorene derivative with 20 mol % 2,1,3-benzothiadiazole (BT) content was synthesized via Suzuki cross-coupling polymerization. The high charge density and cationic oligo(ethylene oxide) side chains endue the polymer with a good water solubility (∼12 mg/mL), leading to a low inherent BT emission background in buffer. Addition of negatively charged heparin into the polymer solution induces polymer aggregation, giving rise to enhanced energy transfer from the fluorene segments to the BT units. With increasing heparin concentrations, the orange BT emission intensity progressively increases at the expense of the blue fluorene emission. In contrast, addition of hyaluronic acid, an analogue of heparin, results in an insignificant enhancement in BT emission. This selective optical signature not only allows distinguishing heparin from hyaluronic acid but also highlights the importance of electrostatic attraction between the polymer and the analyte in an energy transfer process. Heparin quantifi...

Controlled release of heparin from poly( ε-caprolactone) electrospun fibers

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly( ε-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8% w/v PCL in 7:3 dichloromethane:methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury.

Collection of neural inducing factors from PA6 cells using heparin solution and their immobilization on plastic culture dishes for the induction of neurons from embryonic stem cells

Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinson's disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.

Heparin reacts with and inactivates nitric oxide.

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor. S-nitroso-glutathione (SNO-Glu), with an EC(50) of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NO(x) species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NO(x). Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NO(x) species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO(2)/HCl. Negative-ion electro-spray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosamino-glycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.

Troponin T and I Assays Show Decreased Concentrations in Heparin Plasma Compared with Serum: Lower Recoveries in Early than in Late Phases of Myocardial Injury

Heparinized plasma samples allow more rapid analysis than serum samples, but preliminary studies showed lower cardiac troponin T (cTnT) results in plasma. We undertook a multicenter study to characterize this effect for cTnT and cardiac troponin I (cTnI). Blood samples were collected with and without heparin at five hospitals. cTnT was measured by a "third generation" assay (Elecsys((R))), and cTnI was measured by a commercial immunoassay (IMMULITE((R))). Mean cTnT was 15% lower in heparin sampling tubes than in serum. Measured concentrations of cardiac troponins also decreased with increasing heparin concentrations added to sera. Heparin-induced losses were greater in early than in late phases after onset of chest pain. Addition of heparin ( approximately 100 IU/mL) to serial samples from nine acute myocardial infarction patients produced mean cTnT losses of 33% at 1-12 h after onset of chest pain, 17% at 13-48 h, and 7% after 48 h. The changing heparin effects were seen for both cTnT and cTnI during time courses of individual patients with myocardial infarction. We suggest that binding of heparin to troponins decreases immunoreactivity, especially in early phases of myocardial injury. The resulting losses may depend on the antibodies used in each troponin assay.

Apoptotic effects of heparin on lymphoblasts, neutrophils, and mononuclear cells: results of a preliminary in vitro study.

In this study the apoptotic effects of heparin on lymphoblasts, neutrophils, and mononuclear cells were evaluated by flow cytometry for detection of sub-G1 peak, in vitro. Ten children with acute lymphoblastic leukemia (ALL) at diagnosis (Group I), six children with ALL at relapse (Group II), and 10 healthy children (controls) were included in this study. Lymphoblasts in ALL patients, and neutrophils and mononuclear cells in controls, were incubated in increasing heparin concentrations (0, 5, 10, 20 U/ml). Flow cytometric analyses were performed at 0, 1, and 2 hours of incubation in heparin for determination of the apoptotic effects of heparin. In Group I apoptosis was detected in all different levels of heparin concentration except 0 U/ml at 0, 1, and 2 hours. The apoptotic effects of heparin on blast cells peaked at the first hour in 5-, 10-, and 20-U/ml heparin concentrations (p < 0.0001). In Group II similar findings were observed only at zero hour and apoptosis was higher than those in Group I except in 5-U/ml heparin concentration (p < 0.001). Apoptosis was found to increase with heparin levels in both groups (p < 0.02). In the control group, apoptosis was detected only at the 20-U/ml heparin concentration and only at the first and second hours. Lymphoblasts are more sensitive to apoptotic effects of heparin than either neutrophils and mononuclear cells (p < 0.004). It can be suggested that low-dose heparin may cause significant apoptosis of lymphoblasts while inducing no apoptosis on neutrophils and mononuclear cells. The findings of this preliminary study indicate that further and more comprehensive research on the apoptotic effect of heparin on lymphoblasts should be done.

Biointeractive polymers and tissue engineered blood vessels

The regulation of endothelial cell (EC) and smooth muscle cell (SMC) proliferation following vascular interventions is critical to clinical efficacy. Our laboratory has developed a method of impregnating biomaterials with suspensions containing bioactive proteins resulting in the capability of differentially modulating EC and SMC growth in vitro and in vivo following implantation. We have previously reported that 60 μ internodal distance ePTFE grafts impregnated with fibrin glue (FG) containing FGF-1 and heparin develop confluent endothelialization with transiently increased EC and SMC proliferation after 4 weeks in dogs. Thoraco-abdominal implants after 20 weeks were developed significantly thicker (139 μ) inner capsules in response to the FGF. To minimize SMC proliferation we studied the effects of FGF-1, heparin, and thrombin concentrations on SMC growth in vitro. FG caused a 182% increase ( P < 0.001) in DNA synthesis. Heparin within FG diminished this effect in a dose-dependant manner, with complete inhibition of FG-induced growth at 500 U ml −1 (versus FG alone, P < 0.001). FGF-1 within FG without heparin had no effect, but together, FGF-1 caused a dose-dependant growth increase while increasing heparin concentrations initially increased and then decreased proliferation. FGF-1 and heparin in the medium of quiescent SMCs had similar effects. Only thrombin concentrations >3.2 U ml −1 stimulated SMC growth and this stimulation was blocked by heparin. A synergism between FGF and heparin on EC proliferation was also found but without EC growth inhibition in response to higher concentrations of heparin. It is thus possible to modulate the relative proliferative activity of ECs versus SMCs by altering the FGF:heparin ratio. This same system may be useful with other proteins to induce other local affects by the applied protein or systemic affects following release of that protein.

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.

In Vitro Reversal of Heparin Effect with Heparinas

This study was designed to evaluate the potential in vitro use of heparinase to eliminate functionally active heparin prior to performing whole blood (WB) prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. A total of 250 U/kg of heparin for cardiopulmonary bypass (CPB) was administered to 30 cardiac surgical patients in three consecutive, divided doses (20, 80, and 150 U/kg) at 15-min intervals. Blood specimens were obtained prior to heparin administration (baseline) and 10 min after each heparin dose. After collection, blood specimens were fractionated into three aliquots of which the first was used for determination of heparin concentration. After gentle mixing, WB PT and APTT measurements were performed for heparinase (Aliquot 2)- and nonheparinase (Aliquot 3)-treated blood. With consecutive heparin doses of 20 and 80 U/kg, WB PT increased from a baseline of 12.3 +/- 0.1 s to 13.3 +/- 0.2 and 18.5 +/- 1.3 s, while WB APTT increased from a baseline of 28.3 +/- 1.1 s to 89.5 +/- 5.4 after the initial heparin dose (20 U/kg). When compared to baseline (no heparin) results, small, progressive increases in heparinase-treated WB PT (0.7 +/- 0.1, 1.5 +/- 0.1, 2.1 +/- 0.1 s) and APTT (2.3 +/- 0.3, 5.7 +/- 0.4, 9.5 +/- 0.5 s) were seen with increasing heparin concentration (0.23, 1.58, and 3.95 U/mL, respectively). Heparinase was highly effective in eliminating the anticoagulant effects of even large amounts of heparin in plasma from cardiac surgical patients.(ABSTRACT TRUNCATED AT 250 WORDS)