aPTT Assay Research Articles

Bivalirudin and ECLS

Bivalirudin and ECLS

Basic coagulation tests as surrogates of dabigatran levels in a pre-operative setting: Analysis of five activated partial thromboplastin time reagents and thrombin time

BackgroundIn patients who are receiving dabigatran, a direct oral anticoagulant, measuring the anticoagulant effect before surgery may be needed in certain circumstances. Although the dilute thrombin time (dTT) can reliably measure dabigatran levels, it is not consistently available. More commonly used coagulation tests, including the activated partial thromboplastin time (aPTT) and thrombin time (TT) might have clinical utility but their accuracy is uncertain. Methods103 patients stopped dabigatran 1–4 days before an elective surgery/procedure as part of a standardized dabigatran interruption protocol. With a blood sample taken just before surgery, we assessed the accuracy of five aPTT assays (Actin FS, Stago PTT, C.K. PREST, HemosIL aPTT-SP, SynthASil) and TT to measure the residual anticoagulant effect of dabigatran. We determined the sensitivity, specificity and other accuracy indices of these assays to predict a dabigatran level > 30 ng/mL as determined by a reference standard test, the dTT (Hemoclot). ResultsOf five aPTT reagents, four assays had excellent (100%) and one assay had good (93%) sensitivity to detect a level of dabigatran > 30 ng/mL, but all had insufficient specificity (50–74%). A TT > 90 s had good sensitivity (93%) and excellent specificity (100%). ConclusionFive aPTT assays had good sensitivity but poor specificity to detect low levels of dabigatran (≤30 ng/mL) after standardized dabigatran interruption before an elective surgery/procedure, thereby limiting the use of aPTT as an alternative to the dTT in preoperative settings.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

Reversible covalent direct thrombin inhibitors.

IntroductionIn recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.MethodsWe have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.ResultsWe demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.ConclusionCompound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.

Modification of ulvans via periodate-chlorite oxidation: Chemical characterization and anticoagulant activity

Native (F2) and carboxyl-reduced (R) ulvans from Ulva fasciata were sequentially oxidized with periodate-chlorite affording the polycarboxyl ulvans C1, C2 and C3 (1.20, 1.41 and 1.81 mmol g−1 of COOH, respectively; 19.7, 21.3 and 21.0% of NaSO3, respectively) and R-C3 (1.86 mmol g−1 of COOH; NaSO3 = 22.7%), respectively. APTT assay (polysaccharide fractions at 150 μg mL−1) showed clotting time of 45.6 s for F2 fraction. For polycarboxyl ulvans C1, C2, C3 and R-C3 the clotting times were 101.0, 122.2, 222.0 and 227.0 s, respectively. Comparison of the APTT assay results using ulvans chemically modified by carboxyl-reduction, desulfation, periodate oxidation and/or chlorite oxidation showed the anticoagulant activity of polycarboxyl ulvans is dependent of the sulfate groups present in the native polymer. In addition, the increase of the anticoagulant activity was accompanied by the increasing of the carboxyl groups and the content of this acidic substituent seems to be more important than its positioning.

Antithrombotic therapy in pediatric ventricular assist devices: Multicenter survey of the European EXCOR Pediatric Investigator Group.

Mechanical circulatory support for pediatric heart failure patients with the Berlin Heart EXCOR ventricular assist system is the only approved and established bridging strategy for recovery or heart transplantation. In recent years, the burden of thromboembolic events has led to modifications of the recommended antithrombotic therapy. Therefore, we aimed to assess modifications of antithrombotic practice among the European EXCOR Pediatric Investigator Group members. We sent a questionnaire assessing seven aspects of antithrombotic therapy to 18 European hospitals using the EXCOR device for children. Returned questionnaires were analyzed and identified antithrombotic strategies were descriptively compared to "Edmonton protocol" recommendations developed for the US EXCOR pediatric approval study. Analysis of 18 received surveys revealed substantial deviations from the Edmonton protocol, including earlier start of heparin therapy at 6-12 h postoperatively and in 50% of surveyed centers, monitoring of heparin effectiveness with aPTT assay, administering vitamin K antagonists before 12 months of age. About 39% of centers use higher international normalized ratio targets, and platelet inhibition is changed in 56% including the use of clopidogrel instead of dipyridamole. Significant inter-center variability with multiple deviations from the Edmonton protocol was discovered with only one center following the Edmonton protocol completely. Current antithrombotic practice among European EXCOR users representing the treatment of more than 600 pediatric patients has changed over time with a trend toward a more aggressive therapy. There is a need for systematic evidence-based evaluation and harmonization of developmentally adjusted antithrombotic management practices in prospective studies toward revised recommendations.

Measurement of rivaroxaban concentrations demonstrates lack of clinical utility of a PT, dPT and APTT test in estimating levels.

Rivaroxaban concentrations were measured in 127 inpatient samples using an HPLC-MS/MS assay. We compared this measurement with a calibrated anti-Xa assay and performed PT, aPTT and dilute PT tests to assess the value of clot-based assays in clinical decision-making. The correlation between the anti-Xa assay and the HPLC-MS/MS at therapeutic concentrations was strong (R2 =0.98). The PT, RecombiPlasTin 2G, and aPTT, Actin FS, showed a linear dose-response but poor correlation (R2 =0.32 and 0.44, respectively) and at dilutions of 1 in 150 to 1 in 750 the dilute PT assay also showed poor correlation with rivaroxaban concentrations measured by specific assays. A normal PT or aPTT alone did not identify a likely safe rivaroxaban concentration to allow surgery or invasive procedures, but the combination of normal PT and aPTT identified a group of patients with rivaroxaban levels less than 90ng/mL. Combined normal PT and aPTT had specificity and sensitivity of 0.97 (95% CI 0.92-0.99) and 0.37 (95% CI 0.1-0.74) for a rivaroxaban concentration < 32ng/mL. The PT and aPTT show poor correlation with rivaroxaban levels measured by calibrated anti-Xa and HPLC-MS/MS assays. A normal combined PT and APTT identified low rivaroxaban levels with high specificity but lacked sensitivity. The dPT assay at several dilutions could not be used to quantify rivaroxaban in clinical samples. The utility of these PT, aPTT and dilute PT assays in a clinical setting is very limited, and results generated must be interpreted with caution.

The effect of dabigatran on thrombin generation and coagulation assays in rabbit and human plasma

ObjectivesDabigatran etexilate is widely used for stroke prevention in the patients with atrial fibrillation. The anticoagulation activity of dabigatran is not necessary monitored in routine clinical practice. We aimed to study the effect of dabigatran on thrombin generation (TG) and coagulation assays in rabbit and human plasma. MethodsRabbits received different concentrations of dabigatran etexilate (5 mg/kg, 10 mg/kg). Patients with atrial fibrillation took 110 mg dabigatran etexilate twice daily. The concentrations of dabigatran in rabbit and human plasma were detected by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and ecarin chromogenic assay (ECA). The relationship between plasma dabigatran concentrations and the activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), and TG were evaluated. ResultsThere was strong correlation between LC-MS/MS and ECA (P < 0.001). Bland Altman analysis demonstrated that ECA assay could accurately predict human plasma dabigatran concentrations whereas underestimate the plasma concentrations of dabigatran in rabbits. Both the PT and the aPTT assays showed low correlations with dabigatran. The TT assay was highly sensitive to dabigatran levels. Low concentrations of dabigatran paradoxically increased TG. ConclusionsLC-MS/MS is the gold standard for detecting the concentrations of dabigatran. ECA correlates well with LC-MS/MS. The coagulation assays depend more on other factors. Paradoxical enhancement of TG may predict clinically rebound hypercoagulability and warrants further exploration.

UV induced surface modification on improving the cytocompatibility of metallocene polyethylene.

Demand for medical implants is rising day by day as the world becomes the place for more diseased and older people. Accordingly, in this research, metallocene polyethylene (mPE), a commonly used polymer was treated with UV rays for improving its biocompatibility. Scanning electron microscopy (SEM) images confirmed the formation of crests and troughs, which depicts the improvement of surface roughness of mPE substrates caused by UV etching. Accordingly, the contact angle measurements revealed that the wettability of mPE-2.5 J/cm2 (68.09º) and mPE-5 J/cm2 (57.93º) samples were found to be increased compared to untreated mPE (86.84º) indicating better hydrophilicity. Further, the UV treated surface exhibited enhanced blood compatibility as determined in APTT (untreated mPE- 55.3 ± 2.5 s, mPE-2.5 J/cm2 - 76.7 ± 4.1 s and mPE-5 J/cm2 - 112.3 ± 2 s) and PT (untreated mPE - 24.7 ± 1.5 s, mPE- 2.5 J/cm2 - 34.3 ± 1.1 s and mPE-5 J/cm2 - 43 ± 2 s) assay. Moreover, the treated mPE-2.5 J/cm2 (4.88%) and mPE-5 J/cm2 (1.79%) showed decreased hemolytic percentage compared to untreated mPE (15.40%) indicating better safety to red blood cells. Interestingly, the changes in physicochemical properties of mPE are directly proportional to the dosage of the UV rays. UV modified mPE surfaces were found to be more compatible as identified through MTT assay, photomicrograph and SEM images of the seeded 3T3 cell population. Hence UV-modified surface of mPE may be successfully exploited for medical implants.

Green synthesis and characterisation of CuNPs: insights into their potential bioactivity

The current investigation involves the green synthesis of copper nanoparticles (CuNPs) from an aqueous plant extract of Moringa oleifera Lam by two methods: (I) time-based approach and (II) heat treatment of aqueous solution. Prepared CuNPs were characterised via Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and transmission EM. The study also reveals the potential bioactivity of the prepared CuNPs. In vitro anti-microbial efficiency of CuNPs was estimated against bacterial and fungal strains by the agar well diffusion method. Anti-oxidant capacity of CuNPs was determined using ferric reducing ability of plasma (FRAP), lipid peroxidation (LPO) and peroxidase assays, while the antiplatelet potential was determined by measuring two haemostatic parameters (PT & APTT assay). The minimum inhibitory concentration was observed at 60 µg/ml against Streptomyces griseus and Aspergillus niger when NPs were prepared by method II. CuNPs prepared by the method I showed higher FRAP and LPO activities, while increased POX activity was found in CuNPs prepared by method II. CuNPs prepared using method I also showed better anti-oxidant and antiplatelet potential. It was observed that M. oleifera-derived CuNPs exhibits strong anti-microbial, anti-oxidant and APTT potential. This indicates potential utilization of green synthesized NPs for various industrial and therapeutic strategies.

Establishment and assessment of APTT assay based on the combinations of Mg2+ and Ca2+ for lupus anticoagulants measurements

Objective To establish and assess the new method of APTT assay based on the combinations of Mg2+ and Ca2+ for lupus anticoagulants (LA) measurements. Methods This prospective study included 309 trisodium citrate anticoagulated plasma samples from 244 random patients and 65 patients with different autoimmune diseases (AID) to establish and assess the method of LA measurement, respectively. Final concentrations of 0, 2.0, 4.0, 8.0, 16.0 mmol/L Mg2+ were added into 25 mmol/L Ca2+ solution, and Actin reagent was used to measured plasma APTT of 94 patients. The applied concentration of Mg2+ -Ca2+ solution was confirmed through the special and significant alteration of APTT from LA-positive and -negative plasma observed in the presence of Mg2+ (test solution). Based on Actin reagent use, the test solution and 25 mmol/L Ca2+ solution were applied to measure APTT of patients and normal individuals, respectively, and the ratio of Mg2+ -Ca2+ -APTT to Ca2+ -APTT (Mg2+ -Ca2+ -APTT indices) and normalized Mg2+ -Ca2+ -APTT indices (NAR) were calculated, respectively. Mixed plasma NAR was measured, and CV% was calculated to evaluate the repeatability and stability of Mg2+ -Ca2+ -APTT method. APTT of 150 patients was measured with the test solution and Actin reagent to calculate Mg2+ -Ca2+ -APTT indices, and normalized LA ratio was determined with dRVVT method. The applicability of Mg2+ -Ca2+ -APTT assay was assessed through comparisons of the results from the two methods. Finally, NAR and NLR of 65 patients with AID (including 26 SLE patients) were measured with Mg2+ -Ca2+ -APTT assay and dRVVT method, respectively, and ROC curve was also used to assess the efficacy of the two methods for LA measurements. Results In all LA-negative plasma, APTT increased from 28.1±4.5 s to 61.2±7.9 s in normal APTT group , 47.2±8.9 s to 97.5±10.3 s in increased APTT group, and 27.6±5.1 s to 61.2±7.9 s in ACA-positive group when Mg2+ increased from 0 to 8 mmol/L in Mg2+ -Ca2+ solution (F=34.12, 38.9 and 28.35, P 1.034 of Mg2+ /Ca2+ indices, there was 141 patients with increased indices and NLR<1.20, and 9 patients with decreased ones and NLR≥1.20 in all 150 patients. The area under ROC curve of NAR and NLR for LA detection was 0.913 (95% CI: 0.848-0.978) and 0.892(95% CI: 0.817-0.966), respectively, and the cut-off value was 0.87 and 1.13, respectively. The sensitivity and specificity of NAR (85% and 77%) was higher than that of NLR (81% and 74%), respectively. The accordant rate of positive, negative, and total results between NAR and NLR was 94.4%, 98.5%, and 98%, respectively. Conclusion The method of APTT assay based on Mg2+ combining Ca2+ for LA measurements is feasible, and can be used to detect plasma LA of patients.(Chin J Lab Med, 2018, 41: 165-170) Key words: Magnesium; Calcium; Partial thromboplastin time; Lupus coagulation inhibitor

QCM-D surpassing clinical standard for the dose administration of new oral anticoagulant in the patient of coagulation disorders

The study focuses the dose administration of dabigatran to avoid the deaths due to hemorrhagic complications and thromboembolic stroke in clinics worldwide. To target the issue, a novel emerging acoustic technology, namely ''Quartz Crystal Microbalance with Dissipation'' (QCM-D) has been applied, while the acoustic assays namely ''activated Partial Thromboplastin Time'' (aPTT) and ''Prothrombinase complex-induced Clotting Test'' (PiCT) have been compared with the standard methods in parallel. Both techniques have been applied to 300 samples, including 220 plasma samples of patients suffering coagulation disorders and 80 plasma samples of non-patients. In comparison, the coagulation times of the acoustic aPTT and PiCT yielded an excellent correlation with the standard methods with in analytical standard deviation limits. Finally, the acoustic aPTT assay is the ''gold standard'' for a dose administration of the new oral anticoagulant, where the Δf/ΔΓ ratio of the acoustic assay demonstrates that dabigatran with FEIBA 50 combination could be a safe remedy to avoid the deaths in clinics.

A semi-synthetic glycosaminoglycan analogue inhibits and reverses Plasmodium falciparum cytoadherence.

A feature of mature Plasmodium falciparum parasitized red blood cells is their ability to bind surface molecules of the microvascular endothelium via the parasite-derived surface protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This ligand is associated with the cytoadherence pathology observed in severe malaria. As pRBC treated with effective anti-malarial drugs are still able to cytoadhere, there is therefore a need to find an adjunct treatment that can inhibit and reverse the adhesion process. One semi-synthetic, sulfated polysaccharide has been identified that is capable of inhibiting and reversing sequestration of pRBC on endothelial cells in vitro under physiological flow conditions. Furthermore, it exhibits low toxicity in the intrinsic (APTT assay) and extrinsic (PT assay) clotting pathways, as well as exhibiting minimal effects on cell (HUVEC) viability (MTT proliferation assay). These findings suggest that carbohydrate-based anti-adhesive candidates may provide potential leads for therapeutics for severe malaria.

Isolation, purification, structural analysis and coagulatory activity of water-soluble polysaccharides from Ligustrum lucidum Ait flowers

In this study, Ligustrum lucidum flowers as raw material, the extraction, isolation and coagulatory activity of polysaccharides were carried out for the first time. The crude polysaccharide was obtained by hot water extraction and ethanol precipitation, and preliminarily purified by Sevage method and D101 macroporous resin. Then the polysaccharide was further purified by DEAE-52 cellulose and Sephadex G-100 column chromatography, respectively. The structural characteristics were detected by LC, GC, FT-IR and NMR. Furthermore, the coagulatory activity of the polysaccharides were investigated by APTT, TT, PT and FIB assays in vitro. The results demonstrated that four polysaccharides were isolated from flowers of L. lucidum, named as LLP-1a, LLP-1b, LLP-2 and LLP-3, and the yields were 0.039, 0.0054, 0.0055 and 0.017%, respectively based on the weight of the dried flowers. The four polysaccharides components were free of nucleic acids and proteins, and their average molecular weights were 25,912, 64,919, 3,940,246 and 2,975,091 g/mol, respectively. The monosaccharide compositions of LLp-1a were l-rhamnose, l-arabinose, d-xylose, d-glucose and d-galactose (molar ratio of 3.16: 2.46: 1.00: 7.27: 4.22). Only d-galactose was detected from LLp-1b. LLp-2 was composed of l-arabinose, d-glucose and d-galactose (molar ratio of 1.28:1.32:1.00). LLp-3 was composed of l-rhamnose, l-arabinose, d-xylose, d-glucose and d-galactose (molar ratio of 5.85: 2.21: 2.23: 1.00: 2.25). Coagulation assays indicated that LLp-1a and LLp-3 had good anticoagulant effect in vitro, while LLp-1b showed procoagulant activity.

Utility and correlation of known anticoagulation parameters in the management of pediatric ventricular assist devices.

AIMTo assess utility and correlation of known anticoagulation parameters in the management of pediatric ventricular assist device (VAD).METHODSRetrospective study of pediatric patients supported with a Berlin EXCOR VAD at a single pediatric tertiary care center during a single year.RESULTSWe demonstrated associations between activated thromboplastin time (aPTT) and R-thromboelastography (R-TEG) values (rs = 0.65, P < 0.001) and between anti-Xa assay and R-TEG values (rs = 0.54, P < 0.001). The strongest correlation was seen between aPTT and anti-Xa assays (rs = 0.71, P < 0.001). There was also a statistically significant correlation between platelet counts and the maximum amplitude of TEG (rs = 0.71, P < 0.001). Importantly, there was no association between dose of unfractionated heparin and either measure of anticoagulation (aPTT, anti-Xa or R-TEG value).CONCLUSIONThis study suggests that while there is strong correlation between aPTT, anti-Xa assay and R-TEG values for patients requiring VAD support, there is a lack of relevant correlation between heparin dose and degree of effect. This raises concern as various guidelines continue to recommend using these parameters to titrate heparin therapy.

Calibrated automated thrombogram values in infants with cardiac surgery before and after cardiopulmonary bypass

IntroductionImpaired thrombin generation has been associated to increase bleeding after cardiac surgery with cardiopulmonary bypass (CPB), especially in children. The aim of this study was to evaluate standard coagulation assay, thrombin generation by calibrated automated thrombogram (CAT), thromboelastography (TEG) and procoagulant phospholipids (PPL) activity in infants undergoing cardiac surgery with CPB. Materials and methodsProspective observational study performed in children aged <24months undergoing cardiac surgery with CPB. Exclusion criteria were preoperative coagulopathy or anticoagulant therapy. Coagulation was evaluated by standard coagulation assays (prothrombin time, activated partial thromboplastin time, fibrinogen level, platelet count), TEG, CAT and PPL at anaesthesia induction (T1) and after 12h (T2). Perioperative bleeding management was performed according to the institutional guidelines. ResultsForty-nine children aged <24months were enrolled. At T1 ETP and peak height evaluated by CAT were significantly lower in infants aged <6months. Standard coagulation tests, TEG and PPL did not correlate with age. At T2 platelet count, plasmatic fibrinogen level, all TEG parameters, ETP and peak height by CAT were significantly impaired compared to baseline values (T1), despite allogeneic blood product transfusions. ConclusionsThrombin generation is significantly impaired in children affected by congenital heart disease, compared to healthy children and adults. CAT parameters resulted age-dependent, and thrombin generation is lower in infants aged <6months. After cardiac surgery with CPB, a coaugulopathy, revealed by CAT, TEG, but not by PT and aPTT assays, is persistent 12h after surgery despite transfusions of blood products.

Effect of evening primrose oil on primeralary blood coagulation investigation in male rabbits

This study was performed to determine the effects of Evening primrose oil (EPO) on haemostatic parameters following 30 and 60 days administration of 90 mg/kg (EPO) to healthy male rabbits. The laboratory resulting in significant increase in Prothrombin time (PT), activated partial thromboplastin time (aPTT) assays (p&lt;0.01), platelets count significantly decreased (p&lt;0.01) at both times. While fibrinogen concentration are insignificantly decreased. These effects might be due to inactivation or inhibition of factors affecting coagulation.

Anticoagulant and antithrombotic effects of chemically sulfated fucogalactan and citrus pectin.

Citrus pectin (CP14) from Citrus sinensis, and a fucogalactan (E) and a glucan (G16) from Agaricus bisporus were isolated and structurally characterized. CP14 was constituted by (1→4)-linked α-d-GalpA units, E was composed by a (1→6)-linked α-d-Galp main-chain, partially substituted at O-2 by non-reducing end-units of α-l-Fucp or α-d-Galp, and partially methylated at O-3, whereas G16 was composed of (1→6)-linked β-d-Glcp units. The polysaccharides were sulfated giving rise to CP14S, ESL and G16S. The APTT and PT assays showed a decreasing order of anticoagulant activity for ESL, CP14S and G16S, respectively. ESL and CP14S showed greater anticoagulant activity. However, ESL reduced thrombus formation to 32.3% at a dose of 6.0mgkg-1, whereas CP14S inhibited totally the thrombus formation at 3.0mgkg-1, in vivo. NMR and methylation analyses showed that α-d-GalAp units of CP14S were sulfated in 2,3-O-position, whereas ESL was mainly sulfated in 2-O-, 2,3-O- and 2,3,4-O-positions.

Abstract 548: Molecular Mechanism Behind Protein S Deficiency in Obese Patients

Introduction: Protein S is a vitamin K-dependent plasma protein, produced mainly in the liver; Protein S circulates in the blood at a concentration of 450 nM. Protein S is an anticoagulant, serving as a cofactor for APC and TFPI, and as an inhibitor of Factor IX (FIXa). Protein S deficiency causes deep vein thrombosis (DVT), increased risk of inflammation and, because DVT is a complication commonly observed in obese individuals, Protein S deficiency might be associated with obesity. Aim: To identify a correlation between Protein S deficiency and obesity, and identify the probable molecular mechanism behind the Protein S deficiency in the obese subjects. Methods: Immunoblots, ELISA, EMSA, CHIP, aPTT assay, and thrombin generation assay. Results: By ELISA, we measured a decrease in Protein S level in obese mice compared with wild type mice. In obesity, the liver becomes hypoxic, thus, we hypothesized that hypoxia and hypoxia inducible factor 1 alpha (HIF1α) may regulate Protein S expression in obesity. We found that a high fat diet induced HIF1α stability in mice. HIF1α levels were inversely proportional to Protein S levels, suggesting that HIF1α is a negative regulator of Protein S expression. We further identified a putative HIF1α binding site in the Protein S promoter, and, by using in vitro and in vivo assays, we demonstrated that HIF1α binds directly to the Protein S promoter and suppresses transcription. We further confirmed HIF1α-mediated Protein S transcriptional regulation in vivo, Plasma Protein S levels are increased in the liver-specific HIF1α knockout mouse whereas, liver-specific overexpression of HIF1α reduced the concentration of Protein S in the plasma. Conclusion: We conclude that HIF1α regulates Protein S expression in mouse liver and in obesity. Inhibition of HIF1α or intravenous injection of Protein S may reduce the occurrence of DVT in obese individuals.

Plasma Antiphospholipid Antibodies Effects on Activated Partial Thromboplastin Time Assays

BackgroundActivated partial thromboplastin time (aPTT) assays can be affected by plasma antiphospholipid antibodies (aPLs), but the degree of the interference is not easy to predict. This study aimed to investigate the effects on aPTT assay results of different types and combinations of aPLs, including anti-β2-glycoprotein I antibodies, anticardiolipin antibodies and lupus anticoagulant. Materials and MethodsWe retrospectively collected clinical information and laboratory tests from aPL-positive patients. The potential influence of aPLs on aPTT assays was assessed. ResultsThe survey included 589 aPL-positive patients. No significant differences existed in basic characteristics such as sex, age, prothrombin time, fibrinogen and alanine aminotransferase among different cases with 1, 2 or 3 types of positive-aPL markers (P > 0.05). In 113 patients with abnormal aPTT values, multivariable linear regression analysis showed a significant correlation between an abnormal degree of aPTT values and dilute Russell viper venom time (dRVVT) or silica clotting time (SCT) with a correlation coefficient of 0.437 or 0.497 (P < 0.01), whereas age, anticardiolipin antibodies-immunoglobulin G, anticardiolipin antibodies-immunoglobulin M and anti-β2-glycoprotein I antibodies were of no significance (P > 0.05). Among blood samples with 3 types of aPLs positivity, the rate of abnormal aPTT detection values was 55.3%, which was significantly higher than that observed in patients with negative, single-positive or double-positive aPL markers (P < 0.05). Patients with a moderate to strong dRVVT or SCT had a higher proportion of abnormal aPTT assays than did patients with a low dRVVT or SCT (P < 0.05). ConclusionsWhen abnormal aPTT values are obtained, the influence of aPLs should be considered, especially in the presence of a moderate to strong dRVVT or SCT.