Chromogenic Substrate Assay Research Articles

Plasminogen in cerebrospinal fluid originates from circulating blood.

BackgroundPlasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood–CSF barrier may restrict its diffusion from blood.MethodsWe investigated the origin of plasminogen in CSF using Alexa Fluor 488–labelled rat plasminogen injected into rats with systemic inflammation and blood–CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome (n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8).ResultsEndogenous rat plasminogen was detected in higher amounts in the CSF and urine of LPS-treated animals as compared to controls. In LPS-primed rats, circulating Alexa Fluor 488–labelled rat plasminogen was abundantly localized in the choroid plexus, CSF and urine. Plasminogen in human CSF was higher in Guillain-Barré syndrome (median = 1.28 ng/μl (interquartile range (IQR) = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/μl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/μl (IQR = 0.18 to 0.35)).ConclusionsOur findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0154-y) contains supplementary material, which is available to authorized users.

Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories

Discrepancies exist for some of the modified coagulation factors when assayed with different one-stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one-stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate® at 0.05, 0.20 or 0.80 IU mL−1 were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra- and inter-laboratory variation, accuracy and possible rFVIIIFc-specific assay discrepancies. For the one-stage assay, mean recovery was 95% to 100% of expected for both Advate® and rFVIIIFc at 0.8 IU mL−1. Intra-laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate®, and 6.0% to 10.3% for rFVIIIFc. Inter-laboratory CV ranged from 10% for Advate® and 16% for rFVIIIFc at 0.8 IU mL−1, to over 30% at 0.05 IU mL−1 for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate® and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one-stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product-specific rFVIIIFc laboratory standard. Accuracy by the one-stage assay was comparable to that of Advate®, while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay.

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.

Pharmacokinetics Study of Recombinant Hirudin in the Plasma of Rats Using Chromogenic Substrate, ELISA, and Radioisotope Assays

AimTo compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with 125I-recombinant hirudin.Methods2.0 mg/kg 125I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA).ResultsThe chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t1/2 α), the half-life of elimination phase (t1/2 β), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC0–t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA.ConclusionsThe chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.

Practical and cost‐effective measurement of B‐domain deleted and full‐length recombinant FVIII in the routine haemostasis laboratory

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product-specific laboratory standard is recommended when measuring Refacto-AF(R) activity in plasma with a one-stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one-stage and a chromogenic assay calibrated either with pooled normal plasma or a product-specific laboratory standard. Results obtained with the one-stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product-specific laboratory standard and plasma can be established. In patients' plasma, Refacto-AF levels measured with a one-stage FVIII assay calibrated with plasma or a product-specific laboratory standard diverged from -58% to -17% and from -25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full-length rFVIII measured with the one-stage assay calibrated with plasma were 6-49% lower than with the chromogenic assay. In a monocentric setting, the long-term stability of the calibration curves allows the implementation of a practical and cost-effective approach to determine rFVIII:C levels.

Discrepancies between ADAMTS13 activity assays in patients with thrombotic microangiopathies

ADAMTS13 activity assays are sometimes useful in confirming the clinical diagnosis or to distinguish different thrombotic microangiopathies (TMA). We investigated the commonly used clinical assays for ADAMTS13 activity. 159 samples from normal subjects or acquired TMA patients were studied in collagen binding (CBA), Fret and chromogenic peptide substrate assays. Frozen aliquots of pooled normal plasma gave similar values by CBA, Fret-VWF73 peptide, Fret-VWF86 and chromogenic VWF73 ELISA (chr-VWF73). Two lyophilised commercial calibrants gave lower ADAMTS13 activity by CBA than peptide substrate assays. The addition of solid HEPES to normal plasma caused a significant fall in CBA, but not Fret-VWF73 activity and might partly explain the differences, since lyophilised plasmas are often HEPES buffered. Normal plasmas showed good agreement between CBA and Fret assays, although chr-VWF73 gave slightly higher values. In acquired TMA, there was reasonable agreement between assays for samples with <11% ADAMTS13 activity (83% of samples showed agreement between CBA, Fret-VWF73 and chr-VWF73), but samples with moderate deficiency frequently showed lower CBA levels (only 41-52% agreement). However, there were also some discrepancies among the peptide substrate assays, with Fret-VWF86 sometimes giving slightly higher values than the VWF73 substrate assays. An International reference plasma might improve standardisation, but is not the only problem. It is unclear which assay has greatest clinical utility, this may depend on the nature of the sample. If the activity does not match the clinical picture, an alternative method should be performed. Where therapeutic monitoring is required, the same activity assay should be used throughout.

Inhibitory Effect of an Anti-Factor VIII Antibody Fragment On Factor VIII Activity in Different Functional Assays

AbstractAbstract 4387 Background.A single-chain variable antibody fragment (scFv) is an engineered protein corresponding to the antibody’s antigen-recognizing (variable) domains (VH and VL), connected with an artificial linker. Due to the inherited specificity to the respective antigen, scFv are useful in a broad range of applications. A relatively small size (∼30 kDa) of scFv makes them easy to produce using the recombinant DNA technology. A particular scFv, known as KM33, was previously derived from a patient with anti-factor VIII (FVIII) auto-antibodies manifested in a functional deficiency of FVIII (inhibitory Hemophilia A). KM33 was described as a high affinity ligand of FVIII inhibiting its major functions: procoagulant activity and interactions with von Willebrand factor, phospholipids and catabolic receptors (van den Brink E. et al, 2001 and Limburg V. et al, 2005). Objective.Our goal was to develop a methodology for fast and easy production of KM33 and test its applicability for selective inhibition of FVIII in functional assays. Experimental Approach.As an alternative to a previously described expression of KM33 in bacteria, we used insect cells capable to perform majority of posttranslational modifications and produce correctly folded proteins. Previously published KM33 coding sequence (Mertens K. et al, 2008) was codon-optimized to the insect host cells (Spodoptera frugiperda), followed by the gene's chemical synthesis, cloning and expression in a baculovirus system. A secretable form of KM33 was purified from the culture media using nickel-affinity and size-exclusion chromatography. The structure and properties of the expressed protein were tested by binding to FVIII in a surface plasmon resonance assay and by measuring its effect on FVIII activity in a chromogenic substrate (CS) assay, clotting assay (APTT), thrombin-generation test (TG) and video microscopy of clot growth (CG). Results.The insect-cell derived KM33 demonstrated high affinity to FVIII (KD 0.1–0.2 nM) and strong inhibitory effect on the FVIII activity in all assays. At the same time, the inhibition efficiency, assessed by KM33/FVIII molar ratio corresponding to 50% of the inhibitory magnitude, was different between assays. The strongest inhibition was observed in the TG assay, and the weakest in the APTT assay (Fig. 1 and 2). Furthermore, the effect reached an apparent saturation: about 5%, 28% and 42% of FVIII activity remained uninhibited in the TG, CS and APTT assays, respectively. Control experiments performed at highest concentrations of KM33 in the absence of FVIII showed absence of non-specific effects of KM33. Conclusions.Altogether, our data shows highly specific interaction of KM33 and FVIII including the complex milieu of plasma, and applicability of KM33 for the inhibition of FVIII in functional assays. Strong inhibition of the FVIII activity in the TG test is consistent with the expected physiological relevance of this “global hemostasis” assay. In turn, our data demonstrate limitations of the classical APTT-based Bethesda assay typically used to analyze FVIII inhibitors in comparison with the CS and TG methods. The discrepant inhibitory effect on the FVIII activity between the assays may indicate a need for optimization or even revising the current methodology of analysis of FVIII inhibitors in Hemophilia A patients. Future studies are needed to evaluate relative clinical relevance of in vitro tests that quantitate activity of the FVIII inhibitory antibodies. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Determination of Rivaroxaban in Urine Samples From Patients Undergoing Elective Hip- and Knee Replacement Surgery Using a Point of Care Method

AbstractAbstract 1162Rivaroxaban is approved at fixed dose for several indications to prevent thromboembolism. Despite the lack of need to dose adjust rivaroxaban it may be necessary to determine its activity/concentration in specific patient populations. Several assays are available to determine the anticoagulant in plasma samples. A point of care (POC) method may provide immediate results for patients requiring acute medical interventions and facilitate therapeutic decisions.Plasma and urine samples were taken from patients (n=150) after having given written informed consent before and between days 4 to 6 following primary elective total knee (THR) and hip replacement (THR) surgery on antithrombotic therapy with 10mg rivaroxaban od. For laboratory assays, rivaroxaban was purified from commercially available Xarelto®. The compound was characterized by analytical methods and was quantified in plasma samples using the factor Xa specific S2222 chromogenic substrate assay as described (J Thromb Haemost 2012,10:1433–6). In urine rivaroxaban was determined by a POC method incubating the lyophilized reagents on mini-strips followed by incubation with patient‘s urine (patent No GB1019674.9). The development of colour of urine samples 10 min after incubation with reagents was determined quantitatively by optical density (OD) measurement and judged by eye qualitatively according negative and positive colour (three independent readings by SD, SK, CG, and photographic documentation). To determine the positive and negative predictive value (PPV, NPV) of plasma samples, a cutoff value of <25 ng/ml rivaroxaban was defined. The PPV and NPV of the POC method with urine samples were determined according to positive and negative results of colour development judged by eye reading.Before administration of rivaroxaban 127/144 patients displayed plasma concentrations of 11.1+7.1 ng/ml (mean, standard deviation) and 17 patients a concentrations of 40.4+13.1 ng/ml (cut off value >25ng/ml). The NPV was 88.2%. During therapy the concentration of rivaroxaban was 76.7+36.6 ng/ml in 113/135 patients (above cutoff value) and 14.3+5.2 ng/ml in 22/135 patients (below cutoff value). The PPV was 83.7%. Values were negative in 16.3% of these patients. Using the POC method for rivaroxaban in urine all of 117 patients had negative values with OD measurement (1.23+0.14 OD units) and eye measurement. The NPV was 100%. During therapy with rivaroxaban 4/125 patients had negative colour development by eye measurement with an OD of 1.21+0.02. 121/125 showed colour development as judged by eye corresponding to an OD of 0.16+0.09. The PPV was 96.8%. There was no interobserver variability. Limitation of the methods is the lack of sure information about the compliance of the patients. Further validation of the POC method is ongoing.Under real life conditions the POC method and judgement of colour development by eye increased the PPV and NPV from about 85% using plasma samples to more than 95% using urine samples for determination of rivaroxaban. The method is non-invasive, rapid, and specific, can be repeatedly performed and may be used by medical personal and patients. Colour development differs from the POC method for dabigatran from urine samples of patients on treatment. Disclosures:No relevant conflicts of interest to declare.

Point of Care Method for the Determination of Dabigatran in Urine Samples From Patients On Treatment.

AbstractAbstract 2270Dabigatran is effective and safe for prevention of thromboembolism in several indications at daily fixed doses not requiring laboratory dose adjustment. However, it may be necessary to determine its activity/concentration in specific patient populations by specific assays from plasma samples. A point of care (POC) method may offer advantages by providing immediate results to facilitate medical decisions for acute therapeutic interventions.Plasma and urine samples were taken from patients on treatment with 110mg or 150mg dabigatran bid (n=110) and healthy persons (n=144) after having given written informed consent. Dabigatran was purified from commercially available Pradaxa® and its purity was characterized by analytical methods and S2238 thrombin specific chromogenic substrate assay and Hemoclot assay containing dabigatran standards. The S2238 assay was also used for the determination of dabigatran in plasma samples of patients and healthy persons using one purified dabigatran as standard. The lower limit of detection was 0.06μg/ml plasma. In urine dabigatran was determined by a POC method incubating the lyophilized reagents on mini-strips followed by incubation with patient‘s urine (international patent application No PCT/EP2012/002540). The development of colour of urine samples was determined 10 min after incubation with reagents quantitatively by optical density (OD) measurement and qualitatively as judged by eye according negative and positive colour (three independent readings by SD, SK, CG, and photographic documentation). To determine the positive and negative predictive value (PPV and NPV) of plasma samples, a cutoff value of <0.06μg/ml dabigatran was defined. The PPV and NPV of POC method was determined for urine samples of the control and treatment group according positive and negative development of colour as judged by eye reading.Control persons (n=144) displayed plasma concentrations of 0.04+0.03 μg/ml (mean, standard deviation) of whom 128 had values below the cutoff value <0.06 μg/ml. Sixteen patients had values above this cutoff. Accordingly, the NPV for correct negative results in controls was 88.9%. In patients on therapy the concentration of dabigatran was 0.12+0.08 μg/ml (n=107). 102/107 patients had plasma concentrations of dabigatran above the cutoff value of 0.06 μg/ml and 5 patients below the cutoff-value. This results in a PPV of 95.4%. Using the POC method for dabigatran in urine all of 144 control persons had negative values with OD measurement (0.803+0.116 OD units) and eye measurement. The NPV for control persons was 100%. During therapy with dabigatran, all 110 patients had positive colour development as judged by eye corresponding to an OD of 0.219+0.161. Accordingly, the PPV was 100% for patients on treatment with dabigatran. Limitations of the POC methods are the lack of information about the compliance of the patient and severe renal impairment. The validation of the POC assay by patients is ongoing.The POC method of patients on treatment with dabigatran improves the NPV and PPV from about 90% with plasma samples to 100% using urine samples, respectively. The method is non-invasive, rapid, and specific, can be repeatedly performed and may be used by medical personal and patients. The development of colour is different from the POC method for rivaroxaban. Disclosures:No relevant conflicts of interest to declare.

P 3: Insulin sensitivity and related atherogenic conditions in ischemic stroke: comparison between type 2 diabetics and nondiabetics

Background and aims The role and importance of insulin sensitivity (IS) and related atherogenic conditions in ischemic stroke have not yet been elucidated. Therefore, our study was aimed to analyze (a) IS and plasma insulin (PI) (b) lipoproteins (c) plasminogen activator inhibitor 1 (PAI-1) levels (d) inflammatory markers, hs-C reactive protein (hs CRP) and interleukin-6 (IL-6) levels (e) abdominal obesity, in 40 type 2 diabetics (T2D) with ischemic stroke (group A), 35 T2D without ischemic stroke (group B), 35 nondiabetics with ischemic stroke (group C) and 34 healthy controls (group D). Material and Methods Ischemic stroke was confirmed by clinical and neuroimaging criteria. IS levels were determined by the frequently sampled intravenous glucose tolerance (FSIGT) test with minimal model analysis (Si index). Total cholesterol, HDL-cholesterol (HDL-c), and tryglicerides concentracion were determined with the chromatography metod. LDL-cholesterol (LDL-c) concentrations were calculated using the Friedewald formula. Plasma PAI-1 activity was determined by plasminogen chromogenic plasmin substrate assay. Hs-CRP was determined by Olympus Analyzer and interleukin 6 (IL-6) levels were measured by ELISA method. Waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. Results Si levels were significantly lower in group A vs B (1.15+/−0.46 vs 2.81+/−0.65 min-1/mU/lx104; p Conclusion Our results demonstrated that decreased IS are associated with increased LDL-c, decreased PAI-1, together with higher hs-CRP, IL-6 levels and waist circumference, both in T2D and nondiabetics. These results imply that decreased IS in association with compensatory hyperinsulinemia, underlying the development of the ischemic stroke, through potentiation of dislipidemia, hypofibrinolisis, low-grade inflammation and abdominal obesity.

High-Altitude Pulmonary Edema Is Associated With Coagulation and Fibrinolytic Abnormalities

ObjectivesHigh-altitude pulmonary edema (HAPE) can develop in unacclimatized persons after acute ascent to high altitude and is associated with fibrinolytic and coagulation abnormalities. The authors investigated whether fibrinolytic and coagulation abnormalities were associated with the severity of HAPE. MethodsSixty-one patients who developed HAPE after acute ascent to altitudes above 3600m were recruited. Twenty unacclimatized controls who acutely ascended to the same altitude and 20 acclimatized inhabitants served as controls. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) levels were measured using chromogenic substrate assays. Plasma fibrinogen concentration was determined by the sodium sulphite fractionation method. The concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer were measured by enzyme linked immunosorbent assay. ResultsThe plasma concentrations of D-dimer, fibrinogen, FDP and t-PA and PAI-1 were significantly higher in patients with HAPE than controls. In addition, these abnormalities were correlated with the severity of HAPE. The plasma concentrations of D-dimer and fibrinogen recovered to normal upon recovery from HAPE while t-PA, PAI-1 and FDP levels in HAPE patients still remained significantly increased over those of unacclimatized controls. ConclusionThe development of HAPE is associated with abnormalities in the fibrinolysis and coagulation system, and these abnormalities correlate with the severity of HAPE.

Changing characteristic of blood coagulation factors and their correlation with blood coagulation status in different hepatic diseases

To investigate the correlation between pro coagulation factors and anti-coagulation factors synthesized by the liver, and the correlation between fibrin degradation products (FDP) and D-dimer (D-D) concentration and coagulation proteins synthesized by extra-hepatic tissues, in different liver diseases; to explore the relationship between coagulation and bleeding in hepatic diseases. Chronic hepatitis B (CHB) patients, CHB-related liver cirrhosis patients, CHB-related liver failure patients and healthy (normal) controls were selected for study and provided blood samples for analysis. The activity of coagulation factors (F) II, V, VII, VIII, IX, X, XI, and XII was detected using the one-stage clotting method. Coagulogram analysis, including activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT), was conducted by the solidification method. Antithrombin III (AT-III) and protein C (PC) activities were measured by chromogenic substrate assay. FDP concentration was detected using immunoturbidimetry. Tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), von Willebrand factor (vWF), and tissue factor (TF) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). With the exception of FVIII, coagulation factors and anticoagulant proteins synthesized by the liver were decreased and the coagulogram was extended for all patients. Likewise, the FDP and D-D concentrations were increased in blood. CHB patients, however, presented with increased levels of FVIII, TFPI, TM, vWF, and TF. Pairwise comparison indicated statistical differences existed among CHB, CHB-related liver cirrhosis, and liver failure patients: TFPI: 239.3+/-206.4, 315.0+/-258.6, and 319.5+/-298.1 -- higher than normal control: 104.0+/-87.1, F = 5.453, P less than 0.05; vWF: 70.3+/-29.5, 105.5+/-58.0, and 179.3+/-61.7 -- higher than normal control: 21.9+/-7.2, F = 20.104, P less than 0.05; TF: 85.9+/-85.7, 234.2+/-202.9, and 344.7+/-214.6 -- higher than normal control: 12.8+/-8.1, F = 8.619, P less than 0.05; FVIII: 157.2+/-53.4, 206.9+/-86.9, and 335.7+/-117.7 -- higher than normal control: 105.5+/-46.2, F = 13.418, P less than 0.05. In parallel to the progression of liver diseases, pro coagulation and anti-coagulation elements synthesized by the liver were reduced. In contrast, fibrinolysis activity was enhanced, which is expected to lead to an imbalance between blood clotting and anti-clotting factors. This may be an important cause for the bleeding that occurs in end-stage liver disease. Expressions of TFPI, TM, vWF, and TF significantly change in the early stage of liver diseases, as compared to normal (healthy) levels, and may represent a sensitive indicator of vascular injury.

Determination of Rivaroxaban in Human Plasma Samples

Rivaroxaban is one of the novel oral direct factor Xa inhibitors, which is effective in preventing thromboembolic complications at fixed doses (i.e., once daily), without the need for dose adjustment according to laboratory monitoring. Nearly 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. Therefore, as renal elimination plays a pivotal role in the metabolism of this drug, impairment of renal function may be important during anticoagulation with rivaroxaban over long periods of time. The assessment of the anticoagulant effect/concentration of rivaroxaban may thus be useful in special patient populations such as in the elderly and eldest, during acute diseases with concurrent dehydration, before surgery, during bleeding or thrombotic episodes, or to verify adherence to therapy. Rivaroxaban prolongs prothrombin time in a dose-dependent, linear fashion. Activated partial thromboplastin time (APTT) is also prolonged, but in an exponential manner. Substantial differences in test results might be generated by different thromboplastin and APTT reagents. One-step prothrombin-induced clotting time assay is sensitive to low concentrations of rivaroxaban. Chromogenic substrate assays specific for factor Xa are also sensitive to rivaroxaban. Several initiatives are currently ongoing to standardize the various methods to determine rivaroxaban in human plasma samples, some of which will be summarized in this article along with the dose-dependent effects of rivaroxaban on relevant coagulation parameters. Therefore, although rivaroxaban prolongs all coagulation assays used to assess the anticoagulant effects of most anticoagulants, the most specific assay cannot be identified at present. Moreover, clinical trials are needed to determine the relationship of assay results with bleeding or thrombotic complications.

Guidelines on the investigation and management of antiphospholipid syndrome

This guidance updates and replaces the previous guideline on the investigation and management of antiphospholipid syndrome (APS) published in 2000 (Greaves et al, 2000), though where there have not been changes we refer back to them when appropriate. The guidance is updated with reference to relevant publications since 2000. Publications known to the writing group were supplemented with additional papers identified by searching PubMed for publications in the last 11 years using the key words: lupus anticoagulant, anticardiolipin, antiphospholipid, b2–glycoprotein I, antiprothrombin and limits (clinical trial, randomized control trial, meta-analysis, humans, core clinical journals, English language). The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the Royal College of Obstetricians and Gynaecologists (RCOG), and the British Committee for Standards in Haematology (BCSH) Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMEN DATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with antiphospholipid syndrome though individual patient circumstances may dictate an alternative approach.

Determination of Dabigatran in Human Plasma Samples

The oral direct thrombin inhibitor dabigatran effectively prevents arterial and venous thromboembolism using fixed doses without the need for adjustment according to laboratory results. Dabigatran is eliminated from the circulation by ∼80% through the kidneys. However, the in vitro anticoagulant effect of dabigatran may be necessary to determine in special patient populations such as in the elderly, for renal impairment, before operations, bleeding or thrombotic episodes, and to monitor self-compliance. Several clotting and thrombin-specific chromogenic substrate assays are available to analyze the biological activity of dabigatran. All of them are prolonged in the presence of dabigatran. This article reports the effects of dabigatran on clinical routine assays and the potential usefulness for determination in special risk groups of patients when overdose or lack of compliance are suspected.

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

Evidence for a Factor IX-Independent Role for Factor XI in Thrombin Generation

Abstract 2244In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV.We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not.In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX.Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures:Tucker:Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Development of a Continuous Thrombin Generation-Based Test to Measure Potency in Factor VIII Concentrates

Abstract 1206The potency assigned to a Factor (F) VIII-containing product should be predictive of its clinical effectiveness in treating Hemophilia A patients. Potency is assigned using assays that are calibrated against reference standards traceable to an international standard for FVIII concentrates. Therefore, FVIII products are labeled in International Units (IU). Most manufacturers are currently using either a one-stage clotting (OC) or 2-stage chromogenic substrate (CS) assays. However, for some FVIII products, discrepancies in potency values derived from these 2 assays can exceed 50 %. This presents a challenge to manufacturers, regulators and clinicians alike. Recently, a continuous thrombin generation test (TGT) has been introduced in clinical research. The TGT is physiologically more relevant; however, its utility in drug characterization is not yet established.In this report, we describe the development of a FVIII-TGT assay that is suitable for the measurement of FVIII activity in FVIII concentrates. We systematically optimized each of the TGT parameters to obtain the strongest response for a FVIII sample diluted in FVIII-deficient plasma to a concentration of 1 IU/mL. The relative response (ratio of levels of thrombin generation with and without FVIII), and the level of thrombin generation above instrument noise (signal-to-noise ratio) were used as criteria for optimization. In order to limit the variability of the assay, the final parameters were chosen such that small variations in the concentrations of reagents would have a minimal impact on assay outcome.Concentrations of FVIII-deficient plasma, tissue factor (TF), and corn trypsin inhibitor (CTI, inhibits contact activation) had strongest effects on the relative response to FVIII. Concentrations of procoagulant lipids, fluorogenic substrate and calcium ions were more important to achieve an optimal signal-to-noise ratio. Inhibition of contact activation mediated by the contact of plasma with plastic surfaces, control of temperature prior to the start of recording, pipetting technique and internal standards were important for assay reproducibility. Use of low levels of intrinsic factors IXa and XIa in conjunction with TF or omission of CTI allowed for the greatest sensitivity and linearity range of the assay.The performance and utility of the optimized FVIII-TGT were evaluated by measuring the potency of 6 plasma-derived and recombinant FVIII concentrates, and comparing the values to those by the CS assay and on the product label. Most of the products showed parallel dose-response curves between 0.01 and 0.3 IU/mL of plasma FVIII activity in the TGT-FVIII assay. One product had an activity of at least 20 % higher and another product 20 % lower than the labeled potency. The CS assay demonstrated a lack of parallelism in dose-responses for some products but identified the 2 products with the higher- and lower- than-labeled activities.In conclusion, the FVIII-TGT assay can be used to assign and compare the potency of FVIII concentrates. Additionally, the FVIII-TGT, being physiologically more relevant, may act as a reference in understanding the discrepancies in potency assignments in different FVIII concentrates. DISCLOSURE OF INTEREST:MJG, SAS, TKL, and MVO are employees of the U.S. FDA. The findings and conclusions in this presentation have not been formally disseminated by the FDA and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

A Prospective, Multi-Center, Randomized Clinical Trial to Compare the Pharmacokinetic Properties of Human-Cl rhFVIII, a New Human-Cell Line Derived Recombinant Factor VIII, with Those of a Full-Length Recombinant Factor VIII Expressed in Baby Hamster Kidney Cells,

Abstract 3309▪▪This icon denotes a clinically relevant abstract Introduction:human-cl rhFVIII is the first recombinant factor VIII (FVIII) concentrate expressed in a human cell line (Human Embryonic Kidney (HEK) 293F cells). The manufacturing process includes two steps for virus inactivation and removal (solvent/detergent treatment and a nanofiltration). No animal- or human-derived materials are added to the culture medium or during the purification and final formulation. Objective:The primary objective of this study is to compare the pharmacokinetics (PKs) of human-cl rhFVIII, a B-domain deleted rFVIII product expressed in a human cell line, with a commercially available full-length rFVIII product (Kogenate FS), which is expressed in Baby Hamster Kidney (BHK) cells. Methods:This study is an open, multinational, prospective, multicenter clinical phase-II trial. The PK properties of human-cl rhFVIII and Kogenate FS were determined using a randomized two-period, cross-over design. After a 96 hours or more wash-out phase of any previously administered FVIII product, subjects received a single intravenous dose of 50 IU/kg body weight of one of the products. Blood samples were collected over a 48-h period and FVIII coagulant activity (FVIII:C) was measured by validated chromogenic substrate and one-stage clotting assay in a central laboratory, which also assigned drug potency with the same assays. Following completion of the PK segment of the study, subjects will be followed for at least 50 Exposure Days and at least 6 months of treatment with human-cl rhFVIII to evaluate the efficacy and safety of on-demand treatment. The planned interim analysis stipulates the comparison of the complete PK profile of human-cl rhFVIII and Kogenate FS with the bioequivalence of the AUC as the primary endpoint. The full efficacy and safety profile will be reported when the study is completed. Results:Twenty-two adolescent and adult PTPs with severe hemophilia A (FVIII:C <1%) were enrolled at 10 study centers in the United States and Europe. The key PK parameters (AUC, elimination half-life, in-vivo recovery, clearance) are comparable between human-cl rhFVIII and Kogenate FS both for the one-stage and chromogenic assays. The two products were judged to be bioequivalent as the 90% confidence interval of the ratio of the geometric mean values of the AUC fell within 80–125%. human-cl rhFVIII has been well tolerated and no safety or efficacy issues have been reported so far, in particular no inhibitors or non inhibitory antibodies were detected or reported. Conclusion:The PK data from this study indicate that human-cl rh FVIII is bioequivalent to a licensed rFVIII concentrate measured by the one stage and the chromogenic assays. In this study as well as in the other ongoing clinical studies, which examine the efficacy and safety of human-cl rhFVIII during prophylactic treatment in children and adults with severe hemophilia A, the product has been safe and well tolerated so far. Disclosures:Knaub:Octapharma AG: Employment. Valentino:Baxter Bioscience: Consultancy; GTC Biotherapeutics: Consultancy; Bayer Healthcare: Consultancy; NovoNordisk: Consultancy; Inspiration Bioscience: Consultancy; Biogen: Consultancy. Manco-Johnson:Octapharma AG: Consultancy.

A Novel Single Chain Recombinant Factor VIII Fc Fusion Protein Is Active In Vitro and Fully Efficacious in An In Vivo Hema Mouse Bleeding Model

Abstract 1192Recombinant factor VIIIFc (rFVIIIFc) is comprised of a B domain deleted (BDD) rFVIII protein genetically fused to the Fc domain of human immunoglobulin G1 (IgG1). Prior to secretion from HEK 293 cells, most of the rFVIIIFc is processed into a FVIII heavy chain (HC) and light chain (LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrand factor (VWF) and released upon activation in a manner that is indistinguishable from native FVIII. Spontaneous dissociation of the HC and LC is thought to contribute to the loss of FVIII activity in plasma and during storage of FVIII drug products. Here we describe a single chain non-processed isoform of rFVIIIFc (SC rFVIIIFc), which may provide superior manufacturability and enhanced stability compared to native FVIII.SC rFVIIIFc was purified from rFVIIIFc, which contains a fraction of the non-processed isoform. Compared to rFVIIIFc, SC rFVIIIFc showed equivalent chromogenic activity but approximately 60% reduced activity by the one stage (aPTT) assay. In a thrombin generation assay (TGA), SC rFVIIIFc also showed a reduced thrombin potential, peak thrombin and slope compared to rFVIIIFc. However, full activity of SC rFVIIIFc by aPTT was observed in the absence of VWF, suggesting release from VWF may be delayed due to covalent linkage of the a3 acidic region to the HC after Arg 1680 cleavage in SC rFVIIIFc, in contrast to a3 release and dissociation from fully processed FVIII. Delayed dissociation from VWF may explain the reduced activity observed in the aPTT assay and TGA, while full activity was observed in the two-stage chromogenic assay. A reduced rate of activation in the presence of VWF was confirmed in a modified chromogenic substrate assay with limiting thrombin as FVIII activator.In vivo function of SC rFVIIIFc was assessed in the HemA mouse tail vein transection (TVT) model. SC rFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy in this model, with an ED50 of 1.17 μg/kg and 1.23 μg /kg respectively when TVT was performed at 48 hours post infusion. Comparable post TVT survival curves (p ≥ 0.65) were observed for the SC rFVIIIFc and rFVIIIFc at each tested dose level (Figure 1), indicating that SC rFVIIIFc was equally effective as rFVIIIFc despite its lower apparent aPTT activity. The delayed in vitro activation of SC rFVIIIFc in the presence of vWF therefore appears to have no significant impact on its in vivo efficacy. Thus, SC rFVIIIFc represents a novel and efficacious isoform of rFVIIIFc with potential clinical applications. Further studies will be required to demonstrate enhanced product stability in the context of this Fc fusion protein. [Display omitted] Disclosures:Buyue:Biogen Idec Hemophilia: Employment. Liu:Biogen Idec Hemophilia: Employment. Reidy:Biogen Idec Hemophilia: Employment. Patarroyo-White:Biogen Idec Hemophilia: Employment. Kamphaus:Biogen Idec Hemophilia: Employment. Slein:Biogen Idec Hemophilia: Employment. Pierce:Biogen Idec Hemophilia: Employment. Sommer:Biogen Idec Hemophilia: Employment. Peters:Biogen Idec Hemophilia: Employment.