aPTT Assay Research Articles

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Evaluation of partial thromboplastin time, thrombin time and prothrombin time over treated plasma using a fibrinolytic protease

Fibrinolytic proteases are enzymes that degrade the fibrin, a main component of the blood clot. Thus, the present study aimed to understand the evaluation of aPTT, TT and PT on plasma treated with a fibrinolytic protease produced by Mucor subtillissimus. To assess the anticoagulant effect, the protease was used in different concentrations of 0.5-2.5mg/mL. The tests showed that the enzyme promoted a significant prolonged time over the PT clotting time as concentration increased. In the aPTT assay, fibrinolytic protease practically did not prolong the clotting time, even with increased enzyme concentration. In TT it was verified that at all times of contact with thrombin, be it 5-30 minutes, there was no interference on the action of thrombin or fibrinogen on the formation of thrombi. Since only the prothrombin time among all those studied was altered, it is suggested that the fibrinolytic protease under study affects the extrinsic pathway of coagulation. Thus, it was possible to observe that the serine protease promoted a significant prolonged time on the clotting time of the PT as the enzyme concentration increased, but the serine protease did not cause any type of change in TT and aPTT.

Anti-Coagulant and Antimicrobial Recombinant Heparin-Binding Major Ampullate Spidroin 2 (MaSp2) Silk Protein.

Governed by established structure–property relationships, peptide motifs comprising major ampullate spider silk confer a balance of strength and extensibility. Other biologically inspired small peptide motifs correlated to specific functionalities can be combined within these units to create designer silk materials with new hybrid properties. In this study, a small basic peptide, (ARKKAAKA) known to both bind heparin and mimic an antimicrobial peptide, was genetically linked to a protease-resistant, mechanically robust silk-like peptide, MaSp2. Purified fusion proteins (four silk domains and four heparin-binding peptide repeats) were expressed in E. coli. Successful fusion of a MaSp2 spider silk peptide with the heparin-binding motif was shown using a variety of analytical assays. The ability of the fusion peptide to bind heparin was assessed with ELISA and was further tested for its anticoagulant property using aPTT assay. Its intrinsic property to inhibit bacterial growth was evaluated using zone of inhibition and crystal violet (CV) assays. Using this strategy, we were able to link the two types of genetic motifs to create a designer silk-like protein with improved hemocompatibility and antimicrobial properties.

Identification of new pyrazolyl piperidine molecules as factor Xa inhibitors: Design, synthesis, in silico, and biological evaluation

Coagulation factor Xa (FXa), a serine endopeptidase is a common coagulation factor activated as a result of the initiation of both intrinsic and extrinsic blood coagulation pathways. Hence, FXa has been regarded as an important pharmaceutical target for the treatment of thrombotic disorders. In this study, we reported the design and synthesis of pyrazolyl piperidine analogs 4(a–h) as a new class of anticoagulant drug candidates. Among the synthesized analogs 4(a–h), compound 4a consisting of the 4-chlorophenyl substitution displayed the highest in vitro FXa inhibition activity with an IC50 value of 13.4 nM. The PT and aPTT assay indicated that compound 4a showed good anticoagulation activity compared to Heparin. Furthermore, docking studies suggested that the synthesized analogs displayed binding modes similar to the cocrystallized Rivaroxaban ligand. In addition, in-silico ADMET and DFT studies were carried out for all the designed compounds. Together, our study suggests that the compound (4a) displayed anti-coagulant activity through the inhibition of FXa.

Performance Evaluation of Sysmex CN-3000 and Stago STA R Max Coagulation Analyzers and Interference of Hemolysis.

In this study, our purpose was to evaluate the analytical performances of the STA R Max and CN-3000, and compare the results of both for PT, aPTT, fibrinogen, D-dimer, and factor VIII, and also to show the influence of hemolysis on PT, aPTT, and fibrinogen assays. Three hundred ninety-five randomly-selected blood samples from residual material from Istanbul Faculty of Medicine, Central Laboratory workflow comprised the study group. PT, aPTT, fibrinogen, D-dimer, and factor VIII activity were done using both analyzers. Analytical performances were determined through precision, linearity, and comparability studies. Artificial hemolysis was performed through freezing-thawing and mechanical-sheer methods. Intra-assay and between-day CVs% of PT and aPTT were lower than 5% for STA R Max and CN-3000. Only the within-run and between-day CVs% of fibrinogen and the between-day CVs% of D-dimer were higher than 5%, but in acceptable targets. Intra-assay and between-day CVs% of FVIII on the CN-3000 were 3.5% and 12.3% at the low and 2.5% and 5.3% at high level, and 1.8% and 3.7% at the low and 6.3% and 5.9% at high level on the STA R Max. The comparison results of PT, aPTT, fibrinogen, and D-dimer were good (r > 0.91), also good correlations were obtained for FVIII activity > 40 IU/dL and FVIII between 5 - 40 IU/dL (r = 0.89). The results of the hemolysis study were within acceptable limits of the recommended criteria of Fraser and the manufacturer. CN-3000 and STA R Max coagulation analyzers are accurate and highly precise systems for safe use in clinical diagnostic applications. The interferences obtained for both analyzers were found to be within accepted targets.

Hemophilia: The Past, the Present, and the Future.

Hemophilia: The Past, the Present, and the Future.

An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity

Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice.Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16.Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents.Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice.Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. DisclosuresNo relevant conflicts of interest to declare.

USP Standardized Mixtures of Bovine, Ovine and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single Sourced Heparins and May be Interchangeable,

Introduction: Unfractionated heparin (UFH) is the first line anticoagulant for the management of medical indications. UFH complexes with antithrombin to produce strong inhibition of thrombin and factor Xa. The UFHs are standardized using USP compliant amidolytic anti-Xa and IIa methods in defined conditions. Clinically used UFH is solely sourced from porcine mucosal tissue. Because of the shortage of porcine tissue and the African Swine Fever, the supply chain of this anticoagulant is compromised. Thus, there is a need for resourcing of this anticoagulant. Bovine and ovine mucosal sources represent alternate material for production of UFH. Previous studies have shown that bovine and ovine UFH exhibit anticoagulant effects which can be standardized by using the USP method. Additionally, the standardized heparins from various sources can be blended and their potency can be adjusted to exhibit comparable effects as the single sourced UFH. The purpose of this study is to evaluate the pharmacologic profile of the blended heparin and compare these activities to that of the single sourced porcine, ovine and bovine heparins.Methods: Two groups of heparins were evaluated in this study, porcine, ovine, bovine, and the blended heparin in gravimetric measurements (ug/ml) and these same four in potency adjusted measurements (U/ml). The pharmacologic profiles of the heparins in this study were investigated via global anticoagulant assays and anti-protease assays performed in plasma. Clot based assays such as the activated partial thromboplastin time (aPTT) and thrombin time (TT) were used to study the anticoagulant effects of the single source and blended heparins. The amidolytic anti-Xa and IIa assays were used to assess the inhibitory effects of these heparins on these proteases. USP compliant anti-Xa and IIa assays were used to determine potencies of the various heparins. Protamine sulfate (PS) neutralization studies were performed to evaluate the reversal of anticoagulant effects in each of the heparins.Results: The aPTT assay showed that at final concentrations of 5 ug/ml and 2.5 ug/ml porcine heparin significantly (p < .01) prolonged the aPTT compared to ovine, bovine, and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable aPTT values at all concentrations (U/ml). The TT assay showed that porcine and ovine heparins prolonged the TT at 1.25 ug/ml compared to bovine and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable TT values at all concentrations (U/ml). The anti-Xa assay showed that at all final concentrations between 10 ug/ml and 0.625 ug/ml porcine, ovine, and blended heparins produced significantly (p <.001) stronger Xa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-Xa inhibition at all concentrations (U/ml). The anti-IIa assay showed that at final concentrations 2.5 ug/ml, 1.25 ug/ml, and 0.625 ug/ml porcine and ovine heparins produced significantly (p < .05) stronger IIa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-IIa inhibition at all concentrations (U/ml). The USP compliant anti-Xa assay with gravimetric heparins showed potencies of 201, 201, 150, and 184 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-Xa assay with potency adjusted heparins showed comparable potencies for all four heparins. The USP compliant anti-IIa assay with gravimetric heparins showed potencies of 204, 196, 127, and 167 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-IIa assay with potency adjusted heparins showed comparable potencies for all four heparins. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for all of the potency adjusted heparins in the aPTT, TT, anti-Xa, and anti-IIa assays.Conclusion: These studies support the hypothesis that a blended heparin product from bovine, ovine, and porcine tissue, when standardized in USP unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species heparins. These findings suggest that there is a potential for development of blended heparin to stabilize supply chain of this important anticoagulant and warrant clinical validation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of the sensitivity of commercial aPTT tests in measurement of the pharmacodynamic response of milvexian, a novel FXIa inhibitor

Abstract Introduction The activated partial thromboplastin time (aPTT) assay is a test routinely used to evaluate abnormalities or deficiencies in coagulation factors of the intrinsic and common pathways. The composition of the surface activator and phospholipids in the aPTT reagent is known to influence the response of heparin and direct FXa or thrombin inhibitors. Milvexian (formerly referred to as BMS-986177/JNJ-70033093) is an investigational small-molecule Factor XIa (FXIa) inhibitor being studied for the prevention and treatment of major thrombotic conditions. Clinical pharmacology evaluation of milvexian includes aPTT as the primary pharmacodynamic assay, thus underscoring the need for a sensitive reagent to evaluate its anticoagulant activity. Purpose This study evaluated the sensitivity of six commercially available aPTT reagents in plasma from individual donors spiked with milvexian, at concentrations spanning the anticipated clinically relevant exposures. Methods Platelet-poor plasma (PPP) prepared from citrated whole blood collected from consenting, healthy adult volunteers (n=12) was spiked with vehicle (DMSO, 0.5% v/v) or 0.1–10 μM milvexian. The aPTT was measured using six commercially available aPTT reagent kits which included silica; kaolin, or ellagic acid as the contact activator, in combination with natural or synthetic phospholipids. The coagulation automated analysers used for testing were matched to the kit's manufacturer recommendations. All reagents and respective normal or abnormal controls were prepared as instructed by the manufacturer. Results Milvexian exhibited dose-dependent prolongation of aPTT with all reagents tested. Assays performed with aPTT reagents containing kaolin or ellagic acid demonstrated the highest sensitivity, as measured by the concentration that achieved a 2-fold aPTT prolongation (EC2x), and showed the widest dynamic range of response. Coefficient of variability of aPTT measurements in plasma from 12 individual donors was between 5.6–7.9% for Dade® Actin® FS, and 7.1–8% for STA®-C.K. Prest® 5. Conclusions Prolongation of aPTT in PPP spiked with milvexian exhibited a dose-dependent relationship, with statistically significant differences observed among reagents at milvexian concentrations above 0.3 μM. The highest sensitivity measured by changes in the ratio to baseline was obtained with aPTT reagents containing an activator/procoagulant phospholipid combination of ellagic acid + purified soy phosphatides or kaolin + cephalin, which performed similarly. Identification of the reagents with the best combination of sensitivity, precision, and dynamic range may help guide the selection of reagent for assessing milvexian activity. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Janssen Research &amp; Development, LLC

Characterization of a Missense Mutation in the Catalytic Domain and a Splicing Mutation of Coagulation Factor X Compound Heterozygous in a Chinese Pedigree.

Background: Congenital coagulation factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million caused by mutations in the FX-coding gene(F10), leading to abnormal coagulation activity and a tendency for severe hemorrhage. Therefore, identifying mutations in FX is important for diagnosing congenital FX deficiency. Results: Genetic analysis of the proband identified two single-base substitutions: c.794T > C: p.Ile265Thr and c.865 + 5G > A: IVS7 + 5G > A. His FX activity and antigen levels were < 1% and 49.7%, respectively; aPTT and PT were prolonged to 65.3 and 80.5 s, respectively. Bioinformatics analysis predicted the two novel variants to be pathogenic. In-vitro expression study of the missense mutation c.794T > C: p.Ile265Thr showed normal synthesis and secretion. Activation of FXs by RVV, FVII/TF, and FVIII/FIX all showed no obvious difference between the variant and the reference. However, clotting activity by PT and aPTT assays and activity of thrombin generation in a TGA assay all indicated reduced activity of the mutant FX-Ile265Thr compared to FX-WT. Minigene assay showed a normal splicing mode c.865 + 5G > A: IVS7 + 5G > A, which is inconsistent with clinical phenotype. Conclusions: The heterozygous variants c.794T > C: p.Ile265Thr or c.865 + 5G > A: IVS7 + 5G > A indicate mild FX deficiency, but the compound heterozygous mutation of the two causes severe congenital FX deficiency. Genetic analysis of these two mutations may help characterize the bleeding tendency and confirm congenital FX deficiency. In-vitro expression and functional study showed that the low activity of the mutant FX-Ile265Thr is caused by decrease in its enzyme activity rather than self-activation. The minigene assay help us explore possible mechanisms of the splicing mutation. However, more in-depth mechanism research is needed in the future.

Total Phenolic, Flavonoid and Carotinoid Content and Anticoagulation Activities of Sapparin Tablet

Aims: Sapparin tablet, a medicine used for the treatment of blood diseases specially curing blood thickening or impure blood, liver disease in Mongolian Traditional Medicine. The objectives of the study were to determine total biological active substances and analyze the anticoagulation activity of the Sapparin.&#x0D; Study Design: Experimental study.&#x0D; Place and Duration of Study: Department of Chemistry and Technology and Department of Pharmacology, Institute of Traditional Medicine and Technology of Mongolia.&#x0D; Methodology: Quantitative determination of the total active constituents (phenolic, flavonoid, and carotinoids) of the methanol extracts of Sapparin was performed by using Folin-Ciocalteu reagent, aluminium chloride reagent by spectrophotometry.&#x0D; A totally of forty weighing between 220-250 gm were used. Effect of Sapparin was assessed on coagulation parameters following 7, 14, 21 and 28 days administration of 37 mg/kg, 56 mg/kg, 113 mg/kg to healthy rats. The blood coagulation parameters such as prothrombin time (PT) and the activated partial thromboplastin time (aPTT) were measured by means of Quick’s one-stage assay and modified aPTT assay respectively in the rats. Additionally, thrombin activity test was estimated in rats with PT assay using a hemagglutination analyzer. The levels of serum X and von Willebrand factor were measured in Sapparin and control groups by ELISA.&#x0D; Results: The total content of the phenols measured as 5.33±0.0005%, flavonoids as 12.95± 2.21% and carotinoids as 4.31±0.96%.&#x0D; There was significant increase in all assays except fibrinogen, prothrombin time, thrombin, aPTT. Sapparin treatment significantly reduced levels of serum X factor and von Willebrand factor was significantly decreased in rats.&#x0D; Conclusion: Results of this study suggest that Sapparin shows considerable anti-anticoagulant activity in animals and has potential to reduce cardiovascular morbidity and mortality.

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Platelet-Based Factor VIII Activity Is Inhibited By Anti-C2 Domain Antibodies That Don’t Significantly Inhibit Factor VIII Activity in a Standard Assay

The major complication of hemophilia A treatment is the development of inhibitory antibodies against factor VIII (fVIII), causing severe bleeding in spite of infused fVIII. Most antibodies are targeted to the A2 or the C2 domain of fVIII and the degree of inhibition by these antibodies measured in clinical fVIII assays fails to predict the severity of bleeding risk. We investigated whether the degree of inhibition of platelet dependent fVIII activity by a panel of monoclonal anti-C2 domain mAbs deviated from the degree of inhibition in a commercial one-stage assay.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides PAR-1 and PAR-4, and factor XIa without other exogenous activators or vesicles. Results were compared to a commercial aPTT assay (Helena Laboratories, TX) and to results of a novel factor Xase assay developed to recapitulate the conditions in an APT. In the novel assay fresh, density-gradient purified platelets were activated by thrombin followed by inactivation with hirudin and incubated with fVIII and VWF, fIXa, and fX. In a second step, phospholipid vesicles, fVa and prothrombin were added to produce thrombin in proportion to the fXa generated in the first step. For comparison to the aPTT this assay was also done using purified sonicated phospholipid vesicles instead of activated platelets in the first step. The reaction was quenched with EDTA and thrombin generation was determined immediately in a thermosttated kinetics microtiter plate reader (Molecular Devices) at 250C using the chromogenic substrate S-2238 (Diapharma). A panel of 10 defined, epitope mapped mAbs against the fVIIIC2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies.Results: The APT exhibited a log-linear relationship between fVIII and time to fibrin strand formation over a 4-log range, encompassing 0.01% - 100% plasma fVIII. Nine of 10 mAbs inhibited fVIII activity 89-96% while mAb F85 did not inhibit. There was excellent correlation for the degree of inhibition in the platelet-based factor Xase assay for 8 of 10 antibodies with F85 inhibiting the platelet Xase but not the APT and I109 inhibiting the platelet Xase assay less than the APT. In contrast, there was no correlation between inhibition of the aPTT-based assay and the platelet assay. Four mAbs did not inhibit the aPTT assay yet inhibited approximately 90% of platelet-based activity. The novel factor Xase assay, using phospholipid vesicles instead of activated platelets, showed inhibitory results that correlated to the degree of inhibition in the aPTT assay (which also utilizes purified phospholipids). Thus, the novel assay indicates that the degree of inhibition in both platelet-based and vesicle-based clotting assays is a consequence of the membrane type rather than unidentified clotting modifiers in plasma.Conclusions: We have developed a novel factor Xase assay to measure the low levels of residual fVIII activity, at physiologic concentrations, in the presence of inhibitory antibodies. Antibody inhibition of fVIII activity correlated with inhibition measured in the aPTT and APT clotting assays,. Our results confirm that the anti-C2 inhibitory antibodies have substantially different inhibition on a platelet membrane than in an aPTT-based assay and that the differing degree of inhibition is primarily due to fVIII susceptibility to inhibition when functioning on platelet membranes vs. phospholipid vesicles. These results suggest that current inhibitor assays might underestimate the frequency of significant inhibitory antibodies. DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies on a Branded Recombinant Factor VIIa and Its Generic Version to Demonstrate Bioequivalence

Background: Recombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for the same indication (AryoSeven™, AryoGen, Tehran-Iran). These generic versions of rFVIIa are claimed to be biosimilar to the Novoseven. The purpose of this study is to compare several batches of generic versions of rFVIIa namely, Aryoseven with multiple commercially available batches of branded Novoseven to determine bioavailability.Methods: Four commercially available random lots of Novoseven were obtained through US and European sources and compared to 5 clinical batches of Aryoseven obtained from Aryogen (Tehran, Iran). These drugs were diluted with saline to make a stock solution of 1mg/ml. Further dilutions were made to obtain working concentrations of 100, 10, 1 and 0.1 µg/ml. Molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), immunoblotting studies, and procoagulant activity as measured by clot based method were carried out. Thrombin generation studies were carried out after supplementing with NHP at 0-1.0 ug/ml. The pharmacokinetic profile of one representative batch of Novoseven and Aryoseven was studied in primates after a 50 ug/ml bolus IV in terms of factor VIIa levels.Results: The SELDI mass spectrometric profile of all rFVIIa preparations were comparable. The GEP and immunoblotting studies of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 29 KDa. In the clot based functional assays performed in the citrated whole blood and retrieved plasma the two batches of FVIIa produced comparable procoagulant effects in the PT and aPTT assays. In the 0.25 - 1.0 µg/ml range in comparison to the control with saline supplementation, the aPTT did not exhibit any significant shortening whereas there was a sharpening of PT values in the whole blood which was identical in both the AryoSeven™ and NovoSeven® preparations. In the retrieved plasma both products produced comparable effect on the PT and aPTT assays. In platelet rich plasma (PRP) and platelet poor plasma (PPP) AryoSeven and NovoSeven at graded concentration of 0.125 to 1 µg/ml did not produce any measurable change in both PT and aPTT assays. The correction studies carried out in the FVII deficient plasma at a concentration range of 1 to 0.01 µg/ml did not reveal any differences between 2 groups of drugs. The relative correction was comparable in all concentrations for the 2 tissue factor reagents namely Innovin and Recomboplastin 2G. The results were indistinguishable for the 2 groups of drugs. The effect of NovoSeven® and AryoSeven™ on the reversal of anticoagulation in orally anticoagulated patients' were similar in relative correction of the plasma INR for the 2 groups of FVIIa preparations in the range of 0.03 to 1 µg/ml. In the thrombin generation studies both the Novoseven and Aryoseven produced similar effects as measured by increased thrombin generation. In the primate pharmacokinetic studies both groups exhibited similar pharmacokinetic profile.Conclusions: These results demonstrate that four batches of Novoseven and 5 individual clinical batches of Aryoseven were found to be biosimilar. The pharmacokinetic profile of the Novoseven and Aryoseven were studied in primates measuring the factor VIIa level for 6 hours and were found to be comparable. Thus, the generic Aryoseven is biosimilar to branded Novoseven and warrants further validation studies. DisclosuresNo relevant conflicts of interest to declare.

Activated Factor IX As a Candidate for the Initial Protease in the Coagulation Cascade

As currently understood, activated factor VII is the initiating protease of the coagulation process. The identity of the protease(s) that cleaves factor VII to yield its circulating activated form (~1% of the total factor VII protein in normal individuals) remains uncertain. Although, measurement of plasma concentrations of factor VIIa in normal human subjects and those with specific factor deficiencies suggests that factor IX has a factor VIII-independent role in generating circulating factor VIIa (Blood80:25-28, 1992).Previous work using purified plasma proteins yielded evidence that factor IX cleaved only at residue R226 (factor IXaα) was a competent protease, yet did not efficiently bind factor VIIIa (JBC23:14484-14490, 1995). We investigated whether this form of factor IX might activate factor VII, as it seems to have the appropriate characteristics (proteolytic activity that is VIIIa independent). We created and purified recombinant mutant forms of factor IX and factor VII that enabled exploration of the functions of these separate cleavages without the complication of unanticipated proteolysis that is inherent when using plasma derived proteins.Wild-type factor IX and R191H factor IX (the Hemophilia B Chapel Hill variant) were activated to yield factor IXaβ and IXaα, respectively, as demonstrated by SDS-PAGE under reducing and non-reducing conditions, and incorporation of fluoresceinated EGR-chloromethyl ketone into the active sites, confirming that activity correlated with the appropriate molecular mass subunit. As predicted, factor IXaβ and R191H IXaα demonstrated identical catalytic efficiency in the amidolytic cleavage of a small molecular weight substrate, Pefachrome IXa. In contrast, in the presence of factor VIIIa-phospholipid, compared to factor IXaβ, R191H IXaα exhibited greatly reduced ability to cleave the physiologic macromolecular substrate, factor X. Consistent with previous findings, R191H factor IXaα had appreciably lower affinity for factor VIIIa than did factor IXaβ, and this likely explains the difference in factor X activating activity. This difference in activity was also seen in a plasma-based system, where R191H factor IXaα demonstrated only 1.6% of the coagulant activity of factor IXaβ in an aPTT assay in factor IX immunodepleted plasma.There was a distinct difference with a second physiologic macromolecular substrate. Factor R191H factor IXaα and factor IXaβ activated factor VII to VIIa with identical kinetic parameters in a purified system. Intriguingly, the addition of factor VIIIa did not change the kinetic parameters of factor VII activation by either form of factor IXa. This observation is unlikely to be due to factor VIIa contaminating the factor VII preparation, as catalytically incompetent factor VII (S344A) was cleaved at indistinguishable rates by factors R191H IXaα and IXaβ.Experiments with control proteins that also contained mutations at the active site, showed that factor IX R191Q;S411A demonstrated the appropriate sized bands on SDS-PAGE for factor IXaα, but did not incorporate fluoresceinated EGR-chloromethyl ketone, and had no detectable amidolytic, proteolytic, or coagulant activity, consistent with the active site serine mutation. Factor IX R191Q;R226Q was not cleaved by factor VIIa-tissue factor, and factor IX R226Q was cleaved to subunits consistent with factor IXα. Neither of these proteins incorporated fluoresceinated EGR-chloromethyl ketone, and neither demonstrated detectable amidolytic activity.These experiments with recombinant forms of factor IX support the hypothesis that factor IXaα exhibits active site enzymatic competency equal to factor IXaβ, and that cleavage at R191 is required to generate fully competent factor VIIIa binding determinants. Moreover, factor IXaα and factor IXaβ are fully competent to activate factor VII, without influence by factor VIIIa. Given this mechanistic demonstration and the previously noted clinical observations (Blood 80:25-28, 1992), we propose that activated forms of factor IX are responsible for generating circulating plasma factor VIIa, which is available for the initiation phase of coagulation. These data suggest that activated factor IX is as plausible a candidate for the initiating protease of the coagulation cascade as is activated factor VII. DisclosuresNo relevant conflicts of interest to declare.

Measuring the Anticoagulation Effects of Exosite I Thrombin Inhibitors: Utility and Limitations of Clotting Assays, Thromboelastography, and Calibrated Automated Thrombography

The ability to anticoagulate patients at elevated risk for thrombosis without inducing a bleeding diathesis remains an unmet need of patient care both in a hospital setting and at home. Traditional anticoagulants including warfarin and heparin effectively inhibit clotting, but incur a bleeding risk and require continued monitoring after a stable profile has been established. Current generation direct oral anticoagulants (DOACs) including dabigatran, rivaroxaban, and apixaban overcome many of the liabilities of warfarin and heparin (e.g. lack of target specificity and need for monitoring), but are still susceptible to bleeding diathesis. Thus there remains a need for novel anticoagulants that offer an improved therapeutic index. The recombinant human antibody JNJ-64179375 (JNJ-9375) is a novel thrombin inhibitor that binds to the exosite I region with high affinity and selectivity. In contrast to DOACs that inhibit the protease active site, JNJ-9375 inhibits the interaction of thrombin with its substrates including fibrinogen and PAR-1 while retaining active site and exosite II mediated interactions. This targeted and distinct mechanism of action which modulates the actions of thrombin offers the potential for efficacious anticoagulation with the potential for reduced risk of bleeding compared to active site-specific DOACs.To assess the ability to measure exosite I-specific thrombin inhibitors effects in blood, we compared JNJ- 9375 to the active site ligand dabigatran in clotting assays, thromboelastography (TEG), and calibrated automated thrombography (CAT) assays. We focused on clinically relevant concentrations of these inhibitors based on the efficacious doses reported in the RE-LY study for dabigatran (NCT00262600) and a Phase 1 study for JNJ-9375 (NCT02949206). In conventional clotting assays (prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)) dabigatran and JNJ-9375 each prolonged clotting times in a dose-dependent manner. TT assays were the most sensitive to dabigatran and JNJ-9375, with 80 nM dabigatran prolonging the clotting time by 5-fold and 100 nM JNJ-9375 prolonging the clot time 4-fold over vehicle control. PT and aPTT assays were less sensitive than the TT assay, requiring ≥ 300 nM dabigatran and ≥ 1 µM JNJ- 9375 to double the PT or aPTT clotting times. In TEG assays both JNJ-9375 and dabigatran prolonged the reaction time (R), with 200 nM JNJ-9375 prolonging R by 1.3- to 1.5-fold and 100 nM dabigatran prolonging R from 1.6- to 1.8-fold over buffer control. In CAT assays initiated with 5 pM tissue factor (PPP Reagent, Stago), dabigatran inhibited peak thrombin and Endogenous thrombin potential (ETP) with an approximate IC50 of 1 µM. In contrast, titration of JNJ-9375 up to 1 µM increased both the Peak thrombin and ETP ≥ 20% above buffer control. This increase in peak thrombin and ETP with JNJ-9375 was also observed with other exosite I-specific ligands such as hirugen, suggesting a general effect of exosite I binding on the primary endpoints of the CAT assay. This insensitivity of the CAT assay to exosite I ligands likely reflects the inability of these molecules to inhibit proteolysis of the peptidyl substrate z-Gly-Gly-Arg-AMC, which directly binds to the active site and is used as the readout of thrombin activity.In summary, of the assays tested the TT clotting assay offers the greatest sensitivity to detect multiple classes of direct thrombin inhibitors in plasma, while TEG can detect direct thrombin inhibitors in whole blood. In contrast, the CAT assay is insensitive to exosite I ligands such as JNJ-9375. These results highlight the importance of the relationship between the endpoint of a given coagulation assay and the mechanism of action of the anticoagulant being tested. DisclosuresBunce:Janssen Research & Development: Employment. Huang Devine:Janssen Research & Development: Employment. Chintala:Janssen Research & Development: Employment.

Biochemical and Pharmacological Differentiation of APC and Recombinant Thrombomodulin with Reference to the Effect on the Hemostatic and Fibrinolytic Processes

Introduction:Both thrombomodulin and activated protein C play an important role in the regulation of hemostasis. Recombinant versions of both the thrombomodulin (rTM) and activated protein C (rAPC) have been developed for clinical applications. Marked differences in their biochemical and pharmacological profiles exist. Such differences in their effects may be due to target sites and other interactions and may have therapeutic and safety implications.Aim:The aim of this study is to investigate the biochemical and pharmacological differences between a clinically developed rTM (ART-123, Asahi Kasei Pharma, Tokyo, Japan) and a plasma derived APC (Haematologic Technologies, Burlington, VT, USA).Materials and Methods:The anticoagulant profile of both agents was investigated in native whole blood using the ACT, TEG and global anticoagulant profile using PT and APTT assays at a concentration range of 0-10 ug/ml. The global anticoagulant profile in plasma based systems in terms of PT and APTT was also investigated. The effect of both of these agents was studied in the antiprotease (Xa and IIa) inhibitory assays and thrombin generation inhibition methods. A modified TEG system was used to study the modulation of tissue plasminogen activator mediated fibrinolysis by these agents.Results:In the ACT assays APC produced a much stronger anticoagulant effect in a concentration range of 0-10 ug/ml with a doubling time at 3 ug/ml, whereas rTM produced marginal effects on this parameter. The TEG profile of APC exhibited much stronger anticoagulant effects in comparison to rTM. At 3 ug/mL, APC produces much longer prolongation of the R and K time parameters in comparison to rTM. APC also facilitated tPA-mediated fibrinolysis in contrast to rTM. In the anti-IIa assays, rTM produced concentration dependent inhibition of thrombin and no inhibition of factor Xa. APC did not have any effect on either Factor Xa or IIa. In the thrombin generation assays, APC produced a stronger inhibition of thrombin generation (IC50=0.4 ug/mL) whereas rTM was relatively weaker (IC50 = 1.2 ug/mL). In the APTT assay APC produced a much stronger anticoagulant effect whereas rTM produced relatively weaker effects. Both agents did not produce any modification of agonist induced platelet aggregation. In terms of anticoagulant potency the APC exhibited 70 + 6 USP U/mg whereas the rTM was much weaker < 10 USP U/mg.Conclusions:These studies demonstrate that APC is a much stronger anticoagulant in comparison to rTM. In addition both agents produced different modulation of fibrinolytic and hemostatic processes. These observations suggest that in clinical indications such as DIC, these agents may have differential safety/efficacy implications. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

A Bispecific Antibody Lacks Measurability in Routine Coagulation Assays and Comparability to Factor VIII

Background : Novel therapeutic approaches for hemophilia A include the bispecific antibody emicizumab (ACE910), for which clinical phase III results were recently reported (Oldenburg et al. NEJM 2017). During preclinical and clinical development, ACE910's activity was tracked using thrombin generation assay (TGA), chromogenic assay, and activated partial thromboplastin time assay (aPTT) (Shima et al. NEJM 2016, Kitazawa et al. Nat Med. 2012). The most suitable method to test the efficacy of non-factor therapy in a routine setting is not yet known.Aim: To examine the effect of assay type on hemostatic activity monitoring of a sequence identical analogue (SIA) to ACE910, and to assess FVIII-activity equivalence in chromogenic and aPTT assays, and in TGA.Methods : SIA ACE910 was expressed in HEK293 cells, purified as previously described (Sampei et al. PLoS One. 2013), and analyzed using the following five commercial FVIII chromogenic assay kits: Coatest factor VIII, Coamatic factor VIII, Siemens FVIII:C, Technochrom FVIII:C, and Biophen FVIII:C. aPTT or clot waveform was analyzed using five differently composed trigger reagents (Dapttin, Actin FSL, Pathromtin SL, Triniclot, and APTT-SP). In thrombin generation assays, SIA was tested in comparison with recombinant FVIII (ADVATE) using extrinsic (TF) and intrinsic (FXIa) trigger.Depending on method, SIA concentrations ranged from 2-1200 nM.Results: SIA ACE910's lack of cross-reactivity to bovine factors, which are commonly used in chromogenic assay kits, rendered only the Biophen FVIII:C test, which uses human coagulation factors, suitable for analysis. However, as the concentration response curve of SIA ACE910 maxed out and deviated from the FVIII reference curve, the chromogenic assay was not suitable to assess FVIII-equivalence. The aPTT test was highly sensitive to SIA ACE910, resulting in a substantial reduction in clotting time at low and potentially non-therapeutic concentrations. Different aPTT reagents yielded a FVIII-equivalence of 18-69% for 20 nM SIA and >200% at ≥200 nM SIA ACE910 - the plasma concentrations observed in patients in the phase III trial. SIA ACE910 (600 nM) only partially restored thrombin generation (TG) in hemophilia A patient plasma, resulting in a FVIII-equivalence of 4-8% (intrinsic) and 16-36% (extrinsic) based on peak thrombin. Assessment of other TG parameters demonstrated in FVIII-equivalence of <1 to >100% . Assay results for 60, 200, and 600 nM SIA are summarized in Table 1.Conclusions : Analysis of SIA and its equivalence to FVIII is challenged by its strongly differing mechanism, rendering standard FVIII protocols unsuitable.Results are highly influenced by assay type, analytical conditions, and parameters used for calculation. Chromogenic assays, aPTT, and TGA give largely discrepant values. Thus, new methods and likely a product specific standard are required to better predict the hemostatic effect of non-factor therapies. [Display omitted] DisclosuresHartmann:Shire: Employment. Feenstra:Shire: Employment. Knappe:Shire: Employment. Schrenk:Shire: Employment. Scheiflinger:Shire: Employment, Equity Ownership; Baxter: Equity Ownership. Dockal:Baxter: Equity Ownership, Patents & Royalties; Shire: Employment, Equity Ownership; Baxalta: Patents & Royalties.

Is Lupus Anticoagulant a Significant Feature of COVID-19? A Critical Appraisal of the Literature.

The term "lupus anticoagulant (LA)" identifies a form of antiphospholipid antibodies (aPLs) causing prolongation of clotting tests in a phospholipid concentration-dependent manner. LA is one of the laboratory criteria identified in patients with antiphospholipid (antibody) syndrome (APS). The presence of LA in patients with APS represents a significant risk factor for both thrombosis and pregnancy morbidity. There have been several reports of similarities between some of the pathophysiological features of COVID-19 and APS, in particular the most severe form, catastrophic APS. There have also been many reports identifying various aPLs, including LA, in COVID-19 patients. Accordingly, a very pertinent question arises: "Is LA a feature of COVID-19 pathology?" In this review, we critically appraise the literature to help answer this question. We conclude that LA positivity is a feature of COVID-19, at least in some patients, and potentially those who are the sickest or have the most severe infection. However, many publications have failed to appropriately consider the many confounders to LA identification, being assessed using clot-based assays such as the dilute Russell viper venom time, the activated partial thromboplastin time (aPTT), and the silica clotting time. First, most patients hospitalized with COVID-19 are placed on anticoagulant therapy, and those with prior histories of thrombosis would possibly present to hospital already on anticoagulant therapy. All anticoagulants, including vitamin K antagonists, heparin (both unfractionated heparin and low-molecular-weight heparin), and direct oral anticoagulants affect these clot-based assays. Second, C-reactive protein (CRP) is highly elevated in COVID-19 patients, and also associated with severity. CRP can also lead to false-positive LA, particularly with the aPTT assay. Third, persistence of aPL positivity (including LA) is required to identify APS. Fourth, those at greatest risk of thrombosis due to aPL are those with highest titers or multiple positivity. Most publications either did not identify anticoagulation and/or CRP in their COVID-19 cohorts or did not seem to account for these as possible confounders for LA detection. Most publications did not assess for aPL persistence, and where persistence was checked, LA appeared to represent transient aPL. Finally, high titer aPL or multiple aPL positivity were in the minority of COVID-19 presentations. Thus, at least some of the reported LAs associated with COVID-19 are likely to be false positives, and the relationship between the detected aPL/LA and COVID-19-associated coagulopathy remains to be resolved using larger and better studies.

The Global Hemostatic Thrombodynamics Assay in Healthy Children

Background: There is evidence that the concentrations of clotting and anticoagulant factors in children depend on age and differ from those found in adults. The results of APPT, TT, PT, fibrinogen are similar in children and adults in some studies, while PT and APTT show differences in others. Recent studies on global hemostatic assays Thromboelastography (TEG) revealed no significant differences in test results between healthy children and adults, while the thrombin generation test (TGT) showed significant differences. The Thrombodynamics (TD) assay is a new global hemostasis assay that considers the spatial organization of coagulation. Methods: APTT, TT, PR, fibrinogen and TD assays were performed in 102 healthy children between the ages of 1 and 17 years who underwent annual medical examinations and in 91 healthy adult volunteers. The following TD assay parameters were determined: lag time (Tlag), initial clot growth velocity (Vi), stationary clot growth velocity (Vs), clot size 30 minutes after the start of clot growth (CS) and clot Density (D). Written consent was obtained from all participants or their parents after they received complete information about the tests. Results: Age-specific reference values for the TD assay in healthy children aged 1-17 years are presented. No significant differences were observed between different age groups of children (15 years, 6-10 years, and 11-17 years) or between all children (1-17 years) and adults. Significant differences were not observed between genders. Conclusions: The TD assay results revealed no age-specific differences in the parameters between children aged 1-17 years and adults.