Deficient Plasma Research Articles

A novel assay of excess plasma kallikrein-kinin system activation in hereditary angioedema.

Cleaved high-molecular-weight kininogen (HKa) is a disease state biomarker of kallikrein-kinin system (KKS) activation in patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH), the endogenous inhibitor of plasma kallikrein (PKa). Develop an HKa-specific enzyme-linked immunosorbent assay (ELISA) to monitor KKS activation in the plasma of HAE-C1INH patients. A novel HKa-specific antibody was discovered by antibody phage display and used as a capture reagent to develop an HKa-specific ELISA. Specific HKa detection following KKS activation was observed in plasma from healthy controls but not in prekallikrein-, high-molecular-weight kininogen-, or coagulation factor XII (FXII)-deficient plasma. HKa levels in plasma collected from HAE-C1INH patients in a disease quiescent state were higher than in plasma from healthy controls and increased further in HAE-C1INH plasma collected during an angioedema attack. The specificity of the assay for PKa-mediated HKa generation in minimally diluted plasma activated with exogenous FXIIa was demonstrated using a specific monoclonal antibody inhibitor (lanadelumab, IC50 = 0.044 µM). An ELISA was developed for the specific and quantitative detection of HKa in human plasma to support HAE-C1INH drug development. Improved quantification of the HKa biomarker may facilitate further pathophysiologic insight into HAE-C1INH and other diseases mediated by a dysregulated KKS and may enable the design of highly potent inhibitors targeting this pathway.

Methods, precision, and analytical sensitivity of a novel low-plasma-volume assay of fibrinolytic capacity utilizing the euglobulin fraction.

Fibrinolysis is a critical aspect of the hemostatic system, with assessment of fibrinolytic potential being critical to predict bleeding and clotting risk. We describe the method for a novel low-plasma-volume assay of fibrinolytic capacity utilizing the euglobulin fraction (the "modified mini-euglobulin clot lysis assay [ECLA]"), its analytic sensitivity to alterations in key fibrinolytic substrates/regulators, and its initial applications in acute and convalescent disease cohorts. The modified mini-ECLA requires 50 μL of plasma, a maximal read time of 3 h (with most results available within 60 min), and is entirely performed in a 96-well microplate. Assay measurements were obtained in a variety of commercial control and deficient plasmas representing clinically relevant hypo- and hyperfibrinolytic states, and in three distinct adolescent cohorts with acute or convalescent illness: critically ill, following endotracheal intubation; acute COVID-19-related illness; and ambulatory, 3 months following a venous thromboembolic event. In 100% and 75% deficient plasmas, hypofibrinolysis for plasminogen-deficient, fibrinolysis for alpha-2-antiplasmin-deficient, and hyperfibrinolysis for plasminogen activator inhibitor-1-deficient plasmas were observed. The modified mini-ECLA Clot Lysis Time Ratio ("CLTR") demonstrated moderate-strength correlations with the Clot Formation and Lysis (CloFAL) assay, is analytically sensitive to altered fibrinolytic states in vitro, and correlates with clinical outcomes in preliminarily-studied patient populations.

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b+ putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored by divergent endothelial subsets composed of CD32blowCD31high, LYVE1+STAB1+CD32bhighCD31lowTHBD-vWF-, and LYVE1-THBD+vWF+ cells. Wnt2 mediated sinusoidal-to-hepatic intercellular crosstalk potentiates hepatocyte differentiation and branched endothelial network formation. Intravital imaging revealed iLSEP developed fully patent human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enabled correction of in vitro clotting time with Factor V, VIII, IX, and XI deficient patients' plasma and rescued the severe bleeding phenotype in hemophilia A mice upon transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.

Quantification of the contribution of individual coagulation factors to haemostasis using a microchip flow chamber system and reconstituted blood from deficient plasma.

Quantifying the contribution of individual coagulation factors to haemostasis may aid our understanding of the haemostatic function in patients with rare coagulation deficiencies (RCDs) and the exploration of suitable treatments. Reconstituted blood prepared from specific coagulation factor-deficient plasma (factor [F]II; prothrombin, FV, FVII, FVIII, FIX, FX, FXI or FXII) and red blood cell/platelet products were used to simulate the whole blood of patients with RCD. We prepared in vitro treatment models for patients with prothrombin deficiency using coagulation factor agents and fresh frozen plasma. Haemostatic function was measured using a microchip flow chamber system at 600 s-1. The haemostatic function was low, especially in blood samples reconstituted with prothrombin- and FX-deficient plasma. In a plasma transfusion model of prothrombin deficiency, haemostatic function recovered after 10% replacement with normal plasma and reached a plateau at ≧60% replacement. A treatment model of prothrombin deficiency with prothrombin complex concentrates revealed dose-dependent therapeutic effects in the range of 0-50 IU/kg. Microchip flow chamber system-based quantification of haemostatic function using reconstituted blood could predict haemostasis and therapeutic effects of treatments in patients with prothrombin deficiency.

297 Effects of plasma zinc concentration on longissimus thoracis metabolic status of beef steers

Abstract The objective of this study was to examine the role of Zn status on muscle glucose and other metabolites. Angus steers [n = 144; body weight (BW) = 525 ± 30 kg] with varying plasma Zn concentration and implant status served as the initial pool for this secondary experiment. Steers were assigned to one of two implant (IMP) treatments: no implant (NO) or Component TE-200 (TE-200; Elanco, Greenfield, IN) on d 0. Zinc was supplemented as ZnSO4 at 0 mg Zn/kg dry matter (DM; analyzed 54 mg Zn/kg DM), 30 mg Zn/kg DM, or 100 mg Zn/kg DM, starting 60 d before implant day. Steers were fed via GrowSafe bunks (GrowSafe Systems Ltd., Airdie, AB, Canada), and steer was experimental unit. Steers were fed in two blocks with a 14-d stagger for sample logistics. Blood and muscle biopsies of the longissimus thoracis were collected on d 40 after implant, a minimum of 2 h post-feed delivery. Plasma Zn was quantified via ICP-OES and 144 samples were stratified into quantiles by plasma Zn concentration. Samples (n = 12 low and 12 high from NO and TE-200 groups) were identified and designated as low plasma Zn (LOW, 1.1 mg Zn/L average) and high plasma Zn (HI, 1.6 mg Zn/L average) treatments (TRT). Muscle samples from these 48 steers were homogenized and derivatized for subsequent analysis via gas chromatography-mass spectrometry (GCMS) for non-targeted metabolomic analysis. Data were analyzed as a complete randomized design using the MIXED procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC) with fixed effects of TRT, IMP, BLOCK, and TRT×IMP. A TRT×IMP effect was observed for muscle glycine (P = 0.02) where LOW/IMP was similar to HI/NO but had greater glycine concentrations than LOW/NO and HI/IMP. There was a tendency for a TRT×IMP (P = 0.07) effect where LOW/IMP and HI/NO tended to have greater concentrations of hydroxyisovaleric acid. Given the role of glycine in protein synthesis and hydroxyisovaleric acid in leucine metabolism these data suggest differential protein responses to implants in cattle of lesser vs. greater plasma Zn concentrations. Hydroxybutyric acid, a ketone body which can be utilized for energy, tended to be greater in muscle of steers receiving an IMP (P = 0.09). Contrary to our hypothesis that greater plasma Zn would support differential muscle glucose metabolism, no effects of plasma Zn TRT or IMP were noted for glucose, lactate, and other detected metabolites. Both LOW and HI plasma Zn concentrations were still considered adequate, and future research should explore the impact of distinctly deficient and adequate plasma Zn concentration on glucose metabolism pathways in muscle.

Activated protein C resistance in the copresence of emicizumab and activated prothrombin complex concentrates

BackgroundVenous thromboembolic events have been reported in persons with hemophilia A who received emicizumab and activated prothrombin complex concentrate (APCC) concomitantly, but the relevant mechanism(s) remains unclear. We speculated that activated protein C (APC) and antithrombin (AT) resistance might be associated with these adverse events. ObjectivesTo investigate APC and AT resistance in factor (F)VIII–deficient (FVIIIdef) plasma in the presence of emicizumab and APCC. MethodsIn pooled normal plasma or FVIIIdef plasma samples mixed with emicizumab (50 μg/mL) and FVIII-bypassing agents, including recombinant FVIIa (2.2 μg/mL), APCC (1.3 IU/mL), or plasma-derived FVIIa/FX (1.5 μg/mL), the suppression effect of AT (0-2.4 μM) and APC (0-16 nM) was assessed by tissue factor–triggered thrombin generation assay. The APC effects in FVIIIdef plasma with the copresence of emicizumab, FII (1.3 μM), and/or FIXa (280 pM) were also examined. ResultsThe AT resistance in emicizumab and each bypassing agent was not observed. Moreover, APC dose-dependent suppression effect was observed in pooled normal plasma or FVIIIdef plasma mixed with emicizumab and recombinant FVIIa or plasma-derived FVIIa/FX. However, APC-catalyzed inactivation had little effect on thrombin generation assay potential in FVIIIdef plasma spiked with emicizumab and APCC. The addition of FIXa to emicizumab in FVIIIdef plasma could lead to partial APC resistance. Furthermore, FVIIIdef plasma spiked with emicizumab, FIXa, and FII was markedly resistant to APC-mediated inactivation. ConclusionFII and FIXa in APCCs were key clotting factors for APC resistance in FVIIIdef plasma supplemented with emicizumab and APCCs. The APC resistance in persons with hemophilia A receiving emicizumab and APCC may contribute to venous thromboembolic events.

Measurement of recombinant porcine factor VIII in patients with congenital haemophilia A and inhibitors in the presence of emicizumab.

Recombinant porcine factor VIII (rpFVIII) is a treatment option for break-through bleeds in patients with congenital haemophilia A with inhibitors (CHAwI) on emicizumab. However, there are limited data about the measurement of rpFVIII in the presence of emicizumab. To analyse whether rpFVIII can be measured with a chromogenic assay with bovine component (bCSA) in plasma from CHAwI on emicizumab treatment. In the first part of the study, FVIII deficient plasma was spiked with rpFVIII, in the second part, commercial plasma from CHAwI was spiked with emicizumab and rpFVIII, and in the third part, plasma from CHAwI on emicizumab treatment was spiked with rpFVIII. FVIII was then measured with bCSA and a chromogenic assay with human component (hCSA). Thrombin generation (TG) and clot-waveform analysis (CWA) were also carried out. The recovery of rpFVIII measured with bCSA is approximately 80% and is further influenced by the presence of an anti-porcine inhibitor. rpFVIII assessed with hCSA was influenced by emicizumab. CWA and TG showed a weak correlation with baseline emicizumab concentration, but peak thrombin and CWA correlated well with increasing emicizumab concentrations and rpFVIII activities. This study indicates that rpFVIII can be measured in the presence of emicizumab with a bCSA. A calibration curve for the measurement of rpFVIII with bCSA should be established.

A Brief History of Child Nutrition Situation and Programme Interventions in Bangladesh

Since the independence in 1971, Bangladesh has madesteady progress in improving nutrition status of childrenin the country. As per the first nutrition survey afterindependence, “Nutrition Survey of Rural Bangladesh1975-76” carried out by the Institute of Nutrition and FoodScience (INFS), Dhaka University, revealed that 31% of12-23 months children and 28% of 24-35 months oldchildren were acutely malnourished; and 74% of children0-59 months old were stunted (moderate and severe). Nineout of ten families (i.e., 89%) were deficient in vitaminintake. The first nutrition survey in East Pakistan which is nowBangladesh, was conducted in 1962-64 but did not providemuch child-related data but found that over two-thirds ofthe daily diet consisted of rice, which was supplementedby curries, primarily of vegetables or fish, and Lentil.Milk and meat are consumed in small amounts, and fruitintake was only seasonal. Intake of cooking fats and oilswas scanty. The survey revealed that 46% of householdshad inadequate calorie intake. Carbohydrates constitutedabout 83% of the total calorie intake. Moreover, 85% ofthe people did not meet their recommended level of dailyprotein intake where almost 70% of protein was fromcereal proteins (plant sources) and only 14% was fromanimal sources. Eighty-four percent of all householdsconsumed less than the acceptable intake value of VitaminA (3,060 IU per person per day) and 40% of the populationhad deficient or low plasma concentration of Vitamin Aand carotene. As a result, night blindness was commonamong children and about 30,000 children became blindeach year. Also, about 25 percent of deaths under five wereattributable to Vitamin A deficiency.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Forms of Hereditary Angioedema That Are Independent of Kallikrein and Factor XIIa System

Hereditary angioedema (HAE) is an inherited disorder characterized by recurrent episodes of severe soft tissue swelling involving skin and mucous membranes. In most cases, edema is caused by excessive formation of the vasoactive nanopeptide bradykinin (BK) due to dysregulation of the plasma kallikrein-kinin system (KKS). The KKS consists of the zymogens prekallikrein (PK) and factor XII (FXII) and the cofactor/substrate high-molecular-weight kininogen (HK). PK and FXII reciprocally convert each other to the proteases plasma kallikrein (PKa) and FXIIa. PKa cleaves HK after Lys362 and Arg371 to release bradykinin. In most cases of HAE, increased KKS activation is due to low plasma activity of C1-Inhibitor (C1-INH), the main regulator of PKa and FXIIa. However, ~10% of HAE patients have normal C1-INH activity (HAEnC1). A relationship between fibrinolysis and kinin formation was first described in 1971. The fibrinolytic protease plasmin activates FXII and cleaves HK, although it is unclear that this results in significant bradykinin generation. Recently mutations in the PLG gene encoding plasminogen (Lys311Glu) and the KNG1 gene encoding HK and the related protein low-molecular-weight kininogen (LK, Met361Lys) were identified in patients with HAEnC1. Here we describe how the fibrinolytic system contributes to angioedema in patients with these mutations. Human Glu-plasminogen (Plg), HK and LK were expressed in HEK293 cells. Lys311 in plasminogen was changed to glutamic acid, and Met361 in HK and LK were changed to lysine, by site directed mutagenesis. Wild type (Plg-Lys311) and variant (Plg-Glu311) plasminogen were converted to plasmin (Plm-Lys311 and Plm-Glu311) with urokinase. The capacity of plasmin to cleave HK and LK were studied with SDS-PAGE, western blots and kinin ELISA. Cleavage of wild type HK-Met361 and LK-Met361 and variant HK-Lys361 and LK-Lys361 by PKa and plasmin were assessed in a similar manner. The types of kinin released during reactions were determined by mass spectroscopy. Normal plasma was supplemented with Plg-Lys311 or Plg-Glu311, and tPA was added to generate plasmin. Bradykinin release was substantially greater with Plm-Glu311 than Plm-Lys311. This held when reactions were run in plasma lacking FXII or PK, indicating activation of the KKS was not responsible for kinin production. Using plasma-derived proteins, PKa released bradykinin 100-fold faster from HK than LK, and released bradykinin from HK ~50-fold faster than did plasmin. PKa and plasmin released bradykinin at comparable relatively slow rates from LK. Plm-Glu311 released bradykinin from HK 10-fold faster, and from LK 3-fold faster, than in reactions with Plm-Lys311. In experiments in which kininogen deficient plasma was supplemented with either HK or LK to a physiologic concentration, the majority of bradykinin released by Plm-Glu311 was from LK. PKa-catalyzed release of bradykinin from wild type HK-Met361 and variant HK-Lys361 comparably. The same held for LK-Met361 and LK-Lys361. However, plasmin released kinin from HK-Lys361 and LK-Lys361 3.3-fold and 13-fold faster than from HK-Met361 and LK-Met361. While the kinin released from HK-Met361 and LK-Met361 was bradykinin, plasmin released the decapeptide Lys-bradykinin (kallidin) from HK-Lys361 and LK-Lys36. Our studies indicate that angioedema in some patients with HAEnC1 may be due to fibrinolytic activity, and not to KKS activity. Both HK and LK may be substrates in such patients. As the plasma LK concentration is ~4-fold higher than HK, it may be the main pathologic kinin source. The plasmin Lys311Glu substitution converts the protease into a more efficient kininogenase. Fibrinolytic activity is normally brisk in the mouth, perhaps explaining why patients with the Lys311Glu mutation primarily suffer from oral-lingual angioedema. The findings raise the possibility that oral-lingual edema after tPA infusion or ACE inhibitor administration may also be due to plasmin-mediated cleavage of LK and HK. The Met361Lys substitution in HK and LK introduces a novel protease cleavage site immediately upstream from a normal PKa cleavage site. The novel site is preferred over the normal site by plasmin, but not PKa. Several treatments for HAE directed at neutralizing PKa or FXIIa activity have been developed. Our results suggest that such drugs may be less effective in patients with HAEnC1 in which disease mechanisms operate independently of the KKS.

A Novel Plasminogen Activator Anti-Fibrin-uPA Increases the Sensitivity of the Plasmin Generation Assay to Therapeutic Target PAI-1

Introduction.The fibrinolytic system is crucial for clot lysis during wound healing. Clot lysis is initiated by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which convert plasminogen into plasmin. TPA-mediated plasminogen activation requires binding to fibrin, while uPA activates plasminogen independent of fibrin. Both plasminogen activators are inhibited by plasminogen activator inhibitor-1 (PAI-1), although, fibrin-bound tPA is protected from PAI-1. As inadequate regulation of fibrinolysis might lead to thrombotic and bleeding events, there is a clinical need for tools that estimate a patient's fibrinolytic state. We developed anti-fibrin-uPA (aFn-uPA), a construct containing the plasminogen activator uPA, linked to a nanobody directed against fibrin, to support binding of uPA to fibrin, and to increase effectiveness of plasminogen activation. This novel plasminogen activator improves the sensitivity of the calibrated, automated plasmin generation (PG) assay, for PAI-1. Aim.To increase the sensitivity of the calibrated, automated PG assay for detection of (anti-)PAI-1 effects in human plasma. Methods.PG in normal pooled plasma (NPP) or factor deficient plasma was measured by cleavage of a plasmin-specific fluorogenic substrate, using a calibrated, automated method. In brief, fibrinolysis was started by combining plasma and trigger solution in a 96-well plate. The trigger solution consists of tissue factor (TF) or Russell's viper venom-factor X activator (RVV-X), phospholipids, and tPA or aFn-uPA. PG was measured with a fluorometer, equipped with a 390/460 filter set (excitation/emission) (Fluoroscan Ascent, Thrombinoscope, Maastricht, The Netherlands). The following PG parameters were extracted from the data: lag time, time to peak (ttPeak), endogenous plasmin potential (EPP) and peak. Results.High TF, RVV-X, tPA and aFn-uPA were associated with a shorter PG lag time and a shorter PG ttPeak, compared to lower trigger concentrations. For subsequent experiments, 5 pM TF or 10 -5 RVV-X, and 1.25 µg/mL (17.8 nM) tPA or 1 µg/mL (21 nM) aFn-uPA, were selected as optimal. The assay was specific for plasmin as no PG was observed in plasminogen- nor fibrinogen-deficient plasma. The sensitivity of the PG assay to PAI-1 was higher in PG triggered with the novel plasminogen activator aFn-uPA, compared to tPA, regardless of the affinity of both activators for fibrin. PAI-1 dose-dependently increased the lag time and ttPeak, and reduced the EPP and peak in PG stimulated with aFn-uPA in both NPP and PAI-1-deficient plasma. Using an anti-PAI-1-Ab, the inhibition obtained upon 2 µg/mL (46.5 nM) PAI-1, was reduced or completely abolished, depending on the concentration of anti-PAI-1-Ab (Figure). Conclusions.Overall, the calibrated, automated PG assay, in particular when aFn-uPa is used as trigger for fibrinolysis, is a specific and efficient tool to measure PG in human plasma, and is sensitive to the therapeutic target PAI-1. This tool may help identify abnormal fibrinolytic states in bleeding and thrombotic disorders.

FVIII Inhibitors can Accurately be Measured in the Presence of Mim8 in Hemophilia A Plasma

Introduction: Mim8 (Denecimig; Novo Nordisk) is a next generation bispecific antibody being developed for prophylactic treatment of hemophilia A with or without inhibitors. Mim8 complexes with FIXa and FX to generate FXa in the absence of FVIII. There is a clinical need to monitor FVIII inhibitors in hemophilia A patients, however measuring FVIII inhibitors in the laboratory can be challenging. Using aPTT-based Modified Nijmegen-Bethesda Assays in the presence of FVIIIa Mimetics such as Mim8 is not recommended due to interference. While FVIII chromogenic substrate assays (CSA) containing human or human/bovine proteins vary in their sensitivity to FVIII Mimetics. This study investigated if bovine FIXa/FX FVIII CSA can accurately measure FVIII inhibitors in the presence of Mim8 in hemophilia A plasma. Methods: Two FVIII CSA kits using bovine FIXa/bovine FX reagents, Factor VIII Chromogenic Assay (Siemens Healthineers) and Chromogenix Coatest ® SP4 FVIII (Diapharma Group, Inc.) were evaluated to investigate whether Mim8 at 5, 10, 20 and 40 ug/mL (final concentration) interferes with the measurement of FVIII inhibitor (Affinity BioLogicals, Inc.) spiked into pooled congenital FVIII deficient plasma (George-King Bio-Medical, Inc.), at 0.2, 1.0 and 4.8 Bethesda Units/mL (BU/mL, final concentration). CSA were analyzed on BCS ®XP analyzers (Siemens Healthineers) using manufacturer or laboratory developed protocols. Inhibitor samples were prepared from a FVIII inhibitor working solution that was assigned a potency of 11.8 BU/mL (Siemens CSA). Mim8 was added to the FVIII inhibitor samples at 20x the final concentration so that the plasma matrix was 95% inhibitor plasma. CRYOcheck™ Factor VIII Inhibitor kit (Precision BioLogic, Inc.) was used to measure the FVIII inhibitor levels. Plasma containing FVIII inhibitor alone or in combination with Mim8 was heat inactivated at 56 ± 2°C for 30 minutes, followed by centrifugation at 2700xg for 5 minutes. The inactivated plasma was incubated at 37 ± 1°C for 2 hours with Imidazole buffered Pooled Normal Plasma (PNP). By standardizing the amount of FVIII in the PNP (⁓100%), the inhibitor levels were determined according to how much of the added FVIII was inactivated relative to a control tube mix with no inhibitor present. Mim8 interference was defined as a negative result (<0.6 BU/mL) becoming positive for samples containing 0.2 BU/mL of FVIII inhibitor, and a result outside of ±25% (%RE) of the corresponding un-spiked sample for the samples containing 1.0 or 4.8 BU/mL of FVIII inhibitor. Results: The data presented for each CSA is from 3 separate aliquots tested on 3 independent occasions. No Mim8 interference was observed at any FVIII inhibitor levels whichever CSA kit, using bovine FIXa or FX reagent, was used with all results for Mim8 spiked samples falling within ±25.0% of the target value. Using the Siemens CSA inhibitor samples at 0.2 BU/mL remained negative (<0.6 BU/mL) with no results reporting >0.4 BU/mL. Inhibitor samples with target values of 1.0 or 4.8 BU/mL, recovered 0.96, 0.99, 0.98 and 0.97 BU/mL and 5.66, 5.69, 5.66 and 5.62 BU/mL, as Mim8 concentrations increased, compared to the un-spiked samples at 1.01 and 5.74 BU/mL, respectively. The spiked samples at 1.0 and 4.8 BU/mL recovered within -2.0 to -5.0%, and -2.1 to -0.9%, of the un-spiked sample, whilst the un-spiked 1.0 and 4.8 BU/mL samples recovered within 1.0% and 19.6% of target, respectively. The Chromogenix CSA recovered 0.90, 0.95, 0.94 and 0.84, as Mim8 concentrations increased, compared to the un-spiked sample at 0.88 at 1.0 BU/mL. At 4.8 BU/mL the results were 5.22, 5.50, 5.57 and 5.72 BU/mL compared to the un-spiked sample of 5.07 BU/mL. The spiked sample at 1.0 BU/mL and 4.8 BU/mL recovered within -4.5 to 8.0%, and 3.0 to 12.8%, of the un-spiked sample. The un-spiked 1.0 and 4.8 BU/mL samples recovered within -12.0% and 5.6% of target, respectively. Conclusion: This study showed that by using FVIII chromogenic assays containing bovine reagents, FVIII inhibitors levels up to at least ⁓5.0 BU/mL can be accurately determined in severe hemophilia A plasma in the presence of up to 40 µg/mL Mim8.

Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Results from an Association-Scan of the Extended MHC-Class-II Region Using Novel Association-Based Statistical Methods Establish That DQ Allotypes and Race Independently Influence the Risk of Factor VIII Inhibitor Development in Hemophilia-A Patients

Hemophilia-A (HA) is caused by heterogeneous factor (F) VIII gene ( F8) mutations, variably deficient plasma FVIII activity and reduced to absent intrinsic-pathway amplification of coagulation. Infused therapeutic-FVIII-proteins (tFVIIIs) prevent bleeding in all HA patients but ~30% with severe HA and ~7.5% with non-severe HA become refractory with the development of neutralizing anti-tFVIII-antibodies called “FVIII inhibitors (FEIs)”. HLA-class-II (HLAII) molecules are critical for the development of FEIs and several association studies on different HA patient populations clearly implicate an important role for the highly polymorphic encoding HLA-D loci in the variable frequency of immunogenicity observed for tFVIIIs infused in different subjects with HA. However, the results from these studies were often conflicting, and none have undergone true independent confirmation. We use the ImmunoChip to genotype each subject in the PATH study simultaneously for the >900 single-nucleotide-variations (SNVs) distributed across the extended MHC-class-II (MHCII) region-which includes the classical- and non-classical-HLAII-genes and -pseudogenes-and then evaluated each independently for associations with FEI risk while accounting for the non-independence of data due to genetic relatedness and F8 mutational heterogeneity using novel statistical methods. The “lifetime-FEI-status” of the 438 North American HA patients in PATH-whose racial-identity/ethnic-ancestry was self-reported as either black-African (n=200) or white-European (n=238)-was the dependent variable of interest. The F8-mutation data and a genetic-relatedness matrix were incorporated into a binary linear mixed model of genetic association with lifetime-FEI-status (Yes vs. No), with ‘Yes’ designating those patients having FEIs of either any titer (AT), i.e., >0.4 Bethesda Units (BUs) mL -1, or only high-titer (HT), i.e., ≥5.0 BUs mL -1. Following the analytical procedure used in prior studies designed to identify determinants of FEI risk, we conducted the extended-MHCII-region-wide association screen-against the 926 distinct SNVs with high-quality genotypes that passed QC-of lifetime-FEI-status on two groups of FEI-positive patients, i.e., those with AT-FEIs, or those with only HT-FEIs. HA patients in the HT-FEI group are suspected to be more homogeneous with respect to the underlying immunobiology as they appear clinically to have induced full adaptive immunity. In contrast, HA patients in the AT-FEI group are heterogenous as some will have transient FEIs, which spontaneously disappear, and others will have FEIs that may remain low-titer (LT) despite continued infusions of tFVIIIs. We found several SNPs that were not only significantly associated in both FEI groups-or significant and suggestive respectively in the HT- and AT-groups-but also increased in significance (i.e., their p-values decreased) under the HT analysis ( Figure 1). The latter observation indicates that these results correspond to true associations that became more apparent as sources of “noise” were removed upon going from the AT to HT analysis. These included a SNP in the 3'-UTR of DQB1 (rs1049225, p-value=5.7E-7) and two intergenic HLAII region SNPs (rs2647012, p-value=1.1E-5; and rs2858324, p-value=1.1E-5). The DQB1 SNP reached an ImmunoChip-wide significance threshold in the HT analysis (i.e., a p-value <5.9E-7) and extended-MHCII-region-wide significance in the AT analysis (i.e., p-value <5.4E-5). Although the two HLAII-intergenic SNPs were not ImmunoChip-wide significant, they were significant across the MHCII region (i.e., p-value <5.4E-5). We found two SNPs that were significant and suggestive, respectively, in the HT- and AT-FEI analyses (rs9276189, p-value=1.3E-5; and rs2856717, p-value=3.3E-5), as well as one SNP that was significant in the HT-FEI analyses but not significant or suggestive in the AT analysis (rs9271366, p-value=1.9E-6). Our results establish that a novel DQB1 genetic variant is associated with FEI risk and confirm that HLA-DQ-allotypes and race independently influence the rate of FEI development in HA ( Figure 2).

A quantitative assay system for protein C activity, the regulator of blood coagulation, based on a chromogenic method mimicking the blood coagulation cascade

Background and aimsProtein C is a plasma protein, and its active form regulates blood coagulation. The recommended unit of protein C activity is IU/mL; however, some laboratories use percentage. Some deficiencies cannot be detected owing to measurement principles. This study sought to quantify protein C activity levels and overcome the limitations of the current measurements. Materials and methodsOur protein C activity measurement method mimicked the blood coagulation cascade and used a thrombin-specific chromogenic reagent. The control was prepared by adding protein C to the protein C deficient plasma. The calibration curve was plotted as the increase in the absorbance per minute and the concentration of protein C in the control. Statistical tests were performed to compare our method with the current chromogenic method. ResultsA calibration curve was constructed (y = −0.0132x + 0.14, R2 = 0.9987, n = 10). The statistical results of our method suggested non-inferiority when compared to the current chromogenic method (α = 0.05). ConclusionThe quantitative measurement was performed using plasma samples. Our method provides the possibility of expressing protein C activity quantitatively and detecting deficiencies that cannot be detected using the current chromogenic method.

PB1317 Assessment of the Combined Effects of Emicizumab and Fitusiran with In Vitro Spiking of Emicizumab and Simulated Fitusiran into Factor VIII Deficient Plasma Using Thrombin Generation Assay

PB1317 Assessment of the Combined Effects of Emicizumab and Fitusiran with In Vitro Spiking of Emicizumab and Simulated Fitusiran into Factor VIII Deficient Plasma Using Thrombin Generation Assay

PB1323 Impact on Thrombin Generation of Direct Oral Anticoagulants in a FVIII Deficient Plasma Spiked with Emicizumab and Human FVIII

PB1323 Impact on Thrombin Generation of Direct Oral Anticoagulants in a FVIII Deficient Plasma Spiked with Emicizumab and Human FVIII

Toward non-factor therapy in hemophilia: an antithrombin insensitive Gla-domainless factor Xa as tissue factor pathway inhibitor bait

BackgroundGla-domainless factor (F) Xa (GD-FXa) was proposed as a trap to endogenous anticoagulant tissue factor pathway inhibitor (TFPI) to restore thrombin generation in hemophilia. Using computational chemistry and experimental approaches, we previously showed that S195A GD-FXa also binds TFPI and restores ex vivo coagulation in plasma obtained from person(s) with hemophilia. MethodsTo design a GD-FXa variant with improved anti-TFPI affinity, we performed molecular dynamics simulations and identified suitable sites for mutagenesis. The calculations identified residues R150FXa and K96Fxa as cold-spots of interaction between GD-FXa and the K2 domain of TFPI. In the three-dimensional model, both residues face toward TFPI hydrophobic residues and are thus potential candidates for mutagenesis into hydrophobic residues to favor an improved protein–protein interaction. ResultsCatalytically inactive GD-FXa variants containing the S195A mutation and the additional mutations K96Y, R150I, R150G, R150F, and K96YR150F, were produced to experimentally confirm these computational hypotheses. Among these mutants, the R150FFXa and the K96YR150FFXa were slightly more effective than S195A GD-FXa in restoring coagulation in FVIII deficient plasmas. However, in surface plasmon resonance experiments, they showed TFPI binding affinities in the same range and acted similarly as S195A GD-FXa in FXa/TFPI competition assays. In contrast, the R150 mutants completely lost their interactions with antithrombin as observed in the surface plasmon resonance experiments. ConclusionsWe therefore conclude that their antithrombin resistance is responsible for their improved thrombin generation, through an extension of their half-lives.

Vitamin D status modulates innate immune responses and metabolomic profiles following acute prolonged cycling

PurposeThe influence of vitamin D status on exercise-induced immune dysfunction remains unclear. The aim of this study was to investigate the effects of vitamin D status (circulating 25(OH)D) on innate immune responses and metabolomic profiles to prolonged exercise.MethodsTwenty three healthy, recreationally active males (age 25 ± 7 years; maximal oxygen uptake [{dot{text{V}}text{O}}_{{2}}max] 56 ± 9 mL·kg−1·min−1), classified as being deficient (n = 7) or non-deficient n = 16) according to plasma concentrations of 25(OH)D, completed 2.5 h of cycling at 15% Δ (~ 55–60% {dot{text{V}}text{O}}_{{2}}max). Venous blood and unstimulated saliva samples were obtained before and after exercise.ResultsParticipants with deficient plasma 25(OH)D on average had lower total lymphocyte count (mean difference [95% confidence interval], 0.5 cells × 109 L [0.1, 0.9]), p = 0.013) and greater neutrophil:lymphocyte ratio (1.3 cells × 109 L, [0.1, 2.5], p = 0.033). The deficient group experienced reductions from pre-exercise to 1 h post-exercise (− 43% [− 70, − 15], p = 0.003) in bacterial stimulated elastase in blood neutrophils compared to non-deficient participants (1% [− 20, 21], p = 1.000) Multivariate analyses of plasma metabolomic profiles showed a clear separation of participants according to vitamin D status. Prominent sources of variation between groups were purine/pyrimidine catabolites, inflammatory markers (linoleic acid pathway), lactate and tyrosine/adrenaline.ConclusionThese findings provide evidence of the influence of vitamin D status on exercise-induced changes in parameters of innate immune defence and metabolomic signatures such as markers of inflammation and metabolic stress.

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POSTER ABSTRACTS