Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

The effect of factor XIa on thrombin and plasmin generation, clot formation, lysis and density in coagulation factors deficiencies.

INTRODUCTION: Growing evidence supports the importance of factor (F) XI activation for thrombosis and hemostasis as well as inflammation and complement systems. In this study, we evaluated the effect of activated FXI (FXIa) on the detection of factor deficiencies by global hemostasis assays of thrombin generation (TG), plasmin generation (PG), and clot formation and lysis (CFL). MATERIALS AND METHODS: An absorbance and fluorescence microplate assay was used to simultaneously observe TG, PG, and CFL in FV-, FVII-, FVIII-, and FIX-deficient plasmas supplemented with purified factors. Coagulation was initiated with tissue factor with or without FXIa in the presence of tissue plasminogen activator. Thrombin and plasmin peak heights (TPH and PPH), maximal clot density (MCD), times to clotting (CT), thrombin and plasmin peaks (TPT and PPT) and clot lysis (LyT) and a new parameter, clot lifetime (LiT), were evaluated. RESULTS: TG/CFL were elevated by the FXIa at low FV (below 0.1 IU/mL), and at FVIII and FIX above 0.01 IU/mL. FXIa affected PG only at low FV and FVII. At high factor concentrations, FXIa reduced MCD. Thrombin and plasmin substrates had effect on CT, LyT, LiT and MCD parameters. CONCLUSIONS: FXIa reveals new relationships between TG, PG and CFL parameters in factor deficiencies suggesting potential benefits for discrimination of bleeding phenotypes.

Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm.

BACKGROUND: Fluorogenic thrombin generation (TG) is a global hemostasis assay that provides an overall representation of hemostasis potential. However, the accurate detection of thrombin activity in plasma may be affected by artifacts inherent to the assay-associated fluorogenic substrate. The significance of the fluorogenic artifacts or their corrections has not been studied in hemophilia treatment applications. METHODS: We sought to investigate TG in hemophilia plasma samples under typical and worst-case fluorogenic artifact conditions and assess the performance of artifact correction algorithms. Severe hemophilic plasma with or without added Factor VIII (FVIII) was evaluated using commercially available and in-house TG reagents, instruments, and software packages. The inner filter effect (IFE) was induced by spiking elevated amounts of fluorophore 7-amino-4-methylcoumarin (AMC) into plasma prior to the TG experiment. Substrate consumption was modeled by adding decreasing amounts of Z-Gly-Gly-Arg-AMC (ZGGR-AMC) to plasma or performing TG in antithrombin deficient plasma. RESULTS: All algorithms corrected the AMC-induced IFE and antithrombin-deficiency induced substrate consumption up to a certain level of either artifact (edge of failure) upon which TG results were not returned or overestimated. TG values in FVIII deficient (FVIII-DP) or supplemented plasma were affected similarly. Normalization of FVIII-DP resulted in a more accurate correction of substrate artifacts than algorithmic methods. CONCLUSIONS: Correction algorithms may be effective in situations of moderate fluorogenic substrate artifacts inherent to highly procoagulant samples, but correction may not be required under typical conditions for hemophilia treatment studies if TG parameters can be normalized to a reference plasma sample.

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor.

Preclinical evaluation of drugs in animals helps researchers to select potentially informative clinical laboratory markers for human trials. To assess the utility of animal thrombin generation (TG) assay, we studied the sensitivity of animal plasmas to triggers of TG, human Tissue Factor (TF), and Activated Factor XI (FXIa). Pooled human, mouse, rat, guinea pig, rabbit, bovine, sheep, and goat plasmas were used in this study. TF- or FXIa-triggered TG and clotting were measured via fluorescence and optical density, respectively. Thrombin peak height (TPH) and time (TPT), clot time (CT), and fibrin clot density (FCD) were all analyzed. The trigger low and high sensitivity borders (LSB and HSB) for each assay parameter were defined as TF and FXIa concentrations, providing 20 and 80% of the maximal parameter value, unless the baseline (no trigger) value exceeded 20% of the maximal, in which case, LSB was derived from 120% of baseline value. Normal human samples demonstrated lower TPH HSB than most of the animal samples for both TF and FXIa. Animal samples, except mice, demonstrated lower TPT LSB for FXIa versus humans. Most rodent and rabbit samples produced baseline TG in the absence of TG triggers that were consistent with the pre-activation of blood coagulation. FCD was not sensitive to both TF and FXIa in either of the plasmas. Animal plasmas have widely variable sensitivities to human TF and FXIa, which suggests that optimization of trigger concentration is required prior to test use, and this complicates the extrapolation of animal model results to humans.

Thrombogenic potential of picomolar coagulation factor XIa is mediated by thrombin wave propagation.

Inhibitors of coagulation factor XIa (FXIa) are currently being investigated as potential anticoagulant therapies. We hypothesize that circulating FXIa could be a potential target for these therapies. Using previous analyses of FXIa impurities in immune globulin products involved in thrombotic adverse events, we estimated that picomolar levels of FXIa can be thrombogenic. In an in vitro clot-growth assay, 0.1-3 pM of FXIa did not, by itself, activate clotting but increased the size of growing clots. Spatio-temporal reconstruction of thrombin activity inside the clot revealed that FXIa's effect was limited to the clot-plasma interface, in which FXIa produced a taller than standard wave of thrombin. Factor-depleted plasma and a panel of selective anti-FXIa antibodies showed that exogenous FXIa effects are (1) blocked by anti-FXIa antibodies, (2) independent of FXI activation inside the clot, and (3) larger than the contribution of in situ FXIa. In a thrombin generation (TG) assay, picomolar FXIa did not initiate TG but rather promoted TG triggered by tissue factor or thrombin, suggesting that the effect of FXIa on the thrombin wave is mediated by the elevation of thrombin-triggered TG. In circulating bovine blood, low doses of human FXIa did not initiate clotting but increased the size of stenosis-triggered thrombi. FXIa injection in mice enhanced TG in plasma for at least 6 hours ex vivo, confirming the persistence of circulating FXIa. Our findings suggest that picomolar levels of circulating FXIa may not be able to initiate thrombosis but can facilitate thrombus growth through the facilitation of TG inside the clot.

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex.

Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into VL and VH as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals.

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.

Considerations on activity assay discrepancies in factor VIII and factor IX products.

New modified coagulation factor VIII (FVIII) and factor IX (FIX) products have been designed to improve the treatment of individuals with hemophilia A and B by increasing the interval between dosing. Although these FVIII and FIX molecules have been structurally modified to improve the circulation time, the changes have also influenced their behavior in functional assays in comparison with traditional plasma-derived or recombinant coagulation factors. The assignment of potencies for these products can be problematic because discordance in factor activity values between the commonly used one-stage clotting and chromogenic substrate assays is often observed. Discrepancies in potency assay values also exist when different assay kits and reagents are used in the same assay type. Ideally, all FVIII and FIX products should be calibrated against the World Health Organization (WHO) International Standards (IS) because the assignment of potencies in international units (IU) helps maintain treatment tradition and meaningful references for manufacturers, patients, and clinicians. The discrepant measurements, attributed to the modified structural and functional properties of these products, are manifested in their lack of commutability with the WHO IS for FVIII or FIX. Herein, we discuss the considerations upon which an assay is chosen for potency assignment and postadministration monitoring of a new factor product, which include the validity of the assay calibrated with the IS, the meaning of the potency values in IU, standards of care for patients, clinical relevance between the assigned potency value and recovery value from clinical laboratories, and patient safety.

Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples.

INTRODUCTION: The thrombin generation (TG) test is a global hemostasis assay sensitive to procoagulant conditions. However, some TG assays may underestimate elevated TG when the thrombin fluorogenic substrate is depleted or fluorescence is attenuated by the inner filter effect (IFE). OBJECTIVES: We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE. METHODS: We analyzed corrections for substrate depletion and IFE and their effect on TG parameters in plasma samples with elevated blood coagulation factors in the presence or absence of thrombomodulin via commercial calibrated automated thrombogram (CAT) platform and in-house software capable of internal thrombin calibration with or without CAT-like artifact correction. RESULTS: Elevated thrombin peak height (TPH) and endogenous thrombin potential (ETP) were detected with 2× and 4× increases in blood coagulation factors I, V, VIII, IX, X, and XI, or prothrombin in the presence or absence of artifact correction. The effect of the CAT algorithm was evident in TG curves from both low procoagulant (thrombomodulin-supplemented) and procoagulant (factor-supplemented) plasma samples. However, in all samples, with the exception of elevated prothrombin, CAT's correction was small (<10%) and did not affect detection of procoagulant samples versus normal plasma. For elevated prothrombin samples, uncorrected TPH or ETP values were underestimated, and CAT correction produced drastically elevated TG curves. CONCLUSIONS: Our data suggest that correction for substrate consumption and IFE, as offered by the CAT algorithm, is critical for detecting a subset of extremely procoagulant samples, such as elevated prothrombin, but is not necessary for all other conditions, including elevated factors XI and VIII.

Detecting factor XIa in immune globulin products: Commutability of international reference materials for traditional and global hemostasis assays.

BACKGROUND: Activated coagulation factor XIa (FXIa) is an impurity and primary source of procoagulant activity in thrombosis-implicated immune globulin (IG) products. Several assays, of varying quality and precision are used to assess FXIa-like procoagulant activity in units relevant to their respective principles. OBJECTIVES: To advance unified reporting, we sought to employ the World Health Organization reference reagents (RRs) to present the results of differing methodologies in units of FXIa activity and rank the sensitivity and robustness of these methodologies. METHODS: RR 11/236 served as a calibrator in several FXIa-sensitive blood coagulation tests: two commercial chromogenic FXIa assays (CAs); a nonactivated partial thromboplastin time (NaPTT); an in-house fibrin generation (FG) assay; an in-house thrombin generation (TG) assay; and an assay for FXIa- and kallikrein-like proteolytic activities based on cleavage of substrate SN13a. Some assays were tested in either normal or FXI-deficient plasma. RESULTS: Each method demonstrated a sigmoidal dose-response to RRs. NaPTT was the least sensitive to FXIa and the least precise; our in-house TG was the most sensitive; and the two CAs were the most precise. All methods, except for SN13a, which is less specific for thrombotic impurities, gave comparable (within 20% difference) FXIa activity assignments for IG lots. CONCLUSIONS: Purified FXIa reference standards support quantitation of FXIa levels in IG products in all tested assay methodologies. This should help to standardize the measurement of thrombotic potentials in IG products and prevent products exhibiting high procoagulant activity from distribution for patient use. Further research is needed to address the effect of IG product-specific matrixes on assay performance.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products.

BACKGROUND: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. OBJECTIVE: To isolate and characterize the FVIIIFT fraction in rFVIII products. METHODS: Protein fractions unable (FVIIIFT ) and able (FVIIIEL ) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. RESULTS AND CONCLUSIONS: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL , FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Comparative Analysis of Thrombin Calibration Algorithms and Correction for Thrombin-α2macroglobulin Activity.

BACKGROUND: The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-α2macroglobulin (T-α2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility. METHODS: Effects of internal vs. external normalization, IFE and T-α2MG on TG curves in normal plasma supplemented with coagulation factors, thrombomodulin, and tissue factor were studied using the Calibrated Automated Thrombinography (CAT; Diagnostica Stago, Parsippany, NJ, USA) and in-house software. RESULTS: The various calibration methods demonstrated no significant difference in producing TG curves, nor increased the robustness of the TG assay. Several TG parameters, including thrombin peak height (TPH), produced from internal linear calibration did not differ significantly from uncalibrated TG parameters. Further, TPH values from internal linear and nonlinear calibration with or without T-α2MG correction correlated well with TPH from external calibration. Higher coefficients of variation (CVs) for TPH values were observed in both platelet-free and platelet-rich plasma with added thrombomodulin. CONCLUSIONS: Our work suggests minimal differences between distinct computational approaches toward calibrating and correcting fluorescence signals into TG levels, with most samples returning similar or equivalent TPH results.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.

BACKGROUND: Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs. METHODS: CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test. RESULTS: SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing-thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a(+) PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. CONCLUSIONS: Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing-thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.