Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

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.

Applying the science of measurement to biology: Why bother?

Both basic and translational research are continuously evolving, but the principles that underpin research integrity remain constant. These include rational, hypothesis-driven, and adequately planned and controlled science, which is carried out openly, honestly, and ethically. An important component of this should be minimising experimental irreproducibility. Biological systems, in particular, are inherently variable due to the nature of cells and tissues, as well as the complex molecules within them. As a result, it is important to understand and identify sources of variability and to strive to minimise their influence. In many instances, the application of metrology (the science of measurement) can play an important role in ensuring good quality research, even within biological systems that aren't always amenable to many of the metrological concepts applied in other fields. Here, we introduce the basic concepts of metrology in relation to biological systems and promote the application of these principles to help avoid potentially costly mistakes in both basic and translational research. We also call on funders to encourage the uptake of metrological principles, as well as provide funding and support for later engagement with regulatory bodies.

Development of an updated assay for prekallikrein activator in albumin and immunoglobulin therapeutics.

BACKGROUND: Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed. MATERIALS AND METHODS: A simplified one-step method has been developed to replace the existing Ph Eur two-step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one-stage method is simplified by the use of a dedicated online app. RESULTS: The one-stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one-stage method. Improved methodology using true initial rate determinations and use of acid-treated prekallikrein substrate (PKS) helped understand and reduce artefactual results. CONCLUSIONS: The one-stage method and associated app streamline real-time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.

Selective Stabilization and Destabilization of Protein Domains in Tissue-Type Plasminogen Activator Using Formulation Excipients.

Multidomain biotherapeutic proteins present additional behavioral and analytical challenges for the optimization of their kinetic stability by formulation. Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding. Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multidomain proteins. We show that changes in the thermal unfolding profile of tPA were parametrized by the overall thermal midpoint transition temperature, Tm, and the Van't Hoff entropy for unfolding, Δ Svh, which is a measure of unfolding cooperativity. The kinetics of degradation at 45 °C, leading to aggregation, were measured as rates of monomer and activity loss. These two rates were found to be coincident at all pH. Aggregation accelerated at pH 4 due to the early unfolding of the serine protease N-terminal domain (SP-N), whereas at pH 5-8, the fraction unfolded at 45 °C ( f45) was <1%, resulting in a baseline rate of aggregation from the native ensemble. We used a Design of Experiments (DoE) approach to evaluate how formulation excipients impact and control the thermal unfolding profile for tPA and found that the relative stability of each of the tPA domains was dependent on the formulation. Therefore, the optimization of formulations for complex multidomain proteins such as tPA may need to be multiobjective, with careful selection of the desired attributes that improve stability. As aggregation rates (ln v) correlated well to Tm ( R2 = 0.77) and Δ Svh ( R2 = 0.71) but not Tagg ( R2 = 0.01), we analyzed how formulation excipients and pH would be able to optimize Tm and Δ Svh. Formulation excipient behaviors were found to group according to their combined impact on Tm and Δ Svh. The effects of each excipient were often selectively stabilizing or destabilizing to specific tPA domains and changed the stability of particular domains relative to the others. The types of mechanism by which this could occur might involve specific interactions with the protein surface, or otherwise effects that are mediated via the solvent as a result of the different surface hydrophobicities and polarities of each domain.

Bleeding related to disturbed fibrinolysis.

The components and reactions of the fibrinolysis system are well understood. The pathway has fewer reactants and interactions than coagulation, but the generation of a complete quantitative model is complicated by the need to work at the solid-liquid interface of fibrin. Diagnostic tools to detect disease states due to malfunctions in the fibrinolysis pathway are also not so well developed as is the case with coagulation. However, there are clearly a number of inherited or acquired pathologies where hyperfibrinolysis is a serious, potentially life-threatening problem and a number of antifibrinolytc drugs are available to treat hyperfibrinolysis. These topics will be covered in the following review.

Fractal kinetic behavior of plasmin on the surface of fibrin meshwork.

Intravascular fibrin clots are resolved by plasmin acting at the interface of gel phasesubstrate and fluid-borne enzyme. The classic Michaelis.Menten kinetic scheme cannot describe satisfactorily this heterogeneous-phase proteolysis because it assumes homogeneous well-mixed conditions. A more suitable model for these spatial constraints,known as fractal kinetics, includes a time-dependence of the Michaelis coefficient Km(F) = Km0F (1+ t)h, where h is a fractal exponent of time, t. The aim of the present study was to build up and experimentally validate a mathematical model for surface-acting plasmin that can contribute to a better understanding of the factors that influence fibrinolytic rates. The kinetic model was fitted to turbidimetric data for fibrinolysis under various conditions. The model predicted Km0(F) = 1.98 µM and h = 0.25 for fibrin composed of thin fibers and Km0(F) = 5.01 µM and h = 0.16 for thick fibers in line with a slower macroscale lytic rate (due to a stronger clustering trend reflected in the h value) despite faster cleavage of individual thin fibers (seen as lower Km0(F) ). ε-Aminocaproic acid at 1 mM or 8 U/mL carboxypeptidase-B eliminated the time-dependence of Km F and increased the lysis rate suggesting a role of C-terminal lysines in the progressive clustering of plasmin. This fractal kinetic concept gained structural support from imaging techniques. Atomic force microscopy revealed significant changes in plasmin distribution on a patterned fibrinogen surface in line with the time-dependent clustering of fluorescent plasminogen in confocal laser microscopy. These data from complementary approaches support a mechanism for loss of plasmin activity resulting from C-terminal lysine-dependent redistribution of enzyme molecules on the fibrin surface.

Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones.

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator.

Synergism of red blood cells and tranexamic acid in the inhibition of fibrinolysis.

BACKGROUND: Postpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. The World Maternal Antifibrinolytic trial showed that antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. Maternal anemia increases the risk of PPH. The World Maternal Antifibrinolytic-2 trial is now assessing whether TXA can prevent PPH in women with anemia. Low red blood cell (RBC) counts promote fibrinolysis by altering fibrin structure and plasminogen activation. OBJECTIVES: We explored interactions between RBCs and TXA in inhibiting fibrinolysis. METHODS: We used global fibrinolytic assays (ball sedimentation and viscoelasticity) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator. We applied a fluorogenic kinetic assay to measure plasmin generation in fibrin clots and scanning electron microscopy to study fibrin structure. RESULTS: According to parallel-line bioassay analysis of the fibrin lysis-time data, the antifibrinolytic potency of 4-128 µM TXA was increased in the presence of 10% to 40% (v/v) RBCs. Global fibrinolysis assays showed that the joint effect of RBCs and TXA was about 15% larger than the sum of their individual effects in the inhibition of fibrinolysis. In plasminogen activation, TXA added the same increment of inhibition to the effect of RBCs at any cell count in the fibrin clot. Regarding fibrin structure, TXA thickened fibrin fibers, which impaired plasminogen activation, whereas RBCs promoted fine fibers that were more resistant to plasmin. CONCLUSIONS: The antifibrinolytic potency of TXA is enhanced in fibrin formed in the presence of RBCs through inhibition of plasminogen activation and fibrin lysis, which correlates with modifications of fibrin structures.

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.

Kinetic regulation of the binding of prothrombin to phospholipid membranes.

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K(D) = 5.1 µM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K(D) values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K(D) of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K(D) in the 10(-8) M range and (ii) a kinetically rapid, low-affinity interaction with K(D) in the 10(-6 )M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

Lytic resistance of fibrin containing red blood cells.

OBJECTIVE: Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. METHODS AND RESULTS: If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. CONCLUSIONS: RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

Is it possible to make a common reference standard for D-dimer measurements? Communication from the ISTH SSC Subcommittee on Fibrinolysis.

BACKGROUND: D-dimer antigen is a heterogeneous mixture of fibrin degradation products that when present at high levels in plasma indicate ongoing coagulation and fibrinolysis. The heterogeneous nature of the target D-dimer antigen and the variety of assay systems means that it is difficult to compare results from different methods. OBJECTIVES: To identify a universally agreed D-dimer standard that could help harmonize results from different methods. METHODS: A pool of patient plasma with high D-dimer levels was freeze-dried and investigated as a long-term World Health Organization international standard for D-dimer. Fibrin degradation products from clot lysis reactions were also freeze-dried in various formulations and investigated in commutability studies with patient plasma. RESULTS: Problems of instability of D-dimer plasma emerged suggesting loss of reactivity after freeze-drying and storage at -20°C of 10%-18% per year. Freeze-dried fibrin degradation products added to plasma were also unstable, but the sugar trehalose was found to improve stability. However, this preparation was not suitable as a standard in widely used assay platforms. Previous studies suggest fibrin degradation products are prone to structural rearrangements and amyloid formation, which may explain the instability of candidate D-dimer standards. CONCLUSIONS: The known difficulties of D-dimer standardization are compounded by instability of D-dimer antigen after freeze-drying, described in this report. Fibrin degradation products added to plasma and stabilized by trehalose are not suitable as a standard for D-dimer measurement harmonization. Trehalose stabilization of pooled patient plasma containing high D-dimer levels may produce a useful standard, but this requires confirmation.

Citrullinated fibrinogen forms densely packed clots with decreased permeability.

BACKGROUND: Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. OBJECTIVE: To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. METHODS: Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra-fiber structure of fibrin was analyzed with small-angle X-ray scattering (SAXS). RESULTS: SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3-fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. CONCLUSION: The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.

Measuring Fibrinolysis.

Physiological fibrinolysis under normal conditions progresses slowly, in contrast to coagulation which is triggered rapidly to stop bleeding and defend against microbial invasion. Methods to detect fibrinolysis abnormalities are less simple and poorly standardized compared with common coagulation tests. Fibrinolysis can be accelerated by preparing euglobulin from plasma to reduce endogenous inhibitors, or by adding plasminogen activators to normal plasma. However, these manipulations complicate interpretation of results and diagnosis of a "fibrinolysis deficit." Many observational studies on antigen levels of fibrinolysis inhibitors, plasminogen activator inhibitor 1 or thrombin-activatable fibrinolysis inhibitor, zymogen or active enzyme have been published. However, conclusions are mixed and there are clear problems with harmonization of results. Viscoelastic methods have the advantage of being rapid and are used as point-of-care tests. They also work with whole blood, allowing the contribution of platelets to be explored. However, there are no agreed protocols for applying viscoelastic methods in acute care for the diagnosis of hyperfibrinolysis or to direct therapy. The emergence of SARS-CoV-2 and the dangers of associated coagulopathy provide new challenges. A common finding in hospitalized patients is high levels of D-dimer fibrin breakdown products, indicative of ongoing fibrinolysis. Well-established problems with D-dimer testing standardization signal that we should be cautious in using results from such tests as prognostic indicators or to target therapies.

Extracellular Histones Inhibit Fibrinolysis through Noncovalent and Covalent Interactions with Fibrin.

Histones released into circulation as neutrophil extracellular traps are causally implicated in the pathogenesis of arterial, venous, and microvascular thrombosis by promoting coagulation and enhancing clot stability. Histones induce structural changes in fibrin rendering it stronger and resistant to fibrinolysis. The current study extends these observations by defining the antifibrinolytic mechanisms of histones in purified, plasma, and whole blood systems. Although histones stimulated plasminogen activation in solution, they inhibited plasmin as competitive substrates. Protection of fibrin from plasmin digestion is enhanced by covalent incorporation of histones into fibrin, catalyzed by activated transglutaminase, coagulation factor FXIII (FXIIIa). All histone subtypes (H1, H2A, H2B, H3, and H4) were crosslinked to fibrin. A distinct, noncovalent mechanism explains histone-accelerated lateral aggregation of fibrin protofibrils, resulting in thicker fibers with higher mass-to-length ratios and in turn hampered fibrinolysis. However, histones were less effective at delaying fibrinolysis in the absence of FXIIIa activity. Therapeutic doses of low-molecular-weight heparin (LMWH) prevented covalent but not noncovalent histone-fibrin interactions and neutralized the effects of histones on fibrinolysis. This suggests an additional antithrombotic mechanism for LMWH beyond anticoagulation. In conclusion, for the first time we report that histones are crosslinked to fibrin by FXIIIa and promote fibrinolytic resistance which can be overcome by FXIIIa inhibitors and histone-binding heparinoids. These findings provide a rationale for targeting the FXIII-histone-fibrin axis to destabilize fibrin and prevent potentially thrombotic fibrin networks.

An international collaborative study to establish the WHO 3rd International Standard for Thrombin: Communication from the ISTH SSC subcommittee on factor XIII and fibrinogen.

The calibration of thrombin products relies on the World Health Organization (WHO) 2nd International Standard (IS) for Thrombin (01/580) which defines the international unit (IU) for thrombin potency. With stocks of the 2nd IS (01/580) running low, an international collaborative study was organized to calibrate a replacement. Twenty laboratories from 13 countries took part in the study and measured the potency of two candidate replacement standards (coded 01/578 and 19/188) relative to the 2nd IS. In total, 111 valid assays were returned, which were a combination of plasma/fibrinogen clotting assays and chromogenic assays. Variation between and within laboratories was low, with inter- and intra-laboratory geometric coefficient of variation (GCV) generally <5% for all assay methods and substrates. For 01/578, potency estimates by clotting assays (101.1 IU/ampoule) were significantly lower than estimates by chromogenic assays (111.5 IU/ampoule). Mean potency estimates for 19/188 were 90.4 IU/ampoule by clotting assay and 88.1 IU/ampoule by chromogenic assay, which was not a statistically significant difference. The close ratio between clotting and chromogenic assay potency estimates for 19/188 suggests it has a higher α-thrombin content than 01/578 and is equivalent to the current IS (01/580). Accelerated degradation studies predicted excellent long-term stability profiles for preparations 01/580, 01/578, and 19/188. Based on the results of this study, the WHO Expert Committee on Biological Standardization established 19/188 as the 3rd IS for Thrombin with a potency of 90 IU/ampoule in August 2020.

Size- and charge-dependent modulation of the lytic susceptibility and mechanical stability of fibrin-histone clots by heparin and polyphosphate variants.

BACKGROUND: Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms. OBJECTIVES: To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility. METHODS: Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes. RESULTS: Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules. Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. CONCLUSIONS: Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Anti-fibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.