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Inhibitors of coagulation factor (F) XIa are currently investigated as potential anticoagulant therapies. We hypothesized that circulating FXIa may be a potential target for these therapies. Using previous analyses of FXIa impurity 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, where FXIa produced a taller than normal wave of thrombin. Factor-depleted plasmas and a panel of selective anti-FXIa antibodies showed that exogenous FXIa effects are (1) blocked by the anti-FXIa antibodies, (2) independent from 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 with tissue factor or thrombin, suggesting that the effect of FXIa on the thrombin wave is mediated by elevation of thrombin-triggered TG. In flowing 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 persistence of circulating FXIa. Our findings suggest that picomolar levels of circulating FXIa may not be able to start thrombosis but can facilitate thrombus growth through facilitation of TG inside the clot.

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.

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.

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.

Background Pregnant women are at increased risk of thrombotic adverse events. Plasma derived immune globulin (IG) products, which are used in pregnancy for various indications, may contain procoagulant impurity activated coagulation factor XI (FXIa). Procoagulant IG products have been associated with increased thrombogenicity but their effect in pregnancy is unknown. Methods Late pregnant (gestation days 17–20) or early lactation (days 1–3) and control female mice were treated with IGs supplemented with human FXIa then subjected to ferric chloride (FeCl3) vessel injury. Occlusion of blood vessel was assessed by recording blood velocity in the femoral vein for 20 min using doppler ultrasound laser imaging. FXIa dose was selected by the ability to increase thrombin generation in mouse plasma in vitro. Results FXIa produced robust thrombin generation in mouse plasma ex vivo. Following FeCl3 injury, pregnant and non-pregnant mice receiving IG + FXIa exhibited faster reduction of blood velocity in femoral vein compared to IG alone or untreated controls. In vitro, thrombin generation in plasma samples collected after thrombosis in FXIa-treated animals was elevated and could be reduced by anti-FXI antibody. Conclusions Our results suggest that intravenously-administered FXIa may contribute to thrombosis at the site of vascular injury in both pregnant and non-pregnant animals.

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.

This review is focused on the epidemiology of venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), associated with pregnancy. Superficial vein thrombosis, a less hazardous and less studied type of thrombosis in pregnant women, is beyond the scope of this review. This study discusses the VTE incidence rate in women from developed countries for both antepartum and postpartum periods and for subpopulations of women affected by additional risk factors, such as thrombophilias, circulatory diseases, preeclampsia of varying degrees of severity, and Caesarean section. To minimize bias due to historical changes in medical and obstetric practices, lifestyle, diet, etc., this review is generally limited to relatively recent studies, i.e., those that cover the last 35 years. The absolute risk or incidence rate was used to ascertain risk of VTE associated with pregnancy. For the studies where the direct incidence rates of VTE were not reported, we calculated an estimate of the observed but not reported absolute incidence rates using the data presented in respective articles.

Background Thrombin generation test (TGT) is a global haemostasis assay with a potential to predict bleeding tendencies and treatment effects in patients with haemophilia. Despite 15 years of clinical research, the diagnostic value of TGT remains controversial, possibly due to suboptimal sensitivity to coagulation deficiencies, robustness and reproducibility. Objective The goal of this study was to explore the effect of calcium chloride (CaCl2 ) concentration on the TGT's response to intrinsic coagulation factors (F) VIII, IX and XIa. Methods Normal and factor-deficient plasmas supplemented with lacking coagulation factor and different CaCl2 levels were tested by calibrated thrombinography assay. Results Thrombin peak height (TPH) was strongly CaCl2 dependent, increasing sharply from no TG at 5 mm to a peak at 13.8 mm of CaCl2 (95% confidence interval [CI]: 13.0, 14.5) in normal and normalized deficient plasmas and at 11.9 mm (CI: 9.7, 14.2) in deficient plasmas, and then decreasing slowly to a complete inhibition at 30-40 mm. In contrast, TG lag time, time to peak and endogenous thrombin potential were nearly insensitive to CaCl2 concentrations between 10 and 20 mm. The maximal difference between the TPH in deficient and supplemented plasmas was observed at 15.5 mm (CI: 12.8, 18.1). Conclusion Variations in CaCl2 concentration in the assay mixture and sodium citrate concentrations in patient plasma samples may affect TGT responses, sensitivity and result in increased inter- and intra-laboratory variance. Implementation of TGT by clinical and quality control laboratories may require optimization of CaCl2 concentration.

Summary. Background: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. Objective: To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). Methods: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. Results: BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m−2 by shortening lag time and increasing clot size by up to ∼2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m−2, both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. Conclusions: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.

Introduction Replacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma. A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products. Study objective We aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF. Experimental design We generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties. Results The engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII. Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation. Conclusions The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage. The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands. Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. Disclosures No relevant conflicts of interest to declare.

This review is focused on the epidemiology of venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), associated with pregnancy. Superficial vein thrombosis, a less hazardous and less studied type of thrombosis in pregnant women, is beyond the scope of this review. This study discusses the VTE incidence rate in women from developed countries for both antepartum and postpartum periods and for subpopulations of women affected by additional risk factors, such as thrombophilias, circulatory diseases, preeclampsia of varying degrees of severity, and Caesarean section. To minimize bias due to historical changes in medical and obstetric practices, lifestyle, diet, etc., this review is generally limited to relatively recent studies, i.e., those that cover the last 35 years. The absolute risk or incidence rate was used to ascertain risk of VTE associated with pregnancy. For the studies where the direct incidence rates of VTE were not reported, we calculated an estimate of the observed but not reported absolute incidence rates using the data presented in respective articles.

BACKGROUND Thrombotic events (TEs) are serious adverse events that can occur following administration of clotting factors (CFs). OBJECTIVES To evaluate occurrence of same-day TEs for different CF products and potential risk factors. METHODS A retrospective cohort study of individuals exposed to CF products during 2008-2013 was conducted using a large commercial insurance database. CF products were identified by procedure codes, and TEs were ascertained via diagnosis codes. Crude same-day TE rates (per 1000 persons exposed) were estimated overall and by congenital factor deficiency (CFD) status, CF products, age and gender. Multivariable logistic regression analyses were used to control for confounding. Laboratory analysis was used to compare the procoagulant activities of FIX products. RESULTS Of 3801 individuals exposed to CFs, 117 (30.8 per 1000) had same-day TEs recorded. The crude same-day TE rate was higher for CF users without CFD, 70.2 (102 of 1452), as compared with those with CFD, 6.4 (15 of 2349) (RR, 11.0; 95% CI, 6.4-18.9). For individuals without CFD, a significantly increased same-day TE risk was identified for factor IX complex (OR, 6.92; 95% CI, 3.11-15.40), factor VIIa (OR, 9.42; 95% CI, 4.99-17.78) and other products when compared with fibrin sealant. An increased risk of a TE was found with older age (≥ 45 years), history of TEs and underlying health conditions. The laboratory identified elevated procoagulant activity in Profilnine(®) and Benefix(®) . CONCLUSIONS The study shows an increased same-day TE risk for CF users without CFD and suggests substantial off-label CF use. The study findings also show elevated same-day TE rates for different CF products and suggest the importance of product properties and patient factors.

Background: Activated coagulation factor XI (FXIa) was found in the immunoglobulin products that were associated with higher than usual incidence of thrombotic events (TEs). However, TEs after thrombogenic immunoglobulin administration are very rare and are often recorded as late as 48 hours after administration. Objective: This study was aimed to elucidate the mechanisms behind FXIa thrombogenicity. We hypothesized that low amounts of FXIa are not able to activate clotting directly, but can promote on-going coagulation events at a site of vascular lesion. Materials and methods: FXIa activity was characterized by kinetic clotting and thrombin generation (TG) assays. Spatial clot growth and thrombin wave propagation in stagnant plasma were studied using thrombodynamics, a time-lapse videomicroscopy-based approach. To model coagulation events at the site of vascular wall lesion, spatial clot growth was initiated by plastic surfaces covered with immobilized tissue factor (TF). Human plasma deficient in various coagulation factors was used to study pathways of FXI activation. Results: Low plasma FXIa levels of less than 0.3 pM did not induce thrombin generation or clotting, but increased TG in plasma mixed with TF. Higher FXIa levels (>0.3 pM) activated coagulation directly. In stagnant plasma activated by surface with immobilized TF, sub-picomolar amounts of FXIa were also not able to initiate clotting, but was able to enhance TG inside clots. FXIa-induced TG was localized to the propagating edge of the clot and associated with increased clot growth rate. Conclusion: Sub-picomolar amounts of FXIa may promote growth of clot in the vicinity of vascular lesion and have no procoagulant effect in the absence of exposed TF. These in vitro findings suggest that the patient’s condition might contribute to the thrombogenicity of FXIa.

Background In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. Objective To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro . Methods Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600 nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. Results Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6 pmole/m 2 by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C 30%). Conclusions The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.