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.

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.

Combined thrombogenic effects of vessel injury, pregnancy and procoagulant immune globulin administration in mice.

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.

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.

International consensus on a complications list after gastrectomy for cancer.

BACKGROUND: Perioperative complications can affect outcomes after gastrectomy for cancer, with high mortality and morbidity rates ranging between 10 and 40%. The absence of a standardized system for recording complications generates wide variation in evaluating their impacts on outcomes and hinders proposals of quality-improvement projects. The aim of this study was to provide a list of defined gastrectomy complications approved through international consensus. METHODS: The Gastrectomy Complications Consensus Group consists of 34 European gastric cancer experts who are members of the International Gastric Cancer Association. A group meeting established the work plan for study implementation through Delphi surveys. A consensus was reached regarding a set of standardized methods to define gastrectomy complications. RESULTS: A standardized list of 27 defined complications (grouped into 3 intraoperative, 14 postoperative general, and 10 postoperative surgical complications) was created to provide a simple but accurate template for recording individual gastrectomy complications. A consensus was reached for both the list of complications that should be considered major adverse events after gastrectomy for cancer and their specific definitions. The study group also agreed that an assessment of each surgical case should be completed at patient discharge and 90 days postoperatively using a Complication Recording Sheet. CONCLUSION: The list of defined complications (soon to be validated in an international multicenter study) and the ongoing development of an electronic datasheet app to record them provide the basic infrastructure to reach the ultimate goals of standardized international data collection, establishment of benchmark results, and fostering of quality-improvement projects.

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.

Synergy Between Tissue Factor and Exogenous Factor XIa in Initiating Coagulation.

OBJECTIVE: Recent evidence suggests involvement of coagulation factor XIa (FXIa) in thrombotic event development. This study was conducted to explore possible synergies between tissue factor (TF) and exogenous FXIa (E-FXIa) in thrombin generation. APPROACH AND RESULTS: In thrombin generation assays, for increasing concentrations of E-FXIa with low, but not with high TF concentrations, peak thrombin significantly increased whereas lag time and time to peak significantly decreased. Similar dependencies of lag times and rates of thrombin generation were found in mathematical model simulations. In both in vitro and in silico experiments that included E-FXIa, thrombin bursts were seen for TF levels much lower than those required without E-FXIa. For in silico thrombin bursts initiated by the synergistic action of TF and E-FXIa, the mechanisms leading to the burst differed substantially from those for bursts initiated by high TF alone. For the synergistic case, sustained activation of platelet-bound FIX by E-FXIa, along with the feedback-enhanced activation of platelet-bound FVIIIa and FXa, was needed to elicit a thrombin burst. Furthermore, the initiation of thrombin bursts by high TF levels relied on different platelet FIX/FIXa binding sites than those involved in bursts initiated by low TF levels with E-FXIa. CONCLUSIONS: Low concentrations of TF and exogenous FXIa, each too low to elicit a burst in thrombin production alone, act synergistically when in combination to cause substantial thrombin production. The observation about FIX/FIXa binding sites may have therapeutic implications.

Epidemiology of venous thromboembolism (VTE) associated with pregnancy.

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.

Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.

Low-density lipoprotein receptor-related protein 1 (LRP) mediates clearance of blood coagulation factor VIII (FVIII). In LRP, FVIII binds the complement-type repeats (CRs) of clusters II and IV, which also bind a majority of other LRP ligands. No ligand is known for LRP cluster I, and only three ligands, including the LRP chaperone alpha-2 macroglobulin receptor-associated protein (RAP), bind cluster III. Using surface plasmon resonance, we found that in addition to clusters II and IV, activated FVIII (FVIIIa) binds cluster III. The specificity of this interaction was confirmed using an anti-FVIII antibody fragment, which inhibited the binding. Recombinant fragments of cluster III and its site-directed mutagenesis were used to localize the cluster's site for binding FVIIIa to CR.14-19. The interactive site of FVIIIa was localized within its A1/A3'-C1-C2 heterodimer (HDa), which is a major physiological remnant of FVIIIa. In mice, the clearance of HDa was faster than that of FVIII and prolonged in the presence of RAP, which is known to inhibit interactions of LRP with its ligands. In accordance with this, the cluster III site for RAP (CR.15-19) was found to overlap that for FVIIIa. Altogether, our findings support the involvement of LRP in FVIIIa catabolism and suggest a greater significance of the biological role of cluster III compared to that previously known.

Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids.

Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a high-dose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVII-deficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombin-generation experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development.

Immune globulins and same-day thrombotic events as recorded in a large health care database during 2008 to 2012.

BACKGROUND: Thrombotic events (TEs) are rare and serious adverse events after administration of immune globulin (IG) products. Our study evaluated the occurrence of same-day TEs for different IG products and ascertained potential risk factors. STUDY DESIGN AND METHODS: This retrospective cohort study utilized HealthCore's Integrated Research Database (HIRD) to assess individuals exposed to IGs during 2008 to 2012. IG products were identified using recorded procedure codes and TEs were ascertained using ICD-9-CM diagnosis codes. The unadjusted same-day TE rates (per 1000 persons exposed) were estimated overall and by IG products, age, and sex. Multivariable logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for same-day TEs by IG products. RESULTS: Of 14,944 individuals exposed to IG products, 233 (15.6 per 1000 persons) had TE diagnosis code(s) recorded on the same-day as the IG exposure. Compared to Gammagard Liquid, Gammaplex (OR, 20.96; 95% CI, 2.45-179.33) and Vivaglobin (OR, 2.74; 95% CI, 1.19-6.32) users had a significantly increased same-day TE risk. Elevated, but nonsignificant TE risks were identified for Octagam, Gamunex, Privigen, and Lyophilized IG(s). An increased TE risk was also found with older age (≥45 years), prior TEs, and other health conditions. CONCLUSION: Our claims-based cohort study suggests a potentially elevated TE risk with different IG products and shows importance of recipient factors such as older age, previous TE, hypercoagulable state(s), and other health conditions. The results of this study suggest the need for continuous evaluation of procoagulant activity and manufacturing processes for IG products to further assure their safety.

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.

Engineering and evaluation of FXa bypassing agents that restore hemostasis following Apixaban associated bleeding.

Direct oral anticoagulants (DOACs) targeting activated factor Xa (FXa) are used to prevent or treat thromboembolic disorders. DOACs reversibly bind to FXa and inhibit its enzymatic activity. However, DOAC treatment carries the risk of anticoagulant-associated bleeding. Currently, only one specific agent, andexanet alfa, is approved to reverse the anticoagulant effects of FXa-targeting DOACs (FXaDOACs) and control life-threatening bleeding. However, because of its mechanism of action, andexanet alfa requires a cumbersome dosing schedule, and its use is associated with the risk of thrombosis. Here, we present the computational design, engineering, and evaluation of FXa-variants that exhibit anticoagulation reversal activity in the presence of FXaDOACs. Our designs demonstrate low DOAC binding affinity, retain FXa-enzymatic activity and reduce the DOAC-associated bleeding by restoring hemostasis in mice treated with apixaban. Importantly, the FXaDOACs reversal agents we designed, unlike andexanet alfa, do not inhibit TFPI, and consequently, may have a safer thrombogenic profile.

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.

Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII.

A recombinant single-chain variable antibody fragment (scFv) KM33 was previously described as a ligand that can inhibit the function of blood coagulation factor VIII (FVIII). This scFv was previously derived from an individual with anti-FVIII antibodies manifested in FVIII functional deficiency (Hemophilia A) and expressed in bacteria. In the present work, we describe an alternative approach for fast and easy production of KM33 in insect cells (Spodoptera frugiperda). The KM33 gene was codon-optimized and expressed in secreted form using a baculovirus system. The protein was isolated using metal-affinity and size-exclusion chromatography to purity of about 96% and yield of 0.4-1.2 mg per 120 mL of culture, based on several independent expression experiments. In a binding assay using surface plasmon resonance, the insect cell-derived KM33 (iKM33) was qualified as a high-affinity ligand for FVIII. Epitope specificity of iKM33 on FVIII (C1 domain) was confirmed by testing the binding with a relevant mutant of FVIII. In several FVIII functional tests (factor Xa generation, APTT clotting, thrombin generation and video microscopy clot growth assays), iKM33 strongly inhibited FVIII activity in accordance with the clinical effect of the parental antibody. Therefore, the expressed protein was concluded to be fully functional and applicable in various assays with FVIII.

Hyperimmune globulins and same-day thrombotic adverse events as recorded in a large healthcare database during 2008-2011.

Thrombotic events (TEs) are rare serious complications following administration of hyperimmune globulin (HIG) products. Our retrospective claims-based study assessed occurrence of same-day TEs following administration of HIGs during 2008-2011 and examined potential risk factors using HealthCore's Integrated Research Database (HIRD(SM) ) and laboratory testing of products' procoagulant Factor XIa activity by U.S. Food and Drug Administration. Multivariable regression was used to estimate same-day TE risk for different products. Of 101,956 individuals exposed to 23 different HIG product groups, 86 (0.84 per 1,000 persons) had a TE diagnosis code (DC) recorded on the same day as HIG administration. Unadjusted same-day TE DC rates (per 1,000 persons) ranged from 0.4 to 148.9 for different products. GamaSTAN S/D IG >10 cc had statistically significantly higher same-day TE DC risk compared to Tetanus IG (OR = 57.57; 95% CI = 19.72-168.10). Increased TE risk was also observed with older age (≥45 years), prior thrombotic events, and hypercoagulable state(s). Laboratory investigation identified elevated Factor XIa activity for GamaSTAN S/D, HepaGam B, HyperHep B S/D, WinRho SDF, HyperRHO S/D full dose, and HyperTET S/D. Our study, for the first time, identified increase in the same-day TE DC risk with GamaSTAN S/D IG >10 cc and suggests potentially elevated TE risk with other HIGs.

Correction of microplate location effects improves performance of the thrombin generation test.

BACKGROUND: Microplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate. METHODS: In this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment. RESULTS: Location of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment. CONCLUSIONS: Use of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field.

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.