Beta-2 glycoprotein I (β2GPI) is the primary antigen targeted by thrombotic antiphospholipid antibodies and is a plasma protein with a minor inhibitory effect on blood coagulation. The results of one study suggested that β2GPI may exhibit procoagulant properties. It is important to establish the validity of this observation because β2GPI is sometimes a contaminant in therapeutic immunoglobulin (IG) products, including those associated with thrombotic complications. Unlike the previous study showing procoagulant activity in one plasma-derived lot of β2GPI, we investigated four β2GPI preparations from different vendors: three plasma-derived (pd) and one recombinant (r). No procoagulant activity was detected in two plasma-derived preparations or in the recombinant product, whereas one pd-β2GPI preparation exhibited procoagulant activity. The previous report showing procoagulant activity had also used the same vendor that showed procoagulant activity in our study. Comprehensive characterization of the pd-β2GPI associated with procoagulant activity demonstrated contamination with activated coagulation factor XI (FXIa). Furthermore, incubation of this pd-β2GPI preparation with anti-FXIa antibodies reversed the procoagulant effect. Conversely, spiking FXIa into the r-β2GPI induced a procoagulant effect. Taken together our data indicate that β2GPI exhibits no procoagulant activity, but pd-β2GPI could be contaminated with FXIa (which is a procoagulant) during the purification process. This result is consistent with a large body of work showing FXIa contamination in some lots of therapeutic IG products. These lots of IG products are associated with thrombogenicity in the clinic. Our results demonstrate that β2GPI contamination of IG lots is unlikely to be a risk factor for thrombogenicity. β2-glycoprotein I (β2GPI) itself exhibits no intrinsic procoagulant activity. Recombinant β2GPI and the majority of plasma-derived β2GPI preparations did not trigger thrombin generation in vitro. In contrast, thrombin generation was observed in plasma-derived β2GPI preparations contaminated with activated factor XI (FXIa). The procoagulant response was comparable to that induced by FXIa alone and was abrogated by anti-FXIa antibody addition, confirming that FXIa contamination was responsible for the procoagulant effect.

Intravenous immunoglobulin (IGIV), first licensed for the treatment of primary immunodeficiencies, is currently widely used in treatment of primary immunodeficiency and certain autoimmune diseases. Hemolysis is a potential adverse effect related to IGIV administration that occurs in approximately 1.6% of patients. Serious hemolysis complications are rare but may cause renal failure, severe anemia, and even death and are commonly associated with non-O blood group recipients receiving high doses (1-2 g/kg/day). It is known that antibodies to blood groups A and B may copurify with IGIV, depending on the manufacturing method. Limits on anti-A and anti-B in IG products are required, as measured by direct hemagglutination (DHAT), a European Pharmacopeial (EP) method, however hemolytic events continue to occur and trend higher in recent years due to widespread use of high-dose IGIV as well as certain manufacturing advances aimed at enhancing IgG yield but resulting in higher isoagglutinin content in the final products. In blood, both intra- and extravascular hemolysis can occur in various proportions dependent on the patients: via acute complement-dependent lysis of antibody-bound RBCs within the blood circulation or delayed-onset cell-mediated clearance of antibody- bound RBCs by activated monocytes in liver and spleen, respectively. We developed a complement-dependent hemolysis assay (CDHA), to measure hemolytic activity to model of intravascular hemolysis. Our current aims are to develop a hemolysis assay that reflects IGIV-mediated cell-based extravascular hemolysis, to employ both approaches to determine the hemolytic activities, characterize isoagglutinins in IGIV, and compare findings with DHAT. In developing a functional assay to measure cell-mediated extravascular hemolysis, we adapted the monocyte monolayer assay (MMA) developed in Donald Branch's laboratory with certain modifications. Macrophage preparations included primary elutriated monocytes differentiated to macrophages, PBMC, and THP-1, a macrophage cell line. These cells were exposed to Type A RBC opsonized with IGIV or Rho(D) IG. Anti-Rho(D)-opsonized RBC were used as a positive control, and a known clinically hemolytic IGIV lot (DV-1) was used to evaluate hemolytic/hemophagocytic macrophage activity. These controls were used to test critical conditions for the assay. The results were observed by phase-contrast microscope and the phagocytic index (phagocytosed RBCs per 100 monocytes) were calculated. Elutriated monocytes and PBMC showed greater hemophagocytic activity compared to THP-1. Macrophages can largely be categorized into two main types: M1 and M2 macrophages. The M1 type, referred to as classically activated macrophages, plays a large role in the immune response to foreign pathogens, and the M2 type, referred to as alternatively activated macrophages, play an anti-inflammatory role. To determine whether the M1 and M2 type of macrophages play different roles in MMA, we employed elutriated monocytes differentiated in vitro by various stimuli into unique subsets (M1, M2). Preliminary results indicated that M2 contains higher hemophagocytic activity than M1. Finally, the CDHA and MMA were compared with the DHAT (EP) method. Seven IND products were tested in the FDA research laboratory using both DHAT and CDHA methods. All products met the HA industry standard of 1:64. Several IND products demonstrated hemolytic ability similar to that of clinically hemolytic lots. In such cases, FDA provides advice on potential manufacturing solutions to lower hemolytic activity. Using both CDHA and MMA, we have shown that a clinically hemolytic lot (DV-1) exhibited the highest hemolytic activities in both assays. Taken together, we have developed two functional assays in evaluating the intravascular and extramacular hemolytic activities mediated by the IGIV products. Our findings reveal that the functional assays, i.e., CDHA and MMA, are more precise for measuring hemolytic activity than the currently applied EP method for lot release (DHAT). The CDHA and MMA may be useful for investigation of clinically hemolytic lots, and to increase understanding of product and patient risk factors. Further studies would be needed to determine predictive value of these test methodologies.

In intravenous immunoglobulins (IVIG), and some other immunoglobulin products, protein particles have been implicated in adverse events. Role and mechanisms of immunoglobulin particles in vascular adverse effects of blood components and manufactured biologics have not been elucidated. We have developed a model of spherical silica microparticles (SiMPs) of distinct sizes 200–2000 nm coated with different IVIG- or albumin (HSA)-coronas and investigated their effects on cultured human umbilical vein endothelial cells (HUVEC). IVIG products (1–20 mg/mL), bare SiMPs or SiMPs with IVIG-corona, did not display significant toxicity to unstimulated HUVEC. In contrast, in TNFα-stimulated HUVEC, IVIG-SiMPs induced decrease of HUVEC viability compared to HSA-SiMPs, while no toxicity of soluble IVIG was observed. 200 nm IVIG-SiMPs after 24 h treatment further increased ICAM1 (intercellular adhesion molecule 1) and tissue factor surface expression, apoptosis, mammalian target of rapamacin (mTOR)-dependent activation of autophagy, and release of extracellular vesicles, positive for mitophagy markers. Toxic effects of IVIG-SiMPs were most prominent for 200 nm SiMPs and decreased with larger SiMP size. Using blocking antibodies, toxicity of IVIG-SiMPs was found dependent on FcγRII receptor expression on HUVEC, which increased after TNFα-stimulation. Similar results were observed with different IVIG products and research grade IgG preparations. In conclusion, submicron particles with immunoglobulin corona induced size-dependent toxicity in TNFα-stimulated HUVEC via FcγRII receptors, associated with apoptosis and mTOR-dependent activation of autophagy. Testing of IVIG toxicity in endothelial cells prestimulated with proinflammatory cytokines is relevant to clinical conditions. Our results warrant further studies on endothelial toxicity of sub-visible immunoglobulin particles.

ABSTRACT Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998–2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infection in vitro . These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancement in vitro . Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operating in vivo may modulate ZIKV infection outcomes. IMPORTANCE We investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998–2020) to neutralize or enhance Zika virus infection in vitro . West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, both in vitro and in vivo . Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Background. Apolipoprotein-H (APOH), also known as b2-Glycoprotein I (b2GPI), is a plasma protein that was found in immune globulin product lots associated with thrombotic complications. Previously, APOH demonstrated anticoagulant activity, on the contrary, Lackner et al reported that APOH has procoagulant function. In the present study, we show that plasma derived APOH (pdAPOH) can be contaminated with activated coagulation factor XI (FXIa). Methods. In-house modified simultaneous thrombin and fibrin generation assay (TGA) along with FXIa activity chromogenic assay (FXIa:C) were used to detect FXIa function. In parallel, high-performance liquid chromatography (HPLC) was used to demonstrate presence of FXIa in the procoagulant APOH. Results. Functional FXIa activity was detected in plasma derived APOH (pdAPOH-FIT). This activity was comparable to that measured in purified FXIa and procoagulant immune globulin product (IGIV) batches known to contain FXIa. Anti-FXIa antibody blocked procoagulant activity of pdAPOH-FIT in the TGA. An APOH sample from another source pdAPOH-2 and recombinant APOH did not show any activity in the TGA. Presence of FXIa in pdAPOH-FIT was confirmed using the chromogenic assay. Using HPLC, we show that peaks for pdAPOH-FIT and pdAPOH-2 differed. However, procoagulant pdAPOH-FIT demonstrated peaks similar to pdAPOH-2 when spiked with FXIa. Conclusions. APOH does not demonstrate procoagulant activity in TG assay in plasma. plasma derived APOH products may contain procoagulant FXIa contaminant in the amounts sufficient to impact TG which may be misinterpreted as procoagulant activity of APOH.

The recent global COVID-19 pandemic caused by SARS-CoV-2 lasted for over three years. A key measure in combatting this pandemic involved the measurement of the monoclonal antibody (mAb)-mediated inhibition of binding between the spike receptor-binding domain (RBD) and hACE2 receptor. Potency assessments of therapeutic anti-SARS-CoV-2 mAbs typically include binding or cell-based neutralization assays. We assessed the inhibitory activity of five anti-SARS-CoV-2 mAbs using ELISA, surface plasmon resonance (SPR), and four cell-based neutralization assays using different pseudovirus particles and 293T or A549 cells expressing hACE2 with or without TMPRSS2. We assessed the interchangeability between cell-based and binding assays by applying the Bland–Altman method under certain assumptions. Our data demonstrated that the IC50 [nM] values determined by eight neutralization assays are independent of the cell line, presence of TMPRSS2 enzyme on the cell surface, and pseudovirus backbone used. Moreover, the Bland–Altman analysis showed that the IC50 [nM] and KD [nM] values determined by neutralization/ELISA or by SPR are equivalent and that the anti-spike mAb activity can be attributed to one variable directly related to its tertiary conformational structure conformation, rate dissociation constant Koff. This parameter is independent from the concentrations of the components of the mAb:RBD:hACE2 complexes and can be used for a comparison between the activities of the different mAbs.

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.

Zika virus (ZIKV) has become endemic in multiple tropical and subtropical regions and has the potential to become widespread in countries with limited prior exposure to this infection. One of the most concerning sequelae of ZIKV infection is the teratogenic effect on the developing fetus, with the mechanisms of viral spread to and across the placenta remaining largely unknown. Although vaccine trials and prophylactic or therapeutic treatments are being studied, there are no approved treatments or vaccines for ZIKV. Appropriate tests, including potency and in vivo assays to assess the safety and efficacy of these modalities, can greatly aid both the research of the pathophysiology of the infection and the development of anti-ZIKV therapeutics. Building on previous work, we tested reporter ZIKV variants that express nanoluciferase in cell culture and in vivo assays. We found that these variants can propagate in cells shown to be susceptible to the widely used clinical isolate PRVABC59, including Vero and human placenta cell lines. When used in neutralization assays with bioluminescence as readout, these variants gave rise to neutralization curves similar to those produced by PRVABC59, while being better suited for performing high-throughput assays. In addition, the engineered reporter variants can be useful research tools when used in other in vitro and in vivo assays, as we illustrated in transcytosis experiments and a pilot study in guinea pigs.

Background No human rabies post-exposure prophylaxis (PEP) failure has been documented in the United States using modern cell culture-based vaccines. In January 2021, an 84-year-old male died from rabies 6 months after being bitten by a rabid bat despite receiving timely rabies PEP. We investigated the cause of breakthrough infection.Methods We reviewed medical records, laboratory results, and autopsy findings and performed whole-genome sequencing (WGS) to compare patient and bat virus sequences. Storage, administration, and integrity of PEP biologics administered to the patient were assessed; samples from leftover rabies immunoglobulin were evaluated for potency. We conducted risk assessments for persons potentially exposed to the bat and for close patient contacts.Results Rabies virus antibodies present in serum and cerebrospinal fluid were nonneutralizing. Antemortem blood testing revealed that the patient had unrecognized monoclonal gammopathy of unknown significance. Autopsy findings showed rabies meningoencephalitis and metastatic prostatic adenocarcinoma. Rabies virus sequences from the patient and the offending bat were identical by WGS. No deviations were identified in potency, quality control, administration, or storage of administered PEP. Of 332 persons assessed for potential rabies exposure to the case patient, 3 (0.9%) warranted PEP.Conclusions This is the first reported failure of rabies PEP in the Western Hemisphere using a cell culture-based vaccine. Host-mediated primary vaccine failure attributed to previously unrecognized impaired immunity is the most likely explanation for this breakthrough infection. Clinicians should consider measuring rabies neutralizing antibody titers after completion of PEP if there is any suspicion for immunocompromise. A patient died of bat-borne rabies despite receipt of post-exposure prophylaxis. Rabies biologics failure was ruled out; undiagnosed immunosuppression was the most likely explanation for the rare outcome. Post-vaccination serology should be considered for high-risk exposures and suspicion of immunocompromise.

Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

We applied Surface Plasmon Resonance (SPR) technology to develop a method for potency screening and quantification of anti-influenza antibodies in minimally processed human plasma samples and intravenous immunoglobulin (IGIV) products. We found that specific antibodies in human plasma or IGIV capable of inhibiting binding of influenza hemagglutinin to receptor-analogous glycans do so in concentration-dependent manner. We ranked the inhibitory activity of plasma samples from multiple donors and found a good correlation (r = 0.87) of SPR assay measurements and conventional hemagglutination inhibition (HAI) assay results. This method was also applied to screen for specific anti-influenza antibodies in IGIV lots manufactured pre- and post-2009 H1N1 pandemic. The SPR method was also applied to study binding inhibition of the intact A/California/04/2009 H1N1 and B/Victoria/504/2000 influenza viruses to α2,6 or α2,3-linked synthetic glycans. In contrast to recombinant H1 hemagglutinin, which was found to interact primarily with α2,6-linked terminal sialic acids, intact H1N1 or influenza B virus recognized both types of receptor analogs with different observed dissociation rates and the inhibitory activity of plasma antibodies was dependent on the type of sialic acid link. The SPR method can provide a high-throughput, time-saving and semi-automated alternative to conventional assays such as HAI or microneutralization in situations where screening of large numbers of plasma donations to identify high titer units is needed to product highly potent immunoglobulins.

BACKGROUND:Longitudinal patterns of immune globulins (IG) use have not been described in large populations. Understanding IG usage is important given potential supply limitations impacting individuals for whom IG is the sole life-saving/health-preserving therapy. The study describes US IG utilization patterns from 2009 to 2019.STUDY DESIGN AND METHODS:Using IBM MarketScan commercial and Medicare claims data, we examined four metrics overall and by condition-specific categories during 2009-2019: (1) IG administrations per 100,000 person-years, (2) IG recipients per 100,000 enrollees, (3) average annual administrations per recipient, and (4) average annual dose per recipient.RESULTS:In the commercial and Medicare populations respectively: IG administrations per 100,000 person-years increased by 120% (213-470) and 144% (692-1693); IG recipients per 100,000 enrollees grew by 71% (24-42) and 102% (89-179); average annual administrations per recipient rose by 28% (8-10) and 19% (8-9); and average annual dose (grams) per recipient increased by 29% (384-497) and 34% (317-426). IG administrations associated with immunodeficiency (per 100,000 person-years) increased by 154% (from 127 to 321) and 176% (from 365 to 1007). Autoimmune and neurologic conditions were associated with higher annual average administrations and dose than other conditions.DISCUSSION:IG use increased, coinciding with a growth in the IG recipient population in the United States. Several conditions contributed to the trend, with the largest increase observed among immunodeficient individuals. Future investigations should assess changes in the demand for IVIG by disease state or indication and consider treatment effectiveness.

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

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.

Human Cytomegalovirus (HCMV) infection is widespread and can result in severe sequelae in susceptible populations. Primary HCMV infection of naïve individuals results in life-long latency characterized by frequent and sporadic reactivations. HCMV infection elicits a robust antibody response, including neutralizing antibodies that can block the infection of susceptible cells in vitro and in vivo. Thus, antibody products and vaccines hold great promise for the prevention and treatment of HCMV, but to date, most attempts to demonstrate their safety and efficacy in clinical trials have been unsuccessful. In this review we summarize publicly available data on these products and highlight new developments and approaches that could assist in successful translation of HCMV immunotherapies.

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.

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 (FeCl 3 ) 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 FeCl 3 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.