Absence of Hyperactivation of Fibrinolysis Explains the Lack of Hemostatic Efficacy of Prophylactic Tranexamic Acid (TXA) in Hypoproliferative Thrombocytopenia

Background: We previously reported the results of the A-TREAT study (American Trial Using Tranexamic Acid in Thrombocytopenia: NCT02578901). This randomized double-blind placebo-controlled trial demonstrated that TXA administration is not superior to placebo in preventing WHO grade 2 or higher bleeding in severely thrombocytopenic patients requiring supportive platelet transfusion following myeloablative therapy for hematologic disorders (Gernsheimer T., ASH 2020 Plenary Session). Here, we present results of the A-TREAT ancillary study - Fibrinolysis Evaluation in A-TREAT (FEAT). Blood samples were collected from a subset (n=115) of A-TREAT participants just prior to initiation of study drug (when the platelet count was <30,000/µl) and at a later time point when TXA was at a steady state trough level (5 ±2 days following study drug initiation). Using global assays of fibrinolysis in plasma, our a priori hypotheses were that: 1] a baseline 'hyperfibrinolytic' profile would be associated with a higher proportion of grade 2+ bleeding; and 2] trough TXA levels would be associated with a 'hypofibrinolytic' profile and a lower proportion of grade 2+ bleeding. Methods: Fibrinolysis in platelet-free citrated plasma was assessed by 3 global assays: euglobulin clot lysis time (ECLT), plasmin generation (PG), and tPA resistance clot lysis time (tPA-CLT) using previously described methods (Ilich A. RPTH 2020, Miszta A. JTH 2021). Trough plasma TXA concentration was measured using a validated tandem mass spectrometry assay. Individual fibrinolytic analytes (PAI-1, tPA, plasminogen, alpha2-antiplasmin and plasmin-antiplasmin complexes) were quantified by ELISA. Results: Baseline samples did not demonstrate a hyperfibrinolytic profile by ECLT. To the contrary, ECLT values were significantly increased compared to healthy controls (figure 1). Furthermore, none of the measured fibrinolytic parameters (ECLT, tPA-CLT, total PAI-1, tPA, plasminogen, alpha2-antiplasmin or plasmin-antiplasmin complexes) at baseline were associated with a greater risk of grade 2+ bleeding during follow up, regardless of treatment arm. On the follow-up samples, neither pharmacokinetic (trough TXA concentration) nor pharmacodynamic parameters (PG or tPA-CLT) were associated with bleeding severity. A high inter-patient variability of TXA trough concentrations was noted in the treatment arm (min-max: 0.7-10 ug/ml), and drug levels correlated strongly with global fibrinolysis assessment by PG (Spearman r, -0.78, 95% CI -0.88 - -0.62) and tPA-CLT (r, 0.74, 0.56 - 0.85) (figure 2). Conclusions: 1] No evidence of fibrinolytic hyperactivation was observed in these thrombocytopenic patients; 2] trough TXA concentrations varied significantly between patients receiving the same dosing schedule; and 3] tPA-CLT and PG parameters correlated well with TXA plasma concentrations and thus may be used to estimate the extent of fibrinolytic inhibition in patients treated with TXA. Discussion: The absence of hyperactivation of endogenous fibrinolysis in this study is in contrast to our recent findings in trauma. Specifically, we reported that almost half of trauma patients demonstrated evidence of fibrinolytic hyperactivation (by ECLT) on admission (Ilich A. Thromb Res, 2021). Since TXA has been shown to reduce mortality due to bleeding in trauma (CRASH II, Lancet, 2010) and given that baseline hyperfibrinolysis is common in trauma, we hypothesize that the absence of fibrinolytic hyperactivation observed in the A-TREAT study patients likely explains the clinical lack of efficacy of TXA. Figure 1 Figure 1. Disclosures Gernsheimer: Amgen: Honoraria; Novartis: Honoraria; Principia: Research Funding; Rigel: Research Funding; Cellphire: Consultancy; Dova: Consultancy; Sanofi: Consultancy. Triulzi: Fresenius Kabi: Membership on an entity's Board of Directors or advisory committees; Realta: Membership on an entity's Board of Directors or advisory committees. Wolberg: CSL Behring: Consultancy; Bristol Myers Squibb: Research Funding; Takeda: Research Funding. Key: Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

Emizicumab Promotes Factor Xa Generation on Activated Endothelium in a Blood Cell-Independent Manner

Introduction Hemophilia A (HA) is an inherited bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) resulting in severe hemorrhage if untreated. Recombinant and plasma derived FVIII products have long been the standard of care in hemophilia. However, approximately 25-30% of patients with severe HA develop inhibitors, neutralizing alloantibodies to FVIII, a significant complication in the treatment of patients with HA that leads to bleeding despite factor therapy. First approved for bleed prophylaxis in HA with inhibitors in the US by the FDA in 2018, emicizumab (Genentech, USA) has initiated a new era of HA treatment. This drug is a bispecific, monoclonal antibody that binds to activated Factor IX (FIXa) and Factor X (FX), mimicking activated FVIII (FVIIIa) by bringing FIXa and FX into proximity to enable FX activation, even in the presence of inhibitors. Emicizumab prophylaxis drastically reduces bleed episodes. However, thromboses and thrombotic microangiopathy (TMA) were observed in trials, all associated with concomitant use of activated prothrombin complex concentrates (aPCC) (Callaghan et al., 2021). The mechanism of this devastating condition is uncertain, as emicizumab is not known to bind to phospholipid or vascular surfaces. We report that FX is more readily activated by FIXa and emicizumab on endothelium that has been activated by tumor necrosis factor alpha (TNF). This finding may partially explain the development of TMA in these patients. Methods We utilized novel, microfluidic devices that are inexpensive to manufacture and were modified from a technique previously described (Alapan et al. 2016). These devices are fabricated using a laser cut double-sided adhesive film sandwiched between a clear, gas-permeable polymer (Ibidi, Germany) and an acrylic top that is laser cut (Universal Laser Systems Inc., USA) (Figure 1). Human umbilical endothelial cells (HUVEC, Lonza, Switzerland) were harvested at passage 3 to 4 and seeded into the devices. These were then cultured under flow conditions using a non-peristaltic, air-driven pump (Ibidi GmbH, Germany) to achieve a confluent and quiescent endothelial surface. HUVEC are then activated by incubating with 5 nM TNF in serum-free growth medium for 4 hours. This treatment induced markers of endothelial activation without gross apoptosis. Non-activated HUVEC were incubated with endothelial cell growth medium (2% serum) until time of experiments. Factors IXa, X (Haemtech, USA), and/or emicizumab (discarded clinical material) were mixed in HEPES-buffered saline with 5 mM calcium chloride for all experimental conditions. Concentrations used of FIXa (30 nM), FX (170 nM), and emicizumab (55 ug/mL) were constant for all conditions. Combinations of factors and emicizumab were then incubated in the endothelialized device for 30 minutes at 37° C. The entire volume of the mixture was then aspirated (20 uL) and stored at -80° C. FXa activity was assayed on the effluent for 60 minutes using a chromogenic FXa substrate (Pefachrome, Pentapharm, Switzerland). Results No significant generation of Xa was noted in the presence of healthy or activated endothelium with emicuzumab alone, emicizumab and FIXa, emicizumab and FX, or factors IXa and X. Median Xa generation observed with the combination of emicizumab, FIXa, and FX on healthy endothelium was 2 nM. Median Xa generation with the same combination on activated endothelium was 8.1 nM, a four-fold increase (P = 0.028, Mann-Whitney test) (Figure 2). Discussion Emicizumab represents an evolving standard of care for hemophilia A. Considering data showing diminishing FVIII expression in the months to years after AAV gene therapy, (Pasi et al., 2020) it may well be the dominant treatment paradigm in HA for some time. However, much remains to be answered in the use of emicizumab, and the mechanism of thrombosis and TMA with concomitant aPCC use has resulted in the avoidance of aPCC use for breakthrough bleeding in patients on emicizumab therapy, even up to 6 months after cessation. Our data demonstrate that activated endothelial cells promote FX activation more readily than quiescent endothelial cells in the presence of FIXa and emicizumab. These findings demonstrate the potential of thrombotic angiopathy in an in vitro system. Further investigation of the interaction of endothelium with FIXa, FX, and FVIIIa-mimetic antibodies is warranted. Figure 1 Figure 1. Disclosures Ellsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Monroe: Medexus Pharmaceuticals: Consultancy; Takeda: Consultancy; Otello Medical: Current equity holder in publicly-traded company. Hoffman: Takeda: Research Funding; CSL Behring: Consultancy; Sanofi: Consultancy; BPL (Bio Products Laboratory): Consultancy. Key: BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Sanofi: Consultancy.

Bleeding Data across Baseline FIX Expression Levels in People with Hemophilia B: An Analysis Using the 'Factor Expression Study'

Introduction Complications such as spontaneous and trauma-related bleeding events typically experienced by people with hemophilia B (PWHB) are associated with long-term joint damage and chronic pain, and burdensome treatment with intravenous factor IX administration. Gene therapy, designed to enable the endogenous production of the missing clotting factor, has potential for curative benefit in PWHB (Dolan et al, 2018). Due to its link to risk for bleeding episodes, factor expression level (FEL) is commonly used as an endpoint in hemophilia gene therapy trials. However, little data currently exist linking FEL to bleeding risk in PWHB, most notably within the mild range. As such, the aim of this analysis was to examine the relationship between annual bleed rate (ABR) data across baseline FEL in PWHB. Methods Data from adult non-inhibitor PWHB, across Europe and the United States (US) who received clotting factor on-demand (OD), were drawn from the 'Cost of HaEmophilia in adults: a Socioeconomic Survey' (CHESS) studies. The CHESS studies are retrospective, burden-of-illness studies in people with hemophilia A or B, capturing the economic and humanistic burden associated with living with hemophilia. Additional data were collected to supplement the existing CHESS studies, particularly in people with exogenous FEL in the mild and moderate range. ABR was defined as the physician-reported number of bleed events experienced by the patient in the 12 months to study capture. A generalized linear model (GLM) was used to analyze variation in ABR data across FEL, adjusting for covariates age, body mass index (BMI), and blood-borne viruses. Following this, a multivariable restricted cubic spline (RCS) GLM regression was performed to create, model, and test for the potential non-linear relationship between FEL and ABR. The RCS regression employed 3 knots, located at baseline FEL values of 1, 5, and 10, and controlled once again for age, BMI, and blood-borne viruses. Results A total of 407 adult non-inhibitor PWHB, receiving an OD therapy regimen and with information on ABR, were profiled. The GLM provided adequate fit for the modeling of bleed data; the average marginal effect at the mean was computed from the GLM regression outputs. After controlling for the effects of all other model covariates, the regression analysis showed a significant association between FEL and ABR; for every 1% increase in FEL, the average ABR decreased by 0.08 units (p<0.001). The results of the RCS regression found a significant non-linear relationship between FEL and ABR, ceteris paribus (p<0.001). Conclusions The results of this analysis found baseline FEL to be significantly associated with ABR in PWHB; as baseline FEL increased, ABR reduced. This highlights the clinical importance of new hemophilia gene therapies potentially increasing FEL to that of the mild or non-hemophilic range in terms of reducing patient burden through the better prevention of bleeding events in PWHB. Disclosures Ali: UniQure: Current Employment. Li: UniQure: Current Employment. Konkle: Pfizer, Sangamo, Sanofi, Sigilon, Spark, Takeda and Uniqure: Research Funding; BioMarin, Pfizer and Sigilon: Consultancy. O'Mahony: BioMarin Pharmaceutical Inc.: Consultancy; Freeline: Consultancy; Uniqure: Speakers Bureau. Pipe: Apcintex: Consultancy; ASC Therapeutics: Consultancy; Bayer: Consultancy; Biomarin: Consultancy, Other: Clinical trial investigator; Catalyst Biosciences: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy; Freeline: Consultancy, Other: Clinical trial investigator; Novo Nordisk: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy, Other; Sangamo Therapeutics: Consultancy; Sanofi: Consultancy, Other; Takeda: Consultancy; Spark Therapeutics: Consultancy; uniQure: Consultancy, Other; Regeneron/ Intellia: Consultancy; Genventiv: Consultancy; Grifols: Consultancy; Octapharma: Consultancy; Shire: Consultancy.

Rituximab Monotherapy Is Effective for Inhibitor Eradication with Concomitant Porcine Factor VIII Followed By Emicizumab for Bleed Control in Acquired Hemophilia a

Introduction Acquired hemophilia A (AHA) is a rare bleeding disorder in which acquired auto-antibodies to endogenous Factor VIII (FVIII) resulting in decreased FVIII activity. AHA can lead to life-threatening bleeding, with effective treatment requiring both immunosuppressive therapy (IST) and bypassing agents such as recombinant activated Factor VII (rFVIIa) or activated prothrombin complex concentrates (APCC) (Tiede et al. Haematologica 2020). Some, including our group, have begun using emicizumab as well (Knoebl et al. Blood 2020). IST is required for inhibitor eradication, but regimens are heterogenous and have not been systematically compared in the literature. While there is no standard of care IST in these patients, most patients in the literature receive multiple agents, including corticosteroids, mycophenolate mofetil, cyclosporine, and/or rituximab in combination. We report in a prospective cohort that for IST, rituximab monotherapy is an effective strategy. An updated treatment algorithm is offered that has been effective for treatment of these patients at our institution, which adds emicizumab therapy after initial bleed control. Methods We analyzed clinical, pharmacy, and laboratory data from 24 patients treated with rpFVIII at the University of North Carolina for AHA from July 2015 to June 2021. All patients were initially treated according to our previously established dosing algorithm with recombinant porcine FVIII, and the last five patients have received emicizumab after initial factor dosing (see Figure 1). 17 of the patients who received rituximab and were followed at our center subsequently attained inhibitor eradication, six of those received only rituximab therapy. Investigational review board approval was obtained for our data collection and analysis. Patients who did not receive rituximab, failed to reach an inhibitor level <0.5 BU, or who were lost to follow up were excluded from the analysis. For patients that fit the inclusion criteria, the time between date of the first rituximab infusion and the date of inhibitor eradication was calculated. Results All patients in our cohort who we followed until inhibitor eradication (17 of 24 patients) had eradication of inhibitors after a median of 143 days from initiation of immunosuppression. For patients treated with rituximab monotherapy for inhibitor eradication (6 of 17), this goal was reached in a median of 134.5 days (range 76-191 days). For those who received agents in addition to rituximab and have reached inhibitor eradication to date (9 of 17 patients), median days from initiation of immunosuppression to inhibitor eradication was 137.5 days (range 11-485) (P = 0.43 on Mann-Whitney test). Patients were treated as previously reported by our group per an algorithm that starts recombinant porcine FVIII without waiting for a porcine inhibitor and at lower than FDA recommended dosing. Subsequent doses for bleed control are titrated according to one-stage, clot based FVIII activity. This report also includes 5 new patients who, after initial bleed control per our algorithm, were initiated on emicizumab while awaiting inhibitor eradication. There was no correlation between time to rituximab initiation and time to inhibitor eradication in both those who received rituximab monotherapy and those who had multiple IST agents. There was also no significant difference in initial inhibitor titer between groups with median initial inhibitor titer of 104 BU in the rituximab monotherapy group, and 70 BU in the multiple IST agents group (see Figure 3). Conclusions Rituximab monotherapy appears to be an effective strategy for inhibitor eradication in acquired hemophilia A. In the context of bleed treatment with porcine factor, followed by emicizumab, a standardized, algorithmic approach can be effectively employed for these patients. Though any patients have inhibitor recurrence, as is described in the literature, with emicizumab available, bleeding can be avoided with regular monitoring. Emicizumab given while re-eradicating an inhibitor can prevent morbidity of this disease. Figure 1 Figure 1. Disclosures Ellsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Key: Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy. Ma: Accordant: Consultancy; Takeda: Honoraria, Research Funding. OffLabel Disclosure: Emicizumab is not approved for use in Acquired Hemophilia A and this represents an OFF LABEL use of the drug.

Inhibition of Factor XII Attenuates Prothrombotic Complications in Sickle Cell Mice

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice. First, Townes HbSS mice (SS) and non-sickle Townes HbAA controls (AA) (male and female, 16 weeks) received anti-FXII antibody or control IgGκ1 (10 mg/kg, IV) 30 minutes prior to subjecting them to venous thrombosis, initiated by applying positive current (3 volts, 90 sec) to the femoral vein. To visualize platelet and fibrin accumulation, mice were injected with rhodamine 6G and anti-fibrin antibody 59D8 labeled with Alexa Fluor 647, respectively. The femoral vein thrombi were imaged by intravital fluorescence microscopy using time-lapse capture every 10 seconds, to acquire images of fibrin and platelets over 60 min. The accumulation of platelets and fibrin was quantified for relative intensity of each fluorophore over the region of the observed thrombus. As previously shown, thrombi of SS/IgG mice showed an increased fibrin and platelet accumulation compared to AA/IgG group. Importantly, 15D10 treatment significantly attenuated both fibrin (p<0.001) and platelet (p<0.05) deposition over time in SS mice compared to SS/IgG group. The same effect of 15D10 treatment was observed in AA mice. At the end of experiment, clots were collected and stained with hematoxylin and eosin, and clot volume was assessed histomorphometrically (Nikon Ti-2, FIJI Software). Surprisingly, despite higher fibrin content, clots from SS/IgG mice had significantly smaller volume than clots from AA/IgG group (0.32 ± 0.04 versus 0.60 ± 0.11 mm 3, p<0.05). Importantly, administration of 15D10 significantly reduced clot volume in both SS (0.086 ± 0.01 mm 3, p<0.05) and AA mice (0.1 ± 0.02 mm 3, p<0.05). Next, AA and SS mice (male and female, 8-10 weeks) were subjected to brain IR injury induced by middle cerebral artery occlusion for 60 minutes followed by 24 hours of reperfusion (mouse model of ischemic stroke). 15D10 or control IgGκ1 (10 mg/kg, IV) were injected 30 minutes before occlusion and again at 6 hours into the reperfusion period to generate 3 experimental groups: AA/IgG, SS/IgG and SS/15D10. All analyzed parameters of brain IR injury were significantly worse in the SS/IgG group compared to the AA/IgG group. Compared to IgG, pre-treatment of SS mice with 15D10 significantly attenuated neuronal damage determined by volume of brain infarction (11.7 ± 3.7 vs 24.9 ± 2.4%, p<0.001) and improved behavioral deficit assessed by mean stroke score (9.0 ± 0.9 vs 14.6 ± 0.9, p<0.01). These changes were accompanied by a significant increase in leukocytes rolling (1978.0 ± 93.5 vs 1517.0 ± 180.3 rolling leukocytes/sec/mm 2, p<0.001), and significant reduction in the number of adherent leukocytes (367.2 ± 49.0 vs 723.4 ± 48.5, adherent leukocytes/mm 2, p<0.001) observed in the brain microvasculature of SS mice treated with 15D10 compared to SS/IgG group. Together, our data indicates that in the mouse model of SCD FXII contributes to the experimental venous thrombosis and ischemic stroke. Given that targeting the intrinsic pathway can reduce thrombosis without affecting hemostasis, our data suggest that targeting FXII might be a beneficial treatment in reducing inflammatory and thrombotic complications in SCD patients without a risk of bleeding. Disclosures Wallisch: Aronora Inc,: Current Employment. Key: Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Uniqure: Consultancy, Other: Participation as a clinical trial investigator. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Feasibility of Delivering Pediatric-to-Adult Transition Education during Annual Comprehensive Clinic Appointments and Patient-Reported Outcomes: A Quality Improvement Study

Individuals with bleeding disorder diagnoses require developmentally sensitive care across the lifespan, particularly as they gain knowledge and skills necessary to successfully tackle their illness-specific needs as independent adults (Breakey et al., 2010). The current study describes one phase of a larger quality improvement (QI) initiative aimed at improving transition from pediatric to adult care (TAC) at one US Hemophilia and Thrombosis Center (HTC). Our aim was to assess the feasibility of delivering transition specific education to youth-caregiver dyads during youth annual multidisciplinary clinic appointments. Youth-caregiver dyads were selected given previous research revealing that both patients and their parents express worries about related to TAC (Geerts et al., 2008). Education included discussion of the knowledge and skills necessary for autonomous management of one's bleeding disorder (e.g., illness basics, treatment, communication, and healthy living). During an 8-month period, 101 youth-caregiver dyads were approached. Patients were between the ages of 12 and 25 (M age = 17.66, SD = 3.45). Approximately half of patients were diagnosed with hemophilia A (53.5%) and 16.8% were female. Of the 101 patients approached, 90 completed the transition education discussion. On average, these discussions took 12.80 minutes (SD = 8.49) and ranged from 5 to 50 minutes. Social work delivered the bulk of these discussions (78.7%) and spent an average of 10.61 minutes (SD = 5.76) with youth and caregivers. While the intention was to deliver transition education to youth-caregiver dyads, this only occurred in 37 discussions. Other discussions included the patient only (n = 31), caregiver only (n = 20), or had missing data (n = 1). In instances when a youth-caregiver dyad was approached, but a discussion did not take place, barriers to completing the discussion were identified. "Provider" was listed most frequently (n = 5) as a barrier (e.g., youth sent home by medical team prior to transition discussion occurring; miscommunication between members of multidisciplinary team; low staffing of those trained to deliver transition discussion). Even in instances when a transition discussion did take place, barriers to having the discussion were identified. "Patient" barriers were the most frequently listed (n = 13), followed by barriers related to "Time" (n = 11), and barriers related to "Clinic" (n = 3). At the end of the transition discussion, youth and/or caregivers were encouraged to identify a goal for improving their skills or knowledge in one of the four areas discussed during their appointment. Of those having transition discussions, 72 created a transition goal. The majority of participants reported goals related to Treatment (e.g., infusion skills; n = 36) followed by goals related to Communication (n = 18), Healthy Living (n = 11), and Bleeding Disorder Basics (n = 7). There was a statistically significant difference in the type of goal of expressed by youth and/or caregivers when the patient was 17 years old or younger vs those older than 18, X 2 (3, N = 72) = 9.49, p = 0.024. Generally, more youth reported goals related to Treatment (e.g., infusion skills) than predicted by chance in both age groups. Patients or patient caregivers were contacted via phone between 5 and 14 months following their transition discussion. Approximately 1/3 of the patients who completed transition discussion, responded and provided ratings on progress toward meeting their transition goal. Ratings (M = 4.24, SD = 2.63) were made on a Likert-type scale ranging from 1 (no progress made) to 10 (maximum progress made). The information gleaned from this QI initiative revealed that delivery of transition-specific education within the CU-HTC annual multidisciplinary appointments is feasible and in some cases, served as the impetus necessary for accomplishing transition-specific goals. The results from this initiative have been instrumental in subsequent transition-related efforts related to: (a) fostering full engagement across the multidisciplinary team in TAC efforts utilizing HEMO-Milestones Tool (Croteau et al., 2016); (b) adopting specific materials developed to assess TAC in individuals with bleeding disorder diagnoses (i.e., American Society of Hematology Hemophilia Transition Readiness Assessment); and (c) reducing time between patient goal-setting and follow-up from the HTC. Disclosures Wang: Novo Nordisk: Consultancy, Other: Clinical trial investigator; Bioverativ: Consultancy, Other: Clinical trial investigator; Bayer: Consultancy, Other: Clinical trial investigator; Octapharma: Other; uniQure: Consultancy, Other: Clinical trial investigator; Pfizer/Spark: Other: clinical trial investigator; Genentech: Consultancy, Other: Clinical trial investigator; BioMarin: Consultancy, Other: Clinical trial investigator; CSL Behring: Consultancy, Other: Clinical trial investigator; Takeda: Consultancy, Other: Clinical trial investigator; Hema Biologics: Consultancy, Other: Clinical trial investigator. Buckner: CSL Behring: Honoraria; Tremeau Pharmaceuticals: Consultancy, Honoraria; Genetech: Honoraria; Bayer: Honoraria; Spark: Honoraria; Sanofi: Honoraria; Novo Nordisk: Honoraria; Pfizer: Honoraria; BioMarin: Consultancy, Honoraria; Takeda: Honoraria; American Thrombosis: Membership on an entity's Board of Directors or advisory committees; Hemostasis Network: Membership on an entity's Board of Directors or advisory committees; uniQure: Consultancy, Honoraria.

Cleavage of High Molecular Weight Kininogen and Bradykinin Release By Red Blood Cell Microvesicles As a Putative Mechanism for Hypotensive Transfusion Reactions

Background: Hypotensive transfusion reactions are adverse events typified by a sudden decrease in blood pressure that usually occurs within the first minutes after the initiation of a transfusion and resolves shortly after the transfusion is stopped. Due to current passive reporting practices, the incidence is likely underreported, but recent studies estimate an incidence of 1.3 cases per 10000 RBC units. The pathophysiology of these reactions are not fully understood. One hypothesis proposed is that increased bradykinin (BK), a nonapeptide released from cleavage of high molecular weight kininogen (HK), as seen with the use of negatively charged leukoreduction filters and the use of angiotensin-converting enzyme inhibitors, is a major contributor to the pathophysiology. We have recently demonstrated that red blood cell derived microvesicles (RBCMVs) from aging red blood cell (RBC) units are able to trigger thrombin generation via kallikrein activation - a predominant enzyme to cleave high molecular weight kininogen (Noubouossie, Blood, 2020). Thus, we hypothesize that the same RBCMVs would lead to bradykinin generation and might explain these hypotensive events. Objectives: To determine if RBC storage lesion-derived microvesicles are able to facilitate HK cleavage and BK release. Methods: RBCMVs were prepared from 4 recently expired RBC units (42 or 43 day old, AS-3 preserved, prestorage leukoreduced, all A+) via a series of centrifugations and washes. RBCMVs were quantified and characterized using nanoparticle tracking analysis. Obtained RBCMVs were first assessed for the capacity to initiate thrombin generation in microvesicle free human plasma via a substrate cleavage assay. Next, RBCMVs were added to a buffer reaction containing prekallikrein and HK, and kininogen cleavage was assessed via western blot. RBCMVs were also mixed with microvesicle-free human plasma and analyzed for evidence of kallikrein activation, cleavage of high molecular weight kininogen, and bradykinin production by ELISA. Cohn fractionation of plasma was used to enrich for BK. Results: RBCMVs were enumerated and concentrated to 7.5 ± 1.4 x 10 12 per mL (mean±SD size 160 ± 29µm). RBCMVs were able to initiate thrombin generation principally via contact pathway activation, independently of tissue factor. In a buffer system RBCMVs demonstrated activity to generate kallikrein with a sequential high molecular weight kininogen cleavage in a dose-dependent manner. Exclusion of kallikrein from the buffer system or addition of the small molecule inhibitor of kallikrein - ecallantide - halted cleavage of kininogen. A dose-dependent cleavage of high molecular weight kininogen indicated that RBCMVs could cause BK release in plasma; this was confirmed via an independent assay of Cohn -fractionated samples. Conclusions: Results of this current study demonstrate that RBCMVs are leading to high HK cleavage via kallikrein activation in vitro. We suspect that the same mechanisms could lead to BK generation in patients receiving older RBC units, possibly increasing the risk for hypotensive events from transfusion. Disclosures Karafin: Westat, Inc.: Consultancy. Key: Sanofi: Consultancy; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

502. Early COVID-19 Treatment with SARS-CoV-2 Neutralizing Antibody Sotrovimab

Background COVID-19 disproportionately results in hospitalization and death in older patients and those with underlying comorbidities. Sotrovimab is a pan-sarbecovirus monoclonal antibody that binds a highly conserved epitope of the SARS-CoV-2 receptor binding domain and has an Fc modification that increases half-life. Sotrovimab retains activity against UK, S. Africa, Brazil, India, New York and California variants in vitro. Objectives To evaluate the efficacy and safety of treatment with sotrovimab in high-risk, non-hospitalized patients with mild/moderate COVID-19, as part of the COMET-ICE clinical trial. Methods Multicenter, double-blind, phase 3 trial in non-hospitalized patients with symptomatic COVID-19 and ≥1 risk factor for disease progression were randomized 1:1 to an IV infusion of sotrovimab 500 mg or placebo. The primary efficacy endpoint was the proportion of patients with COVID-19 progression, defined as hospitalization > 24 hours or death, due to any cause, ≤29 days of randomization. Results The study met the pre-defined primary efficacy endpoint in a preplanned interim analysis: the risk of COVID-19 progression was significantly reduced by 85% (97.24% CI, 44% to 96%; P = 0.002) in 583 patients. In the final intention-to-treat analysis (N = 1057), the adjusted relative risk reduction was 79% (95% CI, 50% to 91%; p< 0.001) through Day 29 in recipients of sotrovimab (n=528) vs. placebo (n=529). Treatment with sotrovimab (ITT) resulted in a numerical reduction in the need for ER visits for illness management, hospitalization for acute illness management (any duration) or death (any cause) compared to placebo. No participants on sotrovimab required ICU admission, compared to 9 participants on placebo, of whom 4 participants required mechanical ventilation. No participants who received sotrovimab died, compared to 4 participants on placebo. The incidence of adverse events was similar between treatment arms and SAEs were numerically more common in the placebo arm. Conclusion Treatment with sotrovimab 500 mg IV resulted in a clinically and statistically significant reduction in progression of COVID-19 to hospitalization or death in patients with mild/moderate disease and was well-tolerated. Study funding GSK & VIR; NCT04545060 Disclosures Jaynier Moya, MD, VIR Biotechnology (Other Financial or Material Support, Jaynier Moya received non-financial support for serving as a clinical trial investigator for Vir Biotechnology) Diego Rodrigues Falci, MD, MSc, PhD, Gilead Sciences (Grant/Research Support, Scientific Research Study Investigator, Speaker's Bureau)GSK (Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member)MSD (Speaker's Bureau)Pfizer (Speaker's Bureau)United Medical (Speaker's Bureau, Other Financial or Material Support) Joel Solis, MD, VIR Biotechnology (Other Financial or Material Support, Joel Solis received non-financial support for serving as a clinical trial investigator for Vir Biotechnology) Hanzhe Zheng, PhD, VIR Biotechnology (Employee) Nicola Scott, MSc, GlaxoSmithKline (Employee, Shareholder) Andrea L. Cathcart, PhD, Gilead (Shareholder)VIR (Employee, Shareholder) Christy Hebner, PhD, Vir Biotechnology (Employee, Shareholder) Jennifer Sager, PhD, GSK (Other Financial or Material Support)Vir Biotechnology (Employee, Shareholder) Erik Mogalian, PharmD, PhD, Vir Biotechnology (Employee, Shareholder) Daren Austin, PhD, GlaxoSmithKline (Employee, Shareholder) Amanda Peppercorn, MD, GlaxoSmithKline (Employee) Elizabeth L. Alexander, MD, MSc, GlaxoSmithKline (Grant/Research Support, Other Financial or Material Support)VIR Biotechnology (Employee, Shareholder, GSK pharmaceuticals) Wendy W. Yeh, MD, Vir Biotechnology (Employee) Almena Free, MD, Amgen (Scientific Research Study Investigator)Astra Zeneca (Scientific Research Study Investigator)Cardurian (Scientific Research Study Investigator)Coherus (Scientific Research Study Investigator)Freenome (Scientific Research Study Investigator)GlaxoSmithKline/Vir (Scientific Research Study Investigator)Ionis (Scientific Research Study Investigator)Kowa (Scientific Research Study Investigator)New Amsterdam (Scientific Research Study Investigator)Regenacy (Scientific Research Study Investigator)Romark (Scientific Research Study Investigator)Scynexis (Scientific Research Study Investigator) Cynthia Brinson, MD, Abbvie (Scientific Research Study Investigator)BI (Scientific Research Study Investigator)Gilead Sciences Inc. (Scientific Research Study Investigator, Advisor or Review Panel member, Speaker's Bureau, Personal fees)GSK (Scientific Research Study Investigator)Novo Nordisk (Scientific Research Study Investigator)ViiV Healthcare (Scientific Research Study Investigator, Advisor or Review Panel member, Speaker's Bureau) Melissa Aldinger, PharmD, VIR Biotechnology (Employee) Adrienne Shapiro, MD, PhD, Vir Biotechnology (Scientific Research Study Investigator)