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Recent studies have demonstrated that only 30% of patients referred for assessment of a possible bleeding tendency will eventually be diagnosed with a mild bleeding disorder (MBD) such as von Willebrand disease (VWD) or platelet function defect (PFD). Rather, the majority of such patients will be diagnosed with Bleeding Disorder of Unknown Cause (BDUC). There remains an important unmet need to define consensus regarding the clinical and laboratory criteria necessary for a formal BDUC diagnosis. Nevertheless, BDUC already accounts for more than 10% of patients registered in some Haemophilia Comprehensive Care centres. Accumulating recent data suggest that BDUC is also being diagnosed with increasing frequency. Increased BAT scores are widely utilized to differentiate significant from trivial symptoms in patients with mucocutaneous bleeding. Objective assessment of bleeding phenotype using a standardised bleeding assessment tool (BAT) therefore represents a fundamental first step in the diagnosis of BDUC. Since BDUC is a diagnosis by exclusion, accurate quantification of bleeding phenotype is critical as this will be the primary determinant upon which a diagnosis of BDUC is reached. Importantly, BAT scores suggest that patients with BDUC display bleeding phenotypes comparable to those seen in patients with VWD or PFD respectively. Despite the prevalence of BDUC, diagnosis and management of these patients commonly pose significant clinical dilemmas. In this manuscript, we consider these challenges in the context of a number of typical case studies, discuss the available evidence and outline our approach to the management of these patients.

Introduction: Key characteristics of immune thrombocytopenia (ITP) include immune-mediated platelet destruction/impaired production, with resultant thrombocytopenia and increased bleeding risk. Durable response to current therapies remains an unmet need, particularly in the relapsed/refractory setting. Rilzabrutinib is the first oral, reversible, covalent inhibitor of Bruton tyrosine kinase designed to target immune-mediated pathways in ITP without inhibiting normal platelet aggregation. Initial phase I/II results in ITP demonstrated rapid and durable efficacy with rilzabrutinib that was well-tolerated at all dose levels, including the optimal 400 mg bid dose. Interim results on rilzabrutinib effects in patients with relapsed/refractory ITP were previously reported. Here we present long-term data from a larger group of patients who initiated rilzabrutinib at 400 mg bid and are continuing in the long-term extension (LTE) period. Methods: This ongoing, global phase I/II study (NCT03395210) enrolled adult patients from 8 countries with relapsed or refractory ITP who previously responded to ≥1 prior ITP therapy. Eligible patients with 2 baseline platelet counts of Results: As of June 1, 2021 in 60 patients, 45 patients initiated rilzabrutinib 400 mg bid and to date, 16 patients with durable, stable response proceeded to LTE at 400 mg bid. At enrollment for patients initiating rilzabrutinib 400 mg bid, median age was 49 y (range, 19-74), median duration of ITP was 6.1 y (range, 0.4-52.5), and median platelet count was 15×10 9/L (range, 2-33×10 9/L). Patients were heavily pretreated with a median of 6 prior treatment events (range, 1-53), including 24% with prior splenectomy. Overall, 18/45 patients (40%) achieved the primary endpoint. In primary responders, platelet counts of ≥30×10 9/L, ≥30×10 9/L with ≥20×10 9/L above baseline, and ≥50×10 9/L were maintained for a median of 95%, 86%, and 72% of weeks, respectively. Median time to first platelet counts of ≥30×10 9/L, ≥30×10 9/L with ≥20×10 9/L above baseline, and ≥50×10 9/L were 8.5 (7-134), 11.5 (7-134), and 12.5 (8-142) days, respectively. According to subgroup analyses, primary platelet responses were consistently >30% irrespective of baseline platelet counts, the use of concomitant therapy, duration of ITP, or number of prior therapies (Table). LTE patients received rilzabrutinib for an overall median duration of 462 days (range, 303-764). At LTE entry, patients had a median platelet count of 87×10 9/L (range, 16-321×10 9/L). In addition to all LTE patients achieving the primary endpoint during the main treatment period, these patients maintained platelet counts of ≥30×10 9/L, ≥30×10 9/L with ≥20×10 9/L above baseline, and ≥50×10 9/L, for a median of 100%, 96%, and 90%, of weeks, respectively, during the LTE period (Figure). Treatment-related treatment-emergent adverse events (TEAEs; all grade 1/2) were reported in 27/45 patients (60%); the most common were 36% diarrhea and 31% nausea, and all others were Conclusion: Oral rilzabrutinib 400 mg bid was well-tolerated and had durable, clinically significant platelet responses across subgroups and with extended treatment in patients with heavily pretreated ITP. Continued study in the ongoing, randomized phase III LUNA3 trial (NCT04562766) will further assess the magnitude and durability of rilzabrutinib's clinical benefit in ITP. Figure 1 Figure 1. Disclosures Kuter: Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, Bristol Myers Squibb (BMS), Immunovant, Kezar, Principia, Protalex, Rigel, Takeda (Bioverativ), UCB: Research Funding; Up-to-Date: Patents & Royalties: Up-To-Date; Rubius: Current equity holder in publicly-traded company; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BioCryst, Bristol Myers Squibb (BMS), Caremark, CRICO, Daiichi Sankyo, Dova, Genzyme, Immunovant, Incyte, Kyowa-Kirin, Merck Sharp Dohme, Momenta, Novartis, Pfizer, Principia, Protalex, Protalix, Rigel: Consultancy, Other: grant support and consulting fees; Platelet Disorder Support Association: Membership on an entity's Board of Directors or advisory committees. Mayer: Principia: Research Funding. Jansen: 3SBIO, Novartis: Other: Travel, accomodations, expenses; Advisory Board Novartis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Other: Travel, Accommodations, Expenses. McDonald: Bayer, Sobi, Novartis, Amgen, argenx: Honoraria; Grifols: Research Funding. Baker: Roche, Janssen-Celeg: Membership on an entity's Board of Directors or advisory committees; Bayer, Takeda, Pfizer, Daiichi Sankyo, CSL Behring, Roche, Amgen, Celgene, Rigel Pharmaceuticals, Abbvie, Sanofi, MorphoSys AG, Acerta Pharma, Jansen-Cileg, Bristol-Myers Squibb, Boehringer Ingelheim, Portola, Technoclone, Alexion: Research Funding. Bird: Novartis, Amgen: Speakers Bureau. Garg: Amgen Janssen Novartis Sanofi Takeda: Honoraria; Takeda Janssen Novartis Sanofi: Other: Travel Accommodations, Expenses; University Hospital Leicester: Current Employment. Gernsheimer: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: personal fees ; Amgen: Honoraria; Cellphire: Consultancy; Dova: Consultancy, Honoraria; Principia: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees, Research Funding; NHLBI: Research Funding. Ghanima: Amgen, Novartis, Pfizer, Principia Biopharma Inc- a Sanofi Company, Sanofi, SOBI, Griffols, UCB, Argenx: Consultancy; Amgen, Novartis, Pfizer, Bristol Myers Squibb, SOBI, Griffols, Sanofi: Honoraria; Bayer, BMS/Pfizer: Research Funding. Bandman: Sanofi: Ended employment in the past 24 months. Arora: Principia Biopharma Inc, a Sanofi Company: Ended employment in the past 24 months. Burns: Sanofi: Ended employment in the past 24 months. Yao: Sanofi: Current Employment. Daak: Sanofi: Current Employment. Sourdille: Sanofi: Current Employment. Thomas: Chinook and Equillium Biopharma: Current holder of individual stocks in a privately-held company; Chinook: Membership on an entity's Board of Directors or advisory committees; Equillium Biopharma: Current Employment; Principia, a Sanofi Company: Ended employment in the past 24 months; Equillium Biopharma: Current equity holder in publicly-traded company. Neale: Principia Biopharma/Sanofi: Ended employment in the past 24 months; Principia Biopharma: Divested equity in a private or publicly-traded company in the past 24 months. Cooper: Sanofi and Principia: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations expenses; Principia and Sanofi: Consultancy. OffLabel Disclosure: Rilzabrutinib is an investigational therapy being evaluated in a clinical study for the treatment of patients with immune thrombocytopenia.

INTRODUCTION Bleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways. METHODS Blood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA. RESULTS In the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients. Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients. CONCLUSIONS CLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. Disclosures McGregor: Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Platelets are pivotal to the process of arterial thrombosis resulting in ischemic stroke. Occlusive thrombosis is initiated by the interaction of von Willebrand factor (vWf) and platelet glycoprotein (GP) Ibα. Three polymorphisms have been described in GP Ibα (Kozak T/C polymorphism, variable number of tandem repeats [VNTR], and the human platelet antigen 2a [HPA-2a] [Thr] or HPA-2b [Met] at position 145), each of which may enhance the vWf and GP Ibα interaction. This study investigated whether these polymorphisms are candidate genes for first-ever ischemic stroke. A hospital-based case-control study was conducted of 219 cases of first-ever ischemic stroke and 205 community controls randomly selected from the electoral roll and stratified by age, sex, and postal code. The subtypes of stroke were classified, the prevalence of conventional risk factors was recorded, and blood was collected to perform genotyping analysis for Kozak C or T alleles, VNTR, and HPA-2a/b. It was found that the Kozak T/C genotype was over-represented in the stroke group (32.2%) compared with controls (22.8%) (odds ratio [OR], 1.6; 95% confidence interval [CI], 1.03-2.54; P

Introduction: The phase 3 B-LONG study (NCT01027364) demonstrated the safety, efficacy, and pharmacokinetics of recombinant factor IX Fc fusion protein (rFIXFc) for the prevention and treatment of bleeding episodes in adults and adolescents with severe hemophilia B. The ongoing rFIXFc extension study B-YOND (NCT01425723) is evaluating long-term safety and efficacy of rFIXFc in children and adults with hemophilia B. Safety and efficacy data for adult and adolescent subjects from the second B-YOND interim data cut are reported here. Methods: The B-LONG study enrolled males aged ≥12 years with severe hemophilia B (≤2 IU/dL endogenous factor IX [FIX] activity) who had received prior treatment with FIX. Subjects completing B-LONG could enroll in 1 of 4 treatment groups in B-YOND: weekly prophylaxis (WP; 20-100 IU/kg every 7 d), individualized prophylaxis (IP: 100 IU/kg every 8-16 d), modified prophylaxis (MP; for subjects not achieving optimal prophylactic dosing with IP or WP), or episodic treatment (ET). Subjects could change treatment groups at any point in B-YOND. The primary endpoint was development of inhibitors (neutralizing antibody value ≥0.6 BU/mL as measured by the Nijmegen-modified Bethesda assay). Secondary outcomes included annualized bleeding rate (ABR) and rFIXFc exposure days (EDs). The incidence of adverse events (AEs), treatment of bleeding episodes, and weekly dosing and dosing intervals are also reported. Here, data are reported from the second interim data cut of B-YOND (11 September 2015) for subjects treated with ≥1 dose of rFIXFc. Results: 93/115 subjects (81%) who completed B-LONG enrolled in B-YOND. As of the second interim data cut, 21 subjects had completed B-YOND, 61 subjects were ongoing in B-YOND, and 11 subjects had discontinued (1 due to lack of efficacy, 2 lost to follow up, 4 withdrawal by subject, and 4 other reasons). From the start of B-LONG to the second B-YOND interim data cut, subjects had a median 4.1 y of treatment with rFIXFc, and a median 181.0 cumulative rFIXFc EDs. In B-YOND, 68.8% of subjects had ≥100 EDs.No inhibitors were observed. The AE profile was typical of the hemophilia B population in a clinical trial and was generally similar to what is expected in the overall population of adults/adolescents with hemophilia B. There were no reports of serious allergic reactions or anaphylaxis associated with rFIXFc, and no thromboembolic events. 16/93 subjects (17.2%) enrolled in B-YOND changed treatment groups during the course of B-YOND, including 5 subjects who changed treatment groups from the start of B-YOND and then switched back to their original treatment group before the second interim data cut. 4/15 subjects changed from ET to prophylactic treatment. Among subjects treated prophylactically during B-LONG (n=71), 5.6% of subjects lengthened, 11.3% of subjects shortened, and 83.1% of subjects had no change to their dosing interval during B-YOND compared with their dosing interval in B-LONG. Overall, the median (interquartile range [IQR]) average dosing interval in the IP and MP groups was 13.8 (10.1-14.0) days and 6.9 (4.9-7.0) days, respectively. Compared with the end of B-LONG, the median (IQR) total weekly prophylactic dose was similar at the second B-YOND interim data cut (50.0 [30.0-58.3] vs 50.0 [40.0-60.0]). Median (IQR) overall ABRs were low with rFIXFc prophylaxis (Figure). Median (IQR) spontaneous ABRs were also low in each prophylaxis group: WP, 1.2 (0.0-2.7); IP, 0.6 (0.3-1.9); MP, 0.3 (0.0-1.8); and ET, 3.8 (1.2-14.6). The majority of bleeding episodes were controlled with 1-2 intravenous infusions (WP: 96.9%; IP: 96.8%; MP: 96.5%; ET: 98.8%). Conclusion: Interim data from adults and adolescents with severe hemophilia B treated with rFIXFc in B-YOND confirm the long-term safety of rFIXFc and maintenance of low ABRs with prophylactic dosing every 1-2 weeks. This research was funded by Biogen and Sobi. Biogen and Sobi reviewed and provided feedback on the abstract. The authors had full editorial control of the abstract and provided their final approval of all content. Disclosures Matsushita: CSL Behring: Honoraria, Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; KaketsuKen: Honoraria, Research Funding; JB: Honoraria, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mahlangu:Bayer: Research Funding, Speakers Bureau; Biogen: Consultancy, Research Funding, Speakers Bureau; CSL: Consultancy, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding; Amgen: Speakers Bureau; Biotest: Speakers Bureau; Baxalta: Consultancy. Shapiro:Kedrion Biopharma: Consultancy, Research Funding; Selexys: Research Funding; Octapharma: Research Funding; OPKO: Research Funding; ProMetic Life Sciences: Consultancy, Research Funding; PTC Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Biogen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CSL Behring: Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Healthcare: Research Funding; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Pasi:Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Biogen: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ragni:Baxalta: Research Funding; Genentech: Research Funding; Shire: Consultancy; Novo Nordisk: Research Funding; Biogen: Consultancy, Research Funding; CSL Behring: Research Funding; Biomarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Research Funding; SPARK: Research Funding; Tacere Benitec: Consultancy; OPKO: Research Funding; Vascular Medicine Institute: Research Funding. Ozelo:Novo Nordisk: Research Funding, Speakers Bureau; Bayer: Research Funding; Baxter: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CSL Behring: Research Funding; Biogen: Research Funding. Baker:Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: Conference travel support ; Baxter: Other: Conference travel support , Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Astellas: Research Funding; CSL Behring: Research Funding; Biogen: Membership on an entity's Board of Directors or advisory committees. Yuan:Biogen: Employment, Equity Ownership. Ramirez-Santiago:Biogen: Employment, Equity Ownership. Ferrante:Sobi: Employment. Lethagen:Sobi: Employment.

Background Direct Factor Xa (FXa) inhibitors (e.g. rivaroxaban or apixaban) are widely used oral anticoagulants and are known to interfere with dilute Russell's viper venom time (dRVVT) and aPTT-based lupus anticoagulant (LA) testing, often yielding false-positive LA results. A false positive LA result might incorrectly diagnose a patient with the anti-phospholipid antibody syndrome and lead to a wrong decision for long-term anticoagulation to prevent recurrent thrombosis. Andexanet alfa is a recombinant modified human FXa decoy protein that has been specifically designed as an antidote to neutralise the effect of the direct and indirect FXa inhibitors. Aim To determine whether the addition of andexanet alfa can overcome the interference of rivaroxaban and apixaban on LA testing in plasma samples from both LA positive and LA negative patients. Methods After written informed consent, citrated blood (20mL) was collected from 12 patients on therapeutic doses of rivaroxaban (n=6) or apixaban (n=6) with previously known LA status (3/6 LA positive in each group). Samples were double centrifuged at 3,000 x g for 10 minutes and aliquots were frozen at -40oC until analysed. Normal pooled plasma and rivaroxaban and apixaban calibrated plasma was obtained from a commercial source (Diagnostica Stago) or control plasma from healthy volunteers, prepared as above. IgG was purified by a Protein G column from a known LA positive patient and a control healthy volunteer. Rivaroxaban (Bayer) and apixaban (Bristol-Myers Squibb) tablets were crushed and dissolved in DMSO. Andexanet alfa (Portola Pharmaceuticals, Inc.) was dissolved in distilled water and aliquots were frozen at -40oC until used. Plasma samples were spiked with varying concentrations of FXa inhibitors and andexanet alfa. LA-sensitive aPTT, PT, TCT, dRVVT (Siemens Healthcare Diagnostics) and anti-FXa level (Diagnostica Stago) were performed with and without andexanet alfa on the Sysmex CS5100 analyser. Andexanet alfa (at a final concentration of 250µg/mL) was added to LA positive and LA negative plasma samples to assess the impact on the dRVVT normalised ratio. Results Andexanet alfa demonstrated a concentration-dependent increase in LA sensitive aPTT and dRVVT clotting times, with negligible impact on PT or TCT. Andexanet alfa was capable of reversing FXa inhibition in a concentration-dependent manner in plasma samples containing rivaroxaban or apixaban. When rivaroxaban is added to normal plasma it significantly prolongs the dRVVT without phospholipid more than with excess phospholipid leading to a false-positive LA ratio at final concentrations >100ng/mL. This level of rivaroxaban is in the therapeutic range. The addition of andexanet alfa reduced the measurable anti-FXa level and returned the normalised dRVVT ratio to LA negative result ( Andexanet alfa converted the false positive dRVVT rivaroxaban patient plasmas LA ratio to negative. True LA positive patients remained positive after the addition of andexanet alfa. These results were confirmed by adding LA positive and control purified IgG to known concentrations of rivaroxaban and apixaban (200ng/ml) in normal plasma. Conclusion Andexanet alfa corrects the false positive LA ratio in patients on rivaroxaban without affecting the true diagnosis in LA positive patients. Apixaban and andexanet alfa proportionately prolong the dRVVT with and without phospholipid, so the LA ratio remains negative. For this reason true LA positive patients can be diagnosed whilst on apixaban at therapeutic concentrations without the addition of andexanet alfa. Disclosures Baker: Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: Conference travel support; Astellas and CSL Behring: Research Funding; Bristol-Myers Squibb: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Daiichi Sankyo: Research Funding; Portola Pharmaceuticals: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Biogen Idec: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Roche: Other: Conference travel support. McGregor:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Equity Ownership, Other: Conference travel support; Pfizer: Other: Conference travel support; Roche: Honoraria; Abbvie: Equity Ownership; Portola Pharmaceuticals: Equity Ownership.

Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcRgamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcgammaRIIa, also down-regulates GPVI expression. Agonists that signal through FcgammaRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcgammaRIIa also underwent rapid proteolysis in platelets treated with agonists for FcgammaRIIa (VM58/14A2) or GPVI/FcRgamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcgammaRIIa also bound calmodulin and W7 induced FcgammaRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcgammaRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, micro-calpain cleaved an FcgammaRIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRgamma and FcgammaRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcgammaRIIa after immunologic insult.

Background: rVIII-SingleChain, a novel recombinant Factor VIII, has been designed as a B-domain truncated construct with a covalent bond between the heavy and light chain, aiming for a higher binding affinity to von Willebrand Factor. rVIII-SingleChain has a lower clearance, longer half-life and larger area under the curve compared to octocog alfa (Advate®). This sub-study of the AFFINITY program investigated the safety and efficacy of rVIII-SingleChain to control hemostasis in adult and adolescent patients (12 - 65 years of age) with severe Hemophilia A undergoing major surgery. rVIII-SingleChain was used either as continuous infusion or as a bolus injection. Methods: The study was approved by the relevant ethics committees and national authorities and conducted according to GCP and the Declaration of Helsinki. 13 patients underwent a total of 16 major surgical procedures (a surgical procedure that required general, spinal or regional anesthesia). Dosing was guided by the WFH recommendations. In 8 patients, rVIII-SingleChain was used as continuous infusion and in 8 patients rVIII-SingleChain was used as a bolus injection. Results: The following procedures were performed using continuous infusion: knee replacement (5), cholecystectomy, lengthening of the achilles tendon combined with surgical correction of the right toes, and open reduction and internal fixation of right ankle fracture. Furthermore, circumcision (3), extraction of wisdom teeth, abdominal hernia repair, elbow replacement, ankle arthroplasty, and hardware removal of the right ankle were performed after administering a bolus injection of rVIII-SingleChain. In the 8 procedures that were covered by bolus injection rVIII-SingleChain median pre- and intraoperative consumption was 79.2 IU/kg, and in the 8 procedures that were covered by continuous infusion, rVIII-SingleChain median pre- and intraoperative consumption was 92.5 IU/kg. Investigators rated the efficacy of rVIII-SingleChain during surgery as excellent (defined as hemostasis not clinically significant different from normal) in all cases but one (knee replacement under continuous infusion), in which it was rated as good (defined as hemostasis normal or mildly abnormal in terms of quantity and/or quality e.g., slight oozing). After the procedure, patients returned to routine treatment after a median of 9 days. No related AEs or SAEs were observed during the surgery period. Conclusion: rVIII-SingleChain provides very effective and safe control of hemostasis during a wide range of surgical procedures when dosed either by continuous infusion or by bolus injection. Consumption of factor on the day of surgery seems to be comparable in both treatment regimens. Disclosures Mahlangu: NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Baker:Daiichi Sankyo: Research Funding; CSL Behring: Research Funding; Portola Pharmaceuticals: Research Funding; Astellas: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: conference travel support, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support , Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: conference travel support. Leissinger:Biogen: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Santagostino:Pfizer: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Biotest: Speakers Bureau; Octapharma: Speakers Bureau; Kedrion: Speakers Bureau; Biogen/Sobi: Membership on an entity's Board of Directors or advisory committees. Bensen-Kennedy:CSL Behring: Employment. St. Ledger:CSL Behring: Employment. Veldman:CSL Behring: Employment. Pabinger:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

Background: The phase 3 B-LONG study demonstrated the safety, efficacy, and pharmacokinetics of recombinant factor IX Fc fusion protein (rFIXFc) for the prevention and treatment of bleeding episodes in subjects with severe hemophilia B. For people with hemophilia, frequent bleeding events occur in target joints, which are a known precursor to hemophilic arthropathy (chronic joint disease). There is currently limited information available on the outcomes of prophylaxis in subjects with target joints who have severe hemophilia B. Aims: To assess the frequency of bleeding events and the dosing of rFIXFc in subjects who had ≥1 target joint at baseline in the B-LONG study. Methods: B-LONG was a Phase 3, multicenter, open-label study that enrolled males aged ≥12 years with severe hemophilia B (≤2 IU/dL endogenous FIX activity) who had received prior treatment with FIX. Subjects in B-LONG were enrolled into 1 of 4 arms: Arm 1, weekly prophylaxis; Arm 2, individualized interval prophylaxis; Arm 3, episodic treatment; or Arm 4, perioperative management. For the current post-hoc analysis, subjects with ≥1 target joint at baseline (per protocol, a target joint was defined as a major joint with ≥3 bleeding episodes in a 3-month period) and who received on-study rFIXFc prophylaxis (Arms 1 and 2 only) were included. On-study per subject target joint ABR (annualized number of bleeding episodes per year in all target joints combined), including overall, spontaneous, and traumatic target joint ABR, were assessed. In subjects with available prestudy and on-study ABR data, prestudy 12-month bleeding rates were compared with on-study overall ABR. Results: Forty-three subjects from the prophylaxis arms had target joints at entry into B-LONG and data collected during the efficacy period of the study (weekly prophylaxis, n = 35; individualized interval prophylaxis, n = 8). Of these, 40 subjects had both prestudy and on-study bleeding information available. Regardless of prestudy treatment regimen (prophylactic or episodic rFIX), on-study median ABRs were low among subjects with target joints at baseline compared with prestudy ABRs (Fig. 1). The on-study overall target joint, spontaneous target joint, and traumatic target joint median (interquartile range, [IQR]) ABRs were low for subjects in the weekly prophylaxis arm (1.03 [0.00, 3.07], 0.00 [0.00, 1.10], and 0.00 [0.00, 1.11], respectively) and for subjects in the individualized interval prophylaxis arm (2.20 [0.00, 3.75], 2.20 [0.00, 3.75], and 0.00 [0.00, 0.00], respectively; Fig. 2). A total of 17 (48.6%) subjects receiving weekly prophylaxis and 3 (37.5%) subjects receiving individualized interval prophylaxis had no target joint bleeding episodes on-study. The average weekly prophylactic rFIXFc dose for subjects with target joints at baseline was (median [IQR]) 46.26 (37.98, 54.55) IU/kg and 69.48 (57.28, 77.45) IU/kg for those receiving weekly prophylaxis and individualized interval prophylaxis, respectively. The median (IQR) average on-study dosing intervals for these groups were 6.98 (6.88, 7.00) days and 10.25 (9.45, 12.72) days, respectively. Among subjects with target joints at baseline who received prestudy rFIX and on-study rFIXFc prophylaxis (n = 6, weekly prophylaxis arm only; no subjects from the individualized interval prophylaxis arm who met these criteria had available data), the on-study median (IQR) average weekly prophylactic dose of 50.61 (39.61, 60.61) IU/kg with rFIXFc was lower than the prestudy median (IQR) average weekly prophylactic dose of 111.28 (95.56, 116.76) IU/kg with rFIX. Additionally, the on-study median (IQR) dosing interval (6.92 [6.79, 6.97]) with rFIXFc prophylaxis was longer than the pre-study median (IQR) dosing interval among these 6 subjects (3.50 [2.33, 3.50] days). Summary/Conclusion: For subjects in the current analysis with severe hemophilia B who entered B-LONG with target joints, rFIXFc prophylaxis was effective for reducing the frequency of bleeding episodes overall and in target joints. Furthermore, in subjects who received prestudy and on-study prophylaxis, rFIXFc was associated with reduced consumption and prolonged dosing intervals compared with conventional prestudy rFIX products. These results suggest that target joints can be effectively managed and controlled with rFIXFc dosed prophylactically every 1 to 2 weeks. Disclosures Shapiro: Baxalta, Novo Nordisk, Biogen, ProMetic Life Sciences, and Kedrion Biopharma: Consultancy; Biogen: Speakers Bureau; Bayer Healthcare, Baxalta, Biogen, CSL Behring, Daiichi Sankyo, Kedrion Biopharma, Octapharma, OPKO, ProMetic Life Sciences, PTC Therapeutics, and Selexys: Research Funding; Baxalta, Novo Nordisk, Biogen,: Membership on an entity's Board of Directors or advisory committees. Perry:Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biogen: Consultancy, Honoraria. Baker:Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: conference travel support, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Portola Pharmaceuticals: Research Funding; Astellas: Research Funding; CSL Behring: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Bristol- Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: conference travel support. Tsao:Biogen: Employment, Equity Ownership. Mei:Biogen: Employment, Equity Ownership.

Background : The phase 3 A-LONG and Kids A-LONG studies demonstrated low annualized bleeding rates (ABRs) with reduced infusion frequency rFVIIIFc prophylaxis 1-2 times/week in adults, adolescents, and children with severe hemophilia A. Median prestudy and on-study total weekly prophylactic factor consumption was comparable for subjects previously receiving FVIII prophylaxis. Aims: To evaluate the effect of rFVIIIFc on subjects' physical activity across age groups using a subject-reported assessment and examine ABRs according to change in physical activity and prestudy treatment regimen (prophylaxis or episodic) in A-LONG and Kids A-LONG. Methods : Previously treated males with severe haemophilia A (

Background: Prophylactic replacement of coagulation factor IX (FIX) in patients with hemophilia B improves clinical outcomes; however, the short half-life of conventional FIX products often necessitates frequent intravenous infusions (2-3 times/week). Recombinant FIX Fc fusion protein (rFIXFc), which is produced in a human cell line, binds the neonatal Fc receptor and utilizes the natural IgG recycling pathway to prolong the half-life of FIX. Introduction : The phase 3 B-LONG study (NCT01027364) demonstrated the safety, efficacy and prolonged half-life of recombinant factor IX Fc fusion protein (rFIXFc) in adults and adolescents (aged ≥12 years) with hemophilia B. Here, we report interim safety and efficacy data for adults and adolescents enrolled in the ongoing rFIXFc extension study, B-YOND (NCT01425723). Methods : Upon completing B-LONG, eligible subjects could participate in one of 4 treatment groups in B-YOND: weekly prophylaxis (WP; 20 to 100 IU/kg every 7 days), individualized prophylaxis (IP: 100 IU/kg every 8 to 16 days), modified prophylaxis (MP; for subjects in whom optimal prophylactic dosing could not be achieved with either individualized or weekly prophylaxis), or episodic treatment. Subjects could change treatment groups at the start of or at any time during the study; for efficacy analyses, data were summarized according to the treatment group in which they participated, for the time period they were in that treatment group. The primary endpoint was development of inhibitors (a positive result was defined as a neutralizing antibody value ≥0.6 BU/mL as measured by the Nijmegen-modified Bethesda assay at a central laboratory, confirmed upon retesting within 2 to 4 weeks). Secondary outcomes included incidence of adverse events (AEs), annualized bleeding rate (ABR), and rFIXFc exposure days (EDs). Post hoc analyses of subjects' prophylactic dose and dosing interval were performed to evaluate longitudinal changes in prophylactic regimens. Results: Of the 115 subjects who completed B-LONG, 93 (81%) enrolled in B-YOND. As of the interim data cut (17 October 2014), 18 subjects had completed, 7 had discontinued, and 68 remained on study (median time on study: 27.6 [range: 3.2-32.7] months). From the start of B-LONG to the B-YOND interim data cut, subjects had a median of 39.5 (range: 8.1-52.6) months treatment with rFIXFc; 68 subjects (73%) had ≥100 cumulative rFIXFc EDs. As of the B-YOND interim data cut, no inhibitors were reported (n=93 subjects with at least 1 evaluable inhibitor test result). During B-YOND, AEs were generally typical of the hemophilia B population; 76% of subjects reported at least 1 AE and 23% reported at least 1 serious AE (SAE) on study. No subject discontinued due to an AE. 4 subjects each experienced an AE assessed by the investigator as related to rFIXFc treatment, 1 serious and 3 nonserious; all 4 AEs resolved, and none led to study discontinuation. There were no reports of serious hypersensitivity reactions and no vascular thrombotic events associated with rFIXFc. During B-YOND, most subjects remained in the same treatment group in which they had participated during B-LONG; 29/90 subjects (32%) from the B-LONG prophylactic and episodic treatment arms changed treatment groups, including 9/19 subjects who changed from episodic to prophylactic treatment (1 subject changed back to episodic treatment before the interim data cut). Among subjects treated prophylactically in B-LONG (n=71), 78% had either no change to or decreased their total weekly prophylactic dose during B-YOND; 89% maintained or lengthened their prophylactic infusion interval during B-YOND. The median (IQR) average weekly prophylactic dose was 49.5 (39.9, 62.8) IU/kg, 50.2 (48.2, 61.5) IU/kg, and 61.7 (41.5, 81.8) IU/kg in the WP, IP, and MP groups, respectively. The median (IQR) average dosing intervals in the IP and MP groups were 13.7 (10.1, 14.0) days and 6.9 (4.9, 7.0) days, respectively. Median ABRs were low with rFIXFc prophylaxis (Fig. 1). In all prophylaxis groups, the median ABR was Conclusion: Interim data from B-YOND in adults and adolescents confirm the long-term safety of rFIXFc and the maintenance of a low ABR with prophylactic dosing every 1 to 2 weeks. Figure 1. Figure 1. Disclosures Mahlangu: Amgen: Speakers Bureau; Bayer, CSL, Novo Nordisk, and Biogen: Research Funding, Speakers Bureau; Biotest: Speakers Bureau; Roche: Research Funding. Shapiro:Biogen: Speakers Bureau; Baxalta, Novo Nordisk, Biogen,: Membership on an entity's Board of Directors or advisory committees; Baxalta, Novo Nordisk, Biogen, ProMetic Life Sciences, and Kedrion Biopharma: Consultancy; Bayer Healthcare, Baxalta, Biogen, CSL Behring, Daiichi Sankyo, Kedrion Biopharma, Octapharma, OPKO, ProMetic Life Sciences, PTC Therapeutics, and Selexys: Research Funding. Pasi:Biogen, Octapharma, Genzyme, and Pfizer: Consultancy, Honoraria; Octapharma: Research Funding. Ragni:Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Biomarin: Research Funding; SPARK: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Biogen: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Vascular Medicine Institute: Research Funding; Dimension: Research Funding; Genentech Roche: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ozelo:Biogen: Research Funding; Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau. Oldenburg:SOBI: Consultancy. Matsushita:Biogen, Novo Nordisk, Baxalta, Bayer, and Octapharma: Membership on an entity's Board of Directors or advisory committees; Biogen, Novo Nordisk, Baxalta, CSL Behring, Bayer, Chugai, KaketsuKen, JB, and Octapharma: Honoraria; Biogen, Novo Nordisk, Baxalta, CSL Behring, Bayer, Chugai, KaketsuKen, and JB: Research Funding. Baker:Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: conference travel support, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: conference travel support; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: conference travel support, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Portola Pharmaceuticals: Research Funding; Astellas: Research Funding; CSL Behring: Research Funding. Yuan:Biogen: Employment, Equity Ownership. Ramirez-Santiago:Biogen: Employment, Equity Ownership. Pierce:Biogen: Equity Ownership, Other: Former employee. Allen:Biogen: Employment, Equity Ownership. Mei:Biogen: Employment, Equity Ownership.

Background: Uncontrolled complement activation has a major role in the pathogenesis of atypical HUS (aHUS) and the restraint of this process by eculizumab is life saving. However, the evidence of complement dysregulation in the pathogenesis of Thrombotic Thrombocytopenic Purpura (TTP) is still unclear. In this study we examined the presence of complement activation biomarkers in patients with aHUS and TTP and the levels were compared to normal healthy controls . Patients and Methods: Patients with thrombotic microangiopathic thrombocytopenia diagnosed either as TTP with low ADAMTS13 activity less than 10% or aHUS with impaired renal function, Cr> 2mg/dL and normal ADAMTS13 activity were chosen from the Korean TTP registry from February 2012 to June 2014. Prospective plasma and serum samples prior to intervention were collected from newly diagnosed patients with TTP (n=20), aHUS (n=20), and 20 healthy controls and frozen at -700C. Complement activation products (C3a, Bb as alternative pathway; C4d as classic pathway; C5a, C5b-9; terminal pathway) were measured by ELISA. Results: Significantly increased levels of Bb and C5b-9 were observed in TTP (median [range], ng/mL; Bb, 1220 [540.0 – 16560], p=0.048; C5b - 9, 390.1 [238.5 - 938.7], p Conclusion: Complement biomarkers are activated to a similar level in both newly diagnosed cases of TTP and aHUS. Complement activation product levels did not differentiate aHUS from TTP. Disclosures No relevant conflicts of interest to declare.

The anticoagulant Protein S (PS) is coded for by the PROS1 gene and serves as a co-factor to APC in the inactivation of FVa and FVIIIa. It is produced predominately by the liver. Low PS levels are associated with an increased thrombotic risk. Clinically it is well documented that circulating PS levels are reduced in response to heightened estrogen (E2) levels (oral contraceptive use, pregnancy, etc.). However, an explanation as to how this is occurring at the molecular level has not yet been elucidated. Two approaches have been utilized. Firstly, a liver carcinoma cell line, HepG2, was exposed to 5 and 10nM concentrations of testosterone (T) and E2 for 6, 12 and 24h at which point’s total RNA was extracted and quantitated using real-time PCR. Secondly, a reporter plasmid pQH5′[2]DsRed2 was constructed that has 948bp of the PROS1 promoter region (up to but not including the ATG start codon) fused to an enhanced green fluorescent protein (EGFP) reporter gene sequence. A second reporter gene, DsRed2, was incorporated into pQH5′[2]DsRed2, and is constitutively expressed by a cytomegalovirus (CMV) promoter. This provides a means of standardizing any change in expression of the EGFP reporter. pQH5′[2]DsRed2 was transiently transfected into a breast cell carcinoma line MCF7 [chosen for its high levels of oestrogen receptor alpha (ERα)] and subsequently exposed to 5 and 10nM concentrations of E2 and T for 24h. Expression levels were assessed using a COULTER EPICS flow cytometer. The HepG2 6, 12 and 24h PROS1 mRNA hormone response experiment showed an erratic response to E2. However there were consistently higher levels of PROS1 mRNA over time in response to T compared to the vehicle alone. Higher levels still were evident for the 10nM concentration. The transient transfection of pQH5′[2]DsRed2 into MCF7s followed by a 24h exposure of either T or E2 at 5 and 10nM concentrations, showed a significant change in response to both the 5 and 10nM concentrations of E2, but little response to either of the T concentrations These results suggest that E2 is exerting a direct effect on the promoter of PROS1, down regulating transcription, reflecting clinical observations. However, T may be affecting PROS1 mRNA stability via a yet undetermined mechanism. Therefore, hormonal regulation of PS may be occurring at several levels of the overall production process.