Coagulation factor Va (fVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, fVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the fVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of fVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here we report the cryo-EM structure of the fVa-APC complex at 3.15 Å resolution where the protease domain of APC engages R506 in the A2 domain of fVa mainly through electrostatic interactions between positively charged residues in the 30- and 70- loops of APC and an electronegative surface of fVa. The auxiliary Gla and EGF domains of APC are highly dynamic and point to solvent, without making contacts with fVa. Binding of APC displaces a large portion of the A2 domain of fVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch", or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the fVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.

Sickle cell hemoglobin-C (HbSC) disease results from compound heterozygosity of hemoglobin-S (HbS) and hemoglobin-C (HbC), comprising 30% of sickle cell disease (SCD). HbC induces RBC dehydration/xerocytosis, which promotes sickling. HbSC-SCD causes significant morbidity despite being milder than homozygous HbSS-SCD. Current research/treatment strategies have focused on HbSS-SCD, while HbSC patients are deprived of disease-modifying/transformative therapies due to lack of preclinical models. We generated HbSC mice, which resemble human HbSC-SCD: HbSC erythrocytes showed marked xerocytosis. Anemia, hemolysis, inflammation and organ damage were milder than HbSS mice, but hypoxia/reperfusion injury was similar. Retinopathy developed at higher frequency than HbSS mice (66.7% versus 16.7%; p<0.05), like HbSC-SCD patients. While HbSC RBCs sickled at lower pO2 than HbSS RBCs (43mmHg versus 24mmHg; P<0.0001), they did not completely recover deformability after hypoxia-reoxygenation. Using the HbSC mice developed herein, we studied the mechanism by which hydroxyurea causes significant clinical benefit in HbSC-SCD patients, despite minimal/modest increases in fetal-hemoglobin (HbF). We found hydroxyurea had distinct non-HbF and HbF effects. Hydroxyurea did not increase HbF in adult HbSC/HbSS mice, but reduced RBC ROS, Ferryl-hemoglobin, Heinz-body formation, thereby reducing membrane damage; however, RBC hydration was unaffected. When given to unborn pups before they switch off g-globin expression and continued postnatally, we could induce HbF in both HbSC/HbSS mice (higher HbF in HbSS versus HbSC mice). Minimal increases in HbF (~1%) improved HbSC RBC hydration. Peak HbF levels of 7% in HbSC mice abrogated sickling. Overall, this HbSC model will help bridge the knowledge-gap in mechanistic/therapeutic studies in this neglected disease.

Heavy menstrual bleeding (HMB) is a widespread occurrence among women of reproductive age and inflicts a substantial impact on well-being and healthcare expenses. To better characterize the genetic architecture of HMB, we meta-analyzed GWAS summary statistics from five biobanks, involving up to 84,633 HMB cases and 598,195 controls from several ancestries. Out of 21 signals significantly associated with HMB in a discovery GWAS meta-analysis combining four biobanks, 20 had a concordant direction of effect in the remaining cohort, including 10 significantly replicated. By combining the discovery and replication datasets, 15 additional signals were identified in subsequent meta-analyses. These genetic analyses resulted in 36 signals (33 novel) significantly associated with HMB, and subsequent gene prioritization techniques (e.g., TWAS, PoPS) revealed likely causal genes. Notable discoveries included the strong protective effect of the F5-Leiden variant (rs6025-T, OR=0.75, P=6.8×10-33), variants at the FSHB and LHB/CGB loci, both involved in hormone production regulation, and several signals near genes involved in the Wnt/ß-catenin signaling pathway. We also observed strong and significant genetic correlations with disorders of the female genital tract, including uterine fibroids, endometriosis or ovarian cysts. Overall, we identified 33 novel genetic loci associated with HMB, significantly improving our understanding of the genetic etiology of this condition, which may provide new targets for the development of therapeutic strategies.

Chronic graft-versus-host disease (cGVHD) is characterized by a dysregulation of the adaptive immune system including an aberrant B cell homeostasis after allogeneic hematopoietic stem cell transplantation (allo-SCT). It is uncertain, however, whether this B cell dysregulation is a result of manifest cGVHD or develops as a sign of aberrant B lymphopoiesis after allo-SCT before cGVHD becomes apparent. To gain insight into the development of B cell dysregulation before the onset of cGVHD, we analyzed B cell subpopulations by multiparameter flow cytometry on day 90, day 180 and day 356 post allo-SCT in a prospective study design. After completion of follow-up, patients were assigned retrospectively to three groups according to onset of GVHD: 1) no GVHD (n=17), 2) acute GVHD (aGVHD) without subsequent cGVHD (n=32) and 3) cGVHD (n=59). Whereas CD21low CD11c+ B cells were increased in all groups, the frequency of CD20neg CD38hi plasmablasts was significantly elevated already 90 days after allo-SCT in those patients subsequently developing cGVHD, as compared to patients without GVHD or aGVHD only (median 5.9% vs 2.2% vs 2.2% of CD19+ cells; p=0.0016 and p=0.0304, respectively). Detailed molecular analysis of expanded plasmablasts revealed a dominance of the IgA isotype with molecular evidence for recent generation in mucosal sites and markers for intestinal homing. A large fraction of the clonally expanded plasmablasts produced antibodies binding to subgroups of commensals which are known to produce short-chain fatty acids. In summary, our data suggests that dysregulated intestinal antibody responses against commensals contribute to the pathophysiology of cGVHD.

A recent perspective suggested that prior evidence from over a decade ago established that a liberal rather than a restrictive blood transfusion strategy results in better outcomes in patients with anemia and either acute myocardial infarction or stable cardiovascular disease. Their premise was that physiological evidence, and a different interpretation of the TRICC trial should have been sufficient to establish clinical practice. They also suggest that a more personalized approach to the administration of transfusions would have been made possible by including a usual care arm in all transfusion trials. In this Counter-Point perspective, we describe how and why two discrete and common blood transfusion thresholds were selected in the TRICC, FOCUS, REALITY, and MINT trials. We explain why a usual-care-arm would have been uninformative. We also propose that we still do not have evidence to provide firm transfusion recommendations in several specific subpopulations of patients including those with stable atherosclerotic coronary artery disease. Finally, we provide our perspective on the state of existing evidence and on the clinical recommendations that should be adopted in practice.

No prospective study has evaluated the incidence of TA-TMA in adult allogeneic hematopoietic cell transplant (HCT) recipients. The MIDAS (MIcroangiopathy, endothelial Damage in Adults undergoing Stem cell transplantation) consortium conducted the first multi-center study to prospectively screen for TA-TMA. Longitudinal blood samples and detailed clinical data were collected weekly through day +100 and at months 5, 6, 9 and 12 in first allogeneic HCT recipients at three sites (Ohio State University, Moffitt and Roswell Park Comprehensive Cancer Centers). Adjudication of TA-TMA diagnosis was reviewed in real time by three blinded independent reviewers. Incidence of TA-TMA was scored using 6 published criteria: Harmonization, Jodele, Li, BMT-CTN, IWG and Cho, as well as the center-reported diagnosis and MIDAS adjudication categorized as no TMA, non-severe, or severe TA-TMA. Incidence of severe TA-TMA by day +100 was similar across the three centers at 21.8%, with a median time to onset of 14.5 days in 239 patients. Incidence of non-relapse mortality was 42% in severe compared to 8.4% in non-severe and 5.8% in no TMA groups (p<0.001). Rise in serum creatinine as early as day +7 and occurrence of hypertension by day+14 post-HCT were early indicators of severe TA-TMA. Our prospective study of systematic screening for TA-TMA identifies a higher incidence of a clinically impactful phenotype of TA-TMA than previously reported in adult HCT recipients. Our natural history study provides an essential foundation for urgently needed studies of prophylaxis and treatment of TA-TMA in adults and suggests clinical value in our inexpensive screening strategy.

Orchestrating key homeostatic functions, mitochondria likely entail cancer vulnerabilities. Moreover, due to their bacterial ancestry they can release potent immunogenic signals. Here we show that the mitochondrial protease ClpP is both a cell-intrinsic metabolic vulnerability and an actionable immunogenic trigger in multiple myeloma (MM). We found that ClpP mRNA is higher in bone marrow (BM)-purified malignant plasma cells (PC) than in normal or premalignant counterparts and that MM lines rank first for ClpP expression among human cancers. Moreover, we demonstrated that human MM cells are highly vulnerable to ClpP inhibition in vitro and in vivo. Surprisingly, MM cell dependence on ClpP was not accounted for by its acknowledged oxidative phosphorylation surveillance activity. Proteomic discovery of proteolytic targets, metabolomics, and metabolic tracing identified a critical control exerted by ClpP on ornithine aminotransferase abundance to sustain cytosolic biosynthesis of polyamines, essential to MM cells. Transcriptomics and targeted validation also revealed activation of a cyclic GMP-AMP synthase (cGAS)-dependent type-I interferon (IFN-I) response in ClpP-silenced MM cells, whose supernatants boosted dendritic cell activation and ability to stimulate IFNg production by T cells. In vivo, ClpP silencing re-shaped the BM immune environment in immunocompetent mice, significantly expanding IFNg-producing CD4+ and CD8+ T cells and CD4+ T memory cells, while containing exhausted CD4+ T cells and myeloid derived suppressor cells. Thus, ClpP is a novel addiction of MM cells, whose inhibition not only exerts cell-intrinsic toxicity, but also triggers otherwise indolent anti-tumoral immunity. Our findings yield a novel immunogenic chemotherapeutic framework of potential relevance against myeloma.

Previous reports have highlighted that some low VWF patients with significant bleeding were diagnosed based upon an isolated but persistent reduction in plasma VWF activity levels in the 30-50 IU/dL range. These patients had plasma VWF:Ag levels > 50 IU/dL and thus had 'qualitative' rather than 'quantitative' low VWF. Although the clinical importance of functional VWF defects in type 2 VWD is well recognized, the translational implications of mild functional defects in patients with qualitative low VWF (low VWF-QL) have not been defined. To address this clinically important question, we combined low VWF datasets from the low VWF in Ireland cohort and the low VWF in Erasmus MC studies. Overall, we observed that low VWF-QL was common and accounted for approximately 50% of our combined low VWF cohort. Importantly, our findings demonstrate that many of these patients with mild isolated functional VWF defects in the 30-50 IU/dl range had significant bleeding phenotypes, even though their plasma VWF:Ag levels were within the normal range. In addition, we further show that low VWF-QL is a distinct clinic-pathological entity compared to type 2 VWD. Finally, our studies highlight that low VWF-QL is predominantly due to abnormalities in VWF biosynthesis within endothelial cells that are occurring largely independent of identifiable pathological VWF sequence variants. Cumulatively, these novel observations have important clinical implications for the diagnosis and management of patients with mild functional VWF defects.

Modakafusp alfa is a first-in-class immunocytokine directing interferon-α to CD38-positive cells. Our previous phase 1/2 trial identified two potential phase 2 doses of modakafusp alfa in patients with relapsed/refractory multiple myeloma (RRMM): 1.5 or 3 mg/kg every 4 weeks (Q4W). The overall response rate (ORR) among 30 patients treated at 1.5 mg/kg was 43%. This phase 2 dose optimization study (NCT03215030) randomized 147 patients with triple-class refractory disease and ≥3 prior lines of therapy 1:1 to modakafusp alfa 120 mg (n = 71) or 240 mg (n = 75) Q4W (fixed-dose equivalents of 1.5 and 3 mg/kg Q4W). Patients had received a median of 6 prior lines of therapy; 66% were penta-exposed and 45% anti-BCMA-exposed. Modakafusp alfa development was discontinued for strategic reasons by the sponsor and the study was terminated early. At median follow-ups of 7.3 and 7.6 months in the 120 and 240 mg arms, ORRs were 32% and 41%; median progression-free survival was 4.1 and 5.3 months, respectively. ORRs were higher in patients who had not received prior-BCMA therapy (46% vs 29%). The most common treatment-related adverse events (TEAEs) in the 120 and 240 mg arms were thrombocytopenia (75% and 84%; grade ≥3, 55% and 61%) and neutropenia (68% and 73%; grade ≥3, 56% and 68%); 90% and 96% of patients experienced grade ≥3 TEAEs; 39% and 44% experienced serious TEAEs. Pharmacokinetic studies showed a ~2-fold greater exposure with 240 vs 120 mg. Our results confirm the efficacy of single-agent modakafusp alfa in patients with RRMM.