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.