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The increasing need for red blood cells (RBCs) together with the lack of donors have made the in vitro production of RBCs a major medical challenge.[1][1] We have recently developed a method to produce mature RBCs in vitro , starting from bone marrow, peripheral blood, leukapheresis or cord blood

Hereditary combined vitamin K–dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wild-type carboxylase, and their compound heterozygosity can therefore account for functional VKD factor deficiency. The implications for carboxylase mechanism are discussed.

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34+ cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.

Contains fulltext : 96181.pdf (Publisher’s version ) (Open Access) Emerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry-based metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases.

Abstract 1302 Poster Board I-324 Combined vitamin K-dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase (GGCX) which carboxylates VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and due to mutations within either gene. Of note some mutations within the GGCX gene have recently been found associated with the pseudoxanthoma elasticum (PXE) syndrome, suggesting a role for GGCX in skin development. We report a new case of combined VKD coagulation factor deficiency resulting from two mutations in the GGCX gene, and the first identified in a French child, who exhibited impaired function in hemostatic VKD factors. The propositus exhibited bleeding at sites of venipuncture at birth, and at the age of 3 months was admitted in emergency for spontaneous multiple hematomas of the chest and thighs; recently, at the age of 1 year, he exhibited a hematoma of the wrist following a casual fall. Coagulation and VKD factors were low [PT (Control/patient): 12.8/>100 sec; II : 3 % ; VII :2% ; X :3%] while the non-VKD factor V was normal (105%). Vitamin K infusion corrected the bleeding tendency as well as coagulation paramenters [PT (Control/patient): 12.8/14.8 sec; II :72% ; VII+X : 62%; factor V remained normal at 102%]. Family analysis revealed that both parents and one brother were unaffected, both clinically and biologically, but that a brother had died of an unexplained abdominal hemorrhage, in the neonatal period. Taken together the clinical family history is consistent with a recessive trait. No sign of PXE was found in the family. DNA sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but revealed two new heterozygous mutations in the carboxylase gene: a G10233T transversion in exon 11 (G1565U in mRNA) that caused an W493C substitution and a C12078T transition in the last exon (15) (C2196U in mRNA) that caused a premature R704stop, presumably deleting the last 55 C-terminal amino acids. W493 is a highly conserved amino acid and its homozygous mutation (for S493) has recently been reported in a case of VKD coagulation factor deficiency associated with PXE. R704 mutation has never been reported previously. Family analysis showed that W493C was transmitted by the father, R704stop by the mother, that the deceased brother carried both mutations, while the unaffected brother carried neither. Mutational analysis was carried out by site-directed mutagenesis of the GGCX cDNA subsequently subcloned into the BacPak8 baculovirus-based vector, and GGCX was expressed in the virally-infected SF21 insect cell line. Protein expression level of the enzyme was assessed by western blotting, and enzymatic activity was evaluated by measuring the incorporation of [14]C-CO2 within the Boc-Glu-Glu-Leu-OMe peptide. No difference in expression level was detected for either mutant protein compared to the wild type protein. However, while no difference in molecular weight was seen between the W493C mutant and wild type on wertern blotting, the R704stop mutation generated a shorter form of carboxylase than wild type, consistent with the predicted shorter reading frame. W493C mutant exhibited a much lower activity than wild type, suggesting a functional role for W493. The activity of the R704stop mutant is currently being assessed, as well as the activity of the coexpressed mutants. Since PXE has been correlated with a W493S mutation, a role for the W493C mutation in future development of PXE in this young patient will be assessed during follow-up. Disclosures No relevant conflicts of interest to declare.

Combined deficiency in vitamin K-dependent (VKD) coagulation factors is an autosomal recessive bleeding disorder associated with defects in either the VKD carboxylase which converts Glus to Glas in VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such defects are rare, and we now report the fourth case of deficiency caused by mutations in the carboxylase gene. The mutations were identified in a two year old Tunisian girl who exhibited impaired function in several VKD procoagulant and anticoagulant factors that was not restored by vitamin K administration. Sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but, remarkably, revealed three heterozygous mutations in the carboxylase gene, D31N, W157R and T591K within exons 2, 4 and 13, respectively. None of these mutations have previously been reported. Family analysis showed that D31N and T591K were coallelic and transmitted by the mother while W157R was transmitted by the father. The mutations were not found in the genomes of 200 normal subjects, ruling out frequent polymorphisms. Mutational analysis indicated wild type activity for the D31N carboxylase. In contrast, the W157R and T591K enzymes had activities that were, respectively, 7% and 0% that of wild type carboxylase, and their compound heterozygosity can therefore account for defective carboxylation. Residues 157 and 591 are both highly evolutionarily conserved, and residue 157 lies within a region previously suggested to be important for carboxylase binding to VKD Glus or propeptide. However, the hydrophobic nature of this region and inability of vitamin K administration to restore VKD protein function alternatively suggests that residue 157 may be important for vitamin K binding.