Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocellular bone marrow. In 40% of DBA patients, various physical anomalies are also present. Currently two genes are associated with the DBA phenotype--the ribosomal protein (RP) S19 mutated in 25% of DBA patients and RPS24 mutated in approximately 1.4% of DBA patients. Here we report the identification of a mutation in yet another ribosomal protein, RPS17. The mutation affects the translation initiation start codon, changing T to G (c.2T>G), thus eliminating the natural start of RPS17 protein biosynthesis. RNA analysis revealed that the mutated allele was expressed, and the next downstream start codon located at position +158 should give rise to a short peptide of only four amino acids (Met-Ser-Arg-Ile). The mutation arose de novo, since all healthy family members carry the wild-type alleles. The identification of a mutation in the third RP of the small ribosomal subunit in DBA patients further supports the theory that impaired translation may be the main cause of DBA pathogenesis.

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia that is usually diagnosed during early infancy. Apart from defects in red blood cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 are found in 25% of patients, while mutations in other proteins of the small ribosomal subunit-RPS17 and RPS24-have been found in a fraction of patients. Recently, mutations in RPL5, RPL11, and RPL35a of the large ribosomal subunit have also been reported in several DBA patients. Here, we present the identification of mutations in the RPL5 and RPL11 genes in patients from the Czech DBA Registry. Mutations in RPL5 were identified in eight patients from 6 out of 28 families (21.4%), and mutations in RPL11 in two patients from 2 out of 28 families (7.1%). Interestingly, all 10 patients with either an RPL5 or RPL11 mutation exhibited one or more physical anomalies; specifically, thumb anomalies (flat thenar) were always present, while no such anomaly was observed in seven patients with an RPS19 mutation. Moreover, 9 out of 10 patients with either an RPL5 or RPL11 mutation were born small for gestational age (SGA) compared to 3 out of 7 patients from the RPS19-mutated group. These observations may suggest that mutations, at least in RPL5, seem to generally have more profound impact on fetal development than mutations in RPS19. Since RPL5 and RPL11, together with RPL23, are also involved in the MDM2-mediated p53 pathway regulation, we also screened the RPL23 gene for mutations; however, no mutations were identified.

To cite this article: Handrkova H, Schroeder V, Kohler HP. The activation peptide of coagulation factor XIII is vital for its expression and stability. J Thromb Haemost 2015; 13: 1449-58.Summary. Background: The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. Objective: To investigate the role of AP-FXIII in the expression and stability of FXIII. Methods: We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. Results: Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence 8 FGGR 12R plays a substantial role in intersubunit interactions in FXIII-A 2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. Conclusions: We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A 2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A 2 dimer.

Coagulation factor XIII (FXIII) circulates in plasma as a tetramer of two zymogen A-subunits and two carrier B-subunits. Activation of plasma FXIII is initiated by proteolytic cleavage of the peptide bond Arg37-Gly38 by thrombin, followed by conformational changes, subunit dissociation and exposure of the active site. We have previously shown that the activation peptide of FXIII (AP-FXIII), consisting of the 37 N-terminal amino acids of the FXIII A-subunit, is released into plasma upon cleavage by thrombin (Schroeder et al, 2007;Ortner et al, 2010), and we were able to detect in vivo-generated AP-FXIII in patients with acute stroke . The aim of the current study was to investigate for the first time whether free AP-FXIII has any effects on FXIII function.We used synthetic peptides with the amino acid sequence of wildtype AP-FXIIIVal34 (SETSRTAFGGRRAVPPNNSNAA EDDLPTVELQGVVPR) and the common variant AP-FXIIILeu34. As negative controls we used three different scrambled peptides (Scram1, Scram2, Scram3) of the same amino acid composition as in AP-FXIIIVal34 but in random order.FXIII activity was measured using the biotin incorporation assay (Kohler et al, 1998). We loaded normal citrated plasma (NCP) onto fibrinogen-coated microtitre plates, followed by AP-FXIII or Scram (final concentrations 1-100 lg/ml). After incubation with an activation mix containing thrombin, CaCl 2 , dithiothreitol, and biotinamidopentylamine, the reaction was stopped at different time points with EDTA. Incorporated biotin was detected with streptavidine/alkalinephosphatase and p-nitrophenylphosphate. As shown exemplary for AP-FXIIIVal34 and Scram1 in Fig 1A, the addition of AP-FXIII reduced FXIII activity in a dose-dependent manner, whereas Scram had no effect. When we pre-activated FXIII in the absence of any peptide and then added pre-activated FXIII (FXIIIa) and AP-FXIII into the incorporation reaction, AP-FXIII had no effect (not shown).We also used a fluorogenic FXIIIa isopeptidase assay (Dodt et al, 2013). In this assay, coagulation is activated with tissue factor and phospholipids, and generation of FXIIIa is measured by its isopeptidase activity towards a fluorogenic substrate. Again, addition of AP-FXIII, but not Scram, reduced FXIIIa generation (Fig 1B). AP-FXIII reduced the area under the FXIIIa generation curve and peak FXIIIa concentration in a dose-dependent manner and prolonged the time-to-peak.We investigated the effect of AP-FXIII on fibrin crosslinking by gel electrophoresis. As shown in Fig 2A, c-c-chain crosslinks appeared within the first few minutes in the control samples without any peptide and in the presence of Scram. In the presence of AP-FXIII, however, appearance of c-c-chain crosslinks was clearly delayed. Similarly, after 30 min, highmolecular-weight a-chain crosslinks were clearly visible in the samples without peptide and with Scram, but they were hardly visible in the sample with AP-FXIII. Thus, AP-FXIII significantly delayed fibrin crosslinking in this purified system. FXIII is crucial for fibrin formation ...