Subject(s)
Purpura, Thrombotic Thrombocytopenic/epidemiology , ADAMTS13 Protein/deficiency , Adolescent , Adult , Black or African American/statistics & numerical data , Age Factors , Aged , Female , Humans , Male , Middle Aged , Plasma Exchange , Purpura, Thrombotic Thrombocytopenic/therapy , Recurrence , Registries , Retrospective Studies , Symptom Assessment , Treatment OutcomeABSTRACT
OBJECTIVES: Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic. METHODS: Blood from each donor was subjected to 62 different handling conditions prior to complement assays. RESULTS: Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at -80°C for up to 6 years had no significant effect on levels of complement factors. CONCLUSIONS: The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease.
Subject(s)
Complement Activation , Complement System Proteins/metabolism , Specimen Handling/methods , Thrombotic Microangiopathies/blood , Biomarkers/analysis , Biomarkers/metabolism , Blood Preservation/methods , Blood Specimen Collection/methods , Complement System Proteins/analysis , Humans , Temperature , Thrombotic Microangiopathies/immunologyABSTRACT
At2g44920 belongs to a diverse family (Pfam PF00805) of pentapeptide-repeat proteins (PRPs) that are present in all known organisms except yeast. PRPs contain at least eight tandem-repeating sequences of five amino acids with an approximate consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Recent crystal structures show that PRPs adopt a highly regular four-sided right-handed ß-helical structure consisting mainly of type II and type IV ß-turns, sometimes referred to as a repeated five-residue (or Rfr) fold. Among sequenced genomes, PRP genes are most abundant in cyanobacteria, leading to speculation that PRPs play an important role in the unique lifestyle of photosynthetic cyanobacteria. Despite the recent structural characterization of several cyanobacterial PRPs, most of their functions remain unknown. Plants, whose chloroplasts are of cyanobacterial origin, have only four PRP genes in their genomes. At2g44920 is one of three PRPs located in the thylakoid lumen. Here, the crystal structure of a double methionine mutant of residues 81-224 of At2g44920, the naturally processed fragment of one of its full-length isoforms, is reported at 1.7 Å resolution. The structure of At2g44920 consists of the characteristic Rfr fold with five uninterrupted coils made up of 25 pentapeptide repeats and α-helical elements capping both termini. A disulfide bridge links the two α-helices with a conserved loop between the helical elements at its C-terminus. This structure represents the first structure of a PRP protein whose subcellular location has been experimentally confirmed to be the thylakoid lumen in a plant species.
