A novel modified thaw-siphon method for extracting cryoprecipitate: The Bokutoh-siphon method.

BACKGROUND: Cryoprecipitate (CRYO) is neither produced nor supplied by the Japanese Red Cross Society. A novel CRYO extraction method established in-house by modifying a thaw-siphon technique was demonstrated in this study. STUDY DESIGN AND METHODS: A pack of fresh frozen plasma was thawed and equally divided into two bags for CRYO extraction by different methods. CRYO was extracted from the blood plasma using a standard centrifugation method and our modified thaw-siphon method (Bokutoh-siphon method; B method). The two different CRYOs extracted were analyzed to compare the differences in the amount of fibrinogen recovered, clotting factors extracted, and clotting activity. RESULTS: The amount of fibrinogen in the CRYO extracted using the B-siphon method was similar to that obtained using the standard method (recovery of fibrinogen: B-siphon method: 71.2% vs. standard method: 61.0%). The amount of clotting XIII factor extracted using the B-siphon method was significantly lower than those extracted using the standard method. On the other hand, clotting II, V factors, and C1q esterase inhibitor not concentrated in CRYO content from the B-siphon method were significantly higher than that from the standard method. CONCLUSION: A new in-house CRYO preparation method was established by modifying a previously used thaw-siphon method. A coagulation factor-rich CRYO was extracted from plasma frozen at -40°C along with the first fraction of thawed plasma, without using a large-capacity refrigerated centrifuge for blood bags.

Patient rescue and blood utilization in the Ogasawara blood rotation system.

BACKGROUND: In the past, blood products were not transported to the Ogasawara Islands because of the distance; the islands are approximately 1000 km from the mainland and lack an airport. The Ogasawara Blood Rotation system involves the routine, long-distance transportation of Type O, RhD-positive, irradiated red blood cells to rescue patients with acute hemorrhage and severe anemia and to reduce wastage from the expiration of red blood cell solution. STUDY DESIGN AND METHODS: Blood transfusion and utilization in the Ogasawara blood rotation (BR) system were examined from December 2014 to March 2017. Two packs of RBC solution (one pack = 280 mL) were transported in an active transport refrigerator by scheduled ships between Tokyo City and the Ogasawara Islands. RESULTS: Eight packs of red blood cell (RBC) solution were received as transfusions by four patients who had acute upper gastrointestinal bleeding or severe anemia (eight of 232 packs; 3.4%). The blood utilization rate at the time of re-use was 84.6% (196 of 232 packs), with neither adverse reactions nor wastage. Twenty-eight packs of RBC solution expired because of a delay in scheduled ships as the result of a typhoon (12.1%). CONCLUSION: The Ogasawara BR system was effectively capable of delivering RBC solution for transfusion in patients residing in distant islands and contributed to reducing the wastage of RBC solution by facilitating blood utilization at another hospital.

Expression, purification, crystallization and X-ray crystallographic analysis of the periplasmic binding protein VatD from Vibrio vulnificus M2799.

Vibrio vulnificus is a halophilic marine microorganism which causes gastroenteritis and primary septicaemia in humans. An important factor that determines the survival of V. vulnificus in the human body is its ability to acquire iron. VatD is a periplasmic siderophore-binding protein from V. vulnificus M2799. The current study reports the expression, purification and crystallization of VatD. Crystals of both apo VatD and a VatD-desferrioxamine B-Fe(3+) (VatD-FOB) complex were obtained. The crystal of apo VatD belonged to space group P6422, while the crystal of the VatD-FOB complex belonged to space group P21. The difference in the two crystal forms could be caused by the binding of FOB to VatD.

Crystal structure of RVV-X: an example of evolutionary gain of specificity by ADAM proteinases.

Russell's viper venom factor X activator (RVV-X) is a heterotrimeric metalloproteinase with a mammalian ADAM-like heavy chain and two lectin-like light chains. The crystal structure of RVV-X has been determined at 2.9 A resolution and shows a hook-spanner-wrench-like architecture, in which the metalloproteinase/disintegrin region constitutes a hook, and the lectin-like domains constitute a handle. A 6.5nm separation between the catalytic site and a putative exosite suggests a docking model for factor X. The structure provides a typical example of the molecular evolution of multi-subunit proteins and insights into the molecular basis of target recognition and proteolysis by ADAM/adamalysin/reprolysin proteinases.

Crystal structures of catrocollastatin/VAP2B reveal a dynamic, modular architecture of ADAM/adamalysin/reprolysin family proteins.

Catrocollastatin/vascular apoptosis-inducing protein (VAP)2B is a metalloproteinase from Crotalus atrox venom, possessing metalloproteinase/disintegrin/cysteine-rich (MDC) domains that bear the typical domain architecture of a disintegrin and metalloproteinase (ADAM)/adamalysin/reprolysin family proteins. Here we describe crystal structures of catrocollastatin/VAP2B in three different crystal forms, representing the first reported crystal structures of a member of the monomeric class of this family of proteins. The overall structures show good agreement with both monomers of atypical homodimeric VAP1. Comparison of the six catrocollastatin/VAP2B monomer structures and the structures of VAP1 reveals a dynamic, modular architecture that may be important for the functions of ADAM/adamalysin/reprolysin family proteins.

Crystallization and preliminary X-ray crystallographic analysis of two vascular apoptosis-inducing proteins (VAPs) from Crotalus atrox venom.

VAPs are haemorrhagic snake-venom toxins belonging to the reprolysin family of zinc metalloproteinases. In vitro, VAPs induce apoptosis specifically in cultured vascular endothelial cells. VAPs have a modular structure that bears structural homology to mammalian ADAMs (a disintegrin and metalloproteinases). VAP1 is a homodimer with a MW of 110 kDa in which the monomers are connected by a single disulfide bridge. VAP2 is homologous to VAP1 and exists as a monomer with a MW of 55 kDa. In the current study, several crystal forms of VAP1 and VAP2 were obtained using the vapour-diffusion method and diffraction data sets were collected using SPring-8 beamlines. The best crystals of VAP1 and VAP2 generated data sets to 2.5 and 2.15 angstroms resolution, respectively.

Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold.

ADAMs (a disintegrin and metalloproteinase) are sheddases possessing extracellular metalloproteinase/disintegrin/cysteine-rich (MDC) domains. ADAMs uniquely display both proteolytic and adhesive activities on the cell surface, however, most of their physiological targets and adhesion mechanisms remain unclear. Here for the first time, we reveal the ADAMs' MDC architecture and a potential target-binding site by solving crystal structures of VAP1, a snake venom homolog of mammalian ADAMs. The D-domain protrudes from the M-domain opposing the catalytic site and constituting a C-shaped arm with cores of Ca2+ ions. The disintegrin-loop, supposed to interact with integrins, is packed by the C-domain and inaccessible for protein binding. Instead, the hyper-variable region (HVR) in the C-domain, which has a novel fold stabilized by the strictly conserved disulfide bridges, constitutes a potential protein-protein adhesive interface. The HVR is located at the distal end of the arm and faces toward the catalytic site. The C-shaped structure implies interplay between the ADAMs' proteolytic and adhesive domains and suggests a molecular mechanism for ADAMs' target recognition for shedding.

Inferior alveolar nerve paresthesia relieved by microscopic endodontic treatment.

We experienced two cases of inferior alveolar nerve paresthesia caused by root canal medicaments, which were successfully relieved by microscopic endodontic treatment. In the first case, the paresthesia might have been attributable to infiltration of calcium hydroxide into the mandibular canal through the root canals of the mandibular left second molar tooth. In the second case, the paresthesia might have been attributable to infiltration of paraformaldehyde through the root canals of the mandibular right second molar tooth. The paresthesia was relieved in both cases by repetitive microscopic endodontic irrigation using physiological saline solution in combination with oral vitamin B12 and adenosine triphosphate.