Key Points• Clot retraction of sphingomyelin-rich raftdepleted platelets from sphingomyelin synthase knockout mouse is delayed.• Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen g-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunitdeficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-b-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-aIIbb3-myosin complex is formed as a primary axis to promote platelet contraction. (Blood. 2013;122(19):3340-3348) IntroductionMembrane rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of a number of vital cellular processes. 1 The important role of rafts at the cell surface may be their function in signal transduction. A number of studies provide considerable evidence that rafts are integral to the regulation of immune and neuronal signaling. Membrane rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation. 2 The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor. 3 Membrane rafts are also required for platelet aggregation via the collagen receptor GPVI, 4 the adenosine 59-diphosphate (ADP) receptor P2Y12, 5 the Fcg receptor FcgRIIa, 6 and the C-type lectinlike receptor CLEC-2.7 Detergent-resistant membrane (DRM) rafts of platelets show round vesicles of heterogeneous sizes ranging from 20 to 500 nm, which are enriched in CD36 (GPIV). 8,9 Recent reports have demonstrated that membrane rafts are ...

Glucose-6-phosphate dehydrogenase (G6PDH)-deficient cells of Saccharomyces cerevisiae showed increased susceptibility and were unable to induce adaptation to oxidative stress. Historically, mainly in human erythrocytes, it has been suggested and accepted that decreased cellular GSH, due to loss of the NADPH-dependent activity of glutathione reductase (GR), is responsible for the increased sensitivity to oxidative stress in G6PDH-deficient cells. In the present study we investigated whether the increased susceptibility and the inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast is caused by incompleteness of glutathione recycling. We constructed G6PDH- and GR-deficient mutants and analysed their adaptive response to H2O2 stress. Although G6PDH-deficient cells contained comparable amounts of GSH and GR activity to wild-type cells, GSSG was not reduced efficiently, and intracellular GSSG levels and the ratio of GSSG to total glutathione (GSSG/tGSH) were higher in G6PDH-deficient cells than in wild-type. On the other hand, GR-deficient cells showed a susceptibility identical with that of wild-type cells and induced adaptation to H2O2 stress, even though the GSSG/tGSH ratio in GR-deficient cells was higher than in G6PDH-deficient cells. These results indicate that incompleteness of glutathione recycling alone is not sufficient to account for the increased sensitivity and inability to induce adaptation to H2O2 stress of G6PDH-deficient yeast cells. In S. cerevisiae, G6PDH appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH to the GR reaction.

Association between Intention to Stay on the Job and Job Satisfaction among Japanese Nurses in Small and Medium-sized Private Hospitals: Yasushi KUDO, et al. Department of Preventive Medicine and Public Health, KitasatoUniversity School of Medicine-In order to examine the relationship between the intention to stay on the job and job satisfaction among Japanese nurses, and to obtain clues for preventing turnover, we conducted a questionnaire survey. The subjects involved in the survey included 625 female nurses (registered nurses, licensed practical nurses and assistant nurses) working in 4 small and medium-sized private hospitals, excluding directors of nursing. Of the 625 questionnaires distributed, 556 (89.0%) were returned. After excluding the questionnaires with missing values, 480 questionnaires were analyzed (effective response rate, 76.8%).The average age of the respondents was 32.8 yr (range: 20-65). The content of the questionnaire was nurse attributes, job satisfaction (30 items) and intention to stay on the job. For job satisfaction, factor analysis (principal factor method and promax rotation) was performed, and factors with an eigenvalue of ≥1 were extracted. Six factors were extracted by factor analysis. These factors were interpreted as "Work as specialists" (1st factor), "Relationship with superiors" (2nd factor), "Comfortable life" (3rd factor), "Relationship among nurses" (4th factor), "Communication with physicians" (5th factor) and "Working conditions" (6th factor). The factor scores were calculated and used as a scale for the evaluation of job satisfaction. To investigate the factors associated with intention to stay on the job among nurses, the standard partial regression coefficient was computed by multiple linear regression analysis, with intention to stay on the job as the dependent variable, and nurse attributes and job satisfaction (factor scores) as independent variables. Various factors including the organizational culture of each hospital may affect the relationship between job satisfaction and the intention to stay on the job. In order to adjust for these factors, differences among hospitals were included in the statistical model as independent variables. The result of the multiple regression analysis suggests that the intention to stay on the job was higher among nurses who were older and more satisfied with work as specialists (1st factor) and working conditions (6th factor). (J Occup Health 2006; 48: 504-513)

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, ␣ 2 -plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. IntroductionCoagulation factor XIII (FXIII) is a pro-enzyme of plasma transglutaminase (TGase) consisting of 2 enzymatic A subunits (FXIII-A) and 2 noncatalytic B subunits, and plays a critical role in the generation of a stable hemostatic plug. 1-3 FXIII catalyzes intermolecular cross-linking reactions between fibrin monomers, ␣ 2 -plasmin inhibitor, fibronectin, etc. These reactions increase the mechanical strength of the fibrin clot and its resistance to proteolytic degradation, and enhance the assembly of the extracellular matrix.Congenital FXIII deficiency is a rare autosomal recessive disorder, the cases of most of which are caused by defects in the FXIII-A gene. 3 A lifelong bleeding tendency, abnormal wound healing, and recurrent spontaneous miscarriage are common symptoms of FXIII deficiency. 1,4 FXIII-A exists extracellularly in plasma as well as intracellularly as a cytosolic protein in megakaryocytes/platelets and monocytes/macrophages, although the function(s) of intracellular FXIII-A remain(s) unknown. 5,6 In particular, platelets cause clot retraction (CR) by retracting extended filopodia along fibrin strands. 7 There have been conflicting reports about the effects of FXIII deficiency on CR; investigators reported that the absence of FXIII abolished, 7-9 did not affect, 10,11 or rather enhanced 12 CR in patients with congenital FXIII deficiency. However, platelet aggregation induced by various agents is uniformly normal in patients with congenital FXIII deficiency. 8,9,13,14 To understand the precise molecular pathology of FXIII deficiency in vivo, FXIII-A knockout (KO) mice have been analyzed. FXIII-A KO mice demonstrated a severe bleeding tendency. 15 We also reported that FXIII-A KO mice developed severe uterine bleeding, resulting in spontaneous miscarriage in females, and male-specific intrathoracic hemorrhage as well as excessive cardiac fibrosis. 16,17 Because bleeding times in FXIII-A KO mice were longer than in wild-type, 15,18 we hypothesized that FXIII-A KO mice might have a defective platelet-related function(s). Accordingly, we have explored the contribution of FXIII to the process of CR in the present study. Methods AnimalWild-type C57BL/6J mice were obtained from Japan SLC Inc. Genetargeted mice of FXIII-A were generated as previously reported, ...

No abstract