Factor V is activated and cleaved by platelet calpain: comparison with thrombin proteolysis

Platelets are known to process human factor V during secretion and/or membrane binding. We studied the functional and structural changes produced in human factor V by purified human platelet calpain (calcium- activated thiol protease) and compared the alterations with those induced by thrombin. A maximum increase in coagulant activity of 2.5- fold was observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03 U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, whereas the subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Although thrombin activation eventually results in four final components designated C1 (150 kd), D (105 kd), E (71 kd), and F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd within one minute. Further digestion of the 200 kd species by calpain gives rise first to a polypeptide of 160 kd that is converted to a 140 kd and a 120 kd species by two minutes with an increase in coagulant activity. Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10 directed to factor V and the thrombin-generated C1 fragment yields results demonstrating a common epitope in these calpain-generated components of 200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide gives rise to polypeptides of 100 and 65 kd, both undetectable on immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of less certain origin. Thus, platelet calpain generates a complex but reproducible cleavage pattern different from thrombin that may explain the partial activation observed. Nevertheless, calpain processing may play a role in early hemostatic reactions involving platelets before the appearance of the first thrombin molecule.     

Perceived quality of and access to care among poor urban women in Kenya and their utilization of delivery care: harnessing the potential of private clinics?

This paper uses data from a maternal health study carried out in 2006 in two slums of Nairobi, Kenya, to: describe perceptions of access to and quality of care among women living in informal settlements of Nairobi, Kenya; quantify the effects of women's perceived quality of, and access to, care on the utilization of delivery services; and draw policy implications regarding the delivery of maternal health services to the urban poor. Based on the results of the facility survey, all health facilities were classified as 'appropriate' or 'inappropriate'. The research was based on the premise that despite the poor quality of these maternal health facilities, their responsiveness to the socio-cultural and economic sensitivities of women would result in good perceptions and higher utilization by women. Our results show a pattern of women's good perceptions in terms of access to, and quality of, health care provided by the privately owned, sub-standard and often unlicensed clinics and maternity homes located within their communities. In the multivariate model, the association between women's perceptions of access to and quality of care, and delivery at these 'inappropriate' facilities remained strong, graded and in the expected direction. Women from the study area are seldom able to reach not-for-profit private providers of maternal health care services like missionary and non-governmental organization (NGO) clinics and hospitals. Against the backdrop of challenges faced by the public sector in health care provision, we recommend that the government should harness the potential of private clinics operating in urban, resource-deprived settings. First, the government should regulate private health facilities operating in urban slum settlements to ensure that the services they offer meet the acceptable minimum standards of obstetric care. Second, 'good' facilities should be given technical support and supplied with drugs and equipment.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

Single-acquisition chemical-shift imaging of a binary system with use of stimulated echoes

A method for separating binary chemical-shift components with a single image data acquisition by means of stimulated echoes is demonstrated. With a strategy analogous to the modified Dixon method, three stimulated echoes were acquired to form three complex images. In each of the images, the complex pixel intensities were imparted, by design of the pulse sequence, with a phase factor carrying chemical-shift or field inhomogeneity information. With these three images, true fat/water separation can be obtained in biologic tissues. Studies at high field strength (4.7 T) on a toluene phantom, a pseudo-binary chemical-shift system, were used to evaluate the applicability of the method. Its clinical feasibility was demonstrated on a healthy human subject in a 0.6-T whole-body imaging system.     

Persistent Erection (Priapism) for Months with Concomitant Infertility in Rats due to Protein-Energy-Malnutrition

Monatelange Erektion (Priapismus) mit konsekutiver Infertitität bei Ratten als Folge von Protein-Energie-Mangelernährung
Wachsende, männüche Sprague Dawley Ratten wurden 22 Wochen lang mit einer halbsynthetischen, 9% Rohprotein enthaltenden Diät gefüttert, wobei Kasein (Kontrollgruppe; I) oder Weizenbrotkruste (Gruppe II) die alleinigen Proteinquellen darstellten. Das Kontrollfutter in reduzierter Menge, in Anlehnung an den Futterverzehr der Krusten-Ratten, wurde einem dritten Kollektiv verfüttert (pair-fed-Gruppe; III). Nach 6 Versuchswochen zeigten sich in den Gruppen II und III erste pathomorphologische Genitalveränderungen, die sich in einer monatelangen Erektion manifestierten. Bei 97% der Krusten-Ratten, 67% der isoenergetisch gefütterten Kasein-Ratten, jedoch bei keinem Kontrolltier entwickelte sich am Ende des Beobachtungszeitraums eine Dauererektion. Nach der 15. Versuchswoche erzielten die pair-fed-Ratten 17%, die Krusten-Ratten nur 5% des Gewichtszuwachses der Kasein-Kontrollgruppe. Im Reproduktionstest mit gesunden weibl. SD-Ratten erwies sich nur 1 von 6 Krusten-Ratten als zeugungsfähig, nach anschließender 5wöchiger ad lib. Kasein-Wiederauffütterung konnte eine 83% Fertilität beobachtet werden. Der Pathomechanismus der nutritiv induzierten Dauererektion wird auf die ausgeprägte marastische Stoffwechsellage zurückgeführt, bedarf jedoch weiterer Aufklärung.     

Neutral steroid metabolites in patients with uraemia and after renal transplantation: a quantitative and qualitative study in body fluids

Steroid metabolites enriched from urine, haemofiltrate, and CAPD-dialysate (Continuous Ambulatory Peritoneal Dialysis) were identified by gas chromatography-mass spectrometry and quantified by capillary gas chromatography. The study included twenty healthy controls, twenty-six non-dialysed uraemics, thirty-nine patients on regular dialysis treatment, and twenty-two allograft recipients. Compared to the 24 h urinary excretion rates of controls the excretion rates of androsterone and etiocholanolone were in the lower normal range up to significantly decreased in the body fluids of all patients, and those of the corticoid metabolites were also significantly decreased. 11-Oxygenated androstanolones in urine from non-dialysed uraemics correlated significantly decreased. 11-oxygenated androstano-levels and were significantly increased, but normal in haemofiltrate and CAPD-dialysate, while in urine of allograft recipients the values were significantly lower.     

Hyperlipidemia, hypercoagulability, and accelerated thrombosis: studies in congenitally hyperlipidemic rats and in rats and monkeys with induced hyperlipidemia

Inbred Carworth Farms Nelson (CFN) congenitally hyperlipidemic rats had significantly shorter coagulation and prothrombin times and higher levels of coagulation factors, II, V, VII, VIII, and X than did controls. Conversely, congenitally hypolipidemic rats of the same strain had significantly longer coagulation and prothrombin times and lower levels of factors II, V, VII, X and XII and of blood platelets than did controls. A loop-shaped polyethylene cannula was inserted into the aorta to assess the potential for thrombosis. The hyperlipidemic group obstructed this significantly faster and the hypolipidemic group slower than did the controls. Normal CFN rats made hypertensive by unilateral renal artery clip developed hypertension together with significantly elevated serum cholesterol and factor VII and X levels. Rhesus monkeys with diet-induced hyperlipidemia showed shorter prothrombin times and higher factor X levels than did controls on normal diet. By selective breeding, two groups of squirrel monkeys were obtained. Both groups had similar serum cholesterol levels on a normal diet but one group (hyperresponders) showed higher serum cholesterol levels on a cholesterol-containing diet than did the other (hyporesponder) group. Both groups showed significantly elevated levels of factors II, V, VII, IX and X on a cholesterol-containing diet. There was good correlation between the levels of many coagulation factors and serum cholesterol in both rats and monkeys. If thrombosis is important in the genesis of atherosclerosis, these findings could indicate that elevation of plasma lipids may play a role, via the coagulation pathway, in the production of human vascular disease.