Kidney graft outcome using an anti-Xa therapeutic strategy in an experimental model of severe ischaemia-reperfusion injury.

BACKGROUND: Deceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia-reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model. METHODS: The effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4 °C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage. RESULTS: At 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial-mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment. CONCLUSION: Peritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevance The increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome. Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions. This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.

Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model.

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

A new preservation solution (SCOT 15) Improves the islet isolation process from pancreata of non-heart-beating donors: a Murine model.

INTRODUCTION: Due to the organ shortage, there is increased use of organs harvested from non-heart-beating donors (NHBD). These organs have been subjected to a period of warm ischemia that is most deleterious to functional recovery. We have designed a new preservation solution, "Solution de Conservation des Organes et des Tissus" (SCOT 15; Macopharma, Tourcoing, France) which contains an extracellular ionic composition including PEG 20 kD (15 g/L) as a colloid. METHODS: Our objective was to compare SCOT 15 with University of Wisconsin (UW) solution or islet culture medium CMRL 1066 + 1% of Bovine Serum Albumin (BSA), as the working and preservation solution for islet isolation from pancreata subjected to warm ischemia using a murine model. RESULTS: Warm ischemia decreased the islet yield and cellular viability regardless of the preservation solution. Either when the pancreas was or was not subjected to warm ischemia, the best islet yield was obtained with SCOT 15 (P < .05 vs UW or CMRL 1066). The same results were observed for islet viability as assessed using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test; namely, better viability with SCOT 15 as compared with UW or CMRL 1066 (P < .01). CONCLUSION: In a murine model SCOT 15 was a better preservation solution for islet isolation than UW solution or culture medium (CMRL 1066).

Melagatran prevents tissue factor expression in human platelet-monocyte heterotypic complexes.

Platelets form heterotypic complexes with circulating monocytes, inducing the expression of the procoagulant Tissue Factor (TF) that leads to thrombin generation. We investigated the potential preventive effect of melagatran, a direct anti-thrombin drug, on TF expression by platelet/monocyte heterotypic complexes (PMHC) from healthy human donors. Flow cytometry and western blot analysis were performed to characterize surface and total TF protein expression in PMHC in venous blood samples drawn in the presence of heparin or heparin and melagatran (4 microM). Addition of melagatran significantly lowered the percentage of TF positive PMHC (2.6+/-0.3 vs. 5.9+/-0.7 %, p<0.01). This was not due to a melagatran-induced decrease in activation of the platelets associated with monocytes in PMHC. Indeed, melagatran effect on TF expression was accompanied by an increase in cell surface P-selectin expression in PMHC (95.6+/-1.9 vs. 48+/-18 %, p<0.001), suggesting that platelet were actually more activated in PMHC from the melagatran-treated samples. Western blot analysis of PBMC extracts suggested that melagatran specifically targeted a (54kD) form of TF in monocytes. Although further investigation is warranted, these data suggest that melagatran decreases TF expression in PMHC.

Effect of raloxifene -- a selective oestrogen receptor modulator -- on kidney function in post-menopausal women with Type 2 diabetes: results from a randomized, placebo-controlled pilot trial.

AIMS: Epidemiological and experimental data suggest that activation of the oestrogen receptor pathway limits the incidence and the progression of diabetic nephropathy. We tested the hypothesis that raloxifene protects against increasing urinary albumin excretion in post-menopausal women with Type 2 diabetes in a randomized pilot clinical trial. METHODS: We included 39 post-menopausal women with Type 2 diabetes and micro- or macro-albuminuria in a 6-month, double-blind, placebo-controlled trial: 20 received placebo and 19 received 60 mg raloxifene per day. The albumin : creatinine ratio (ACR) in urine was determined on three consecutive days during the week before randomization and during the week before the final visit. RESULTS: One patient in each group dropped out in the first 3 weeks, leaving 37 patients for the analysis (19 on placebo and 18 on raloxifene). From randomization to the final visit, mean ACR was unchanged in the placebo group {277 microg/mg (67; 651) [median (interquartile range)] vs. 284 microg/mg (79; 1508)} but decreased slightly in the raloxifene group [376 microg/mg (67; 615) vs. 243 microg/mg (103; 549)]. This corresponds to a change of +24 (-37; +517) for the placebo group vs. -10 microg/mg (-36; +16) for the raloxifene group (P = 0.11). In multivariate analysis, raloxifene treatment (P(adjusted) = 0.013), baseline low-density lipoprotein (LDL) cholesterol (P(adjusted) = 0.023) and change in LDL cholesterol (P(adjusted) = 0.008) were related to the absolute change in ACR. Adverse effects were similar in the two groups. CONCLUSIONS: These results suggest that raloxifene may limit the progression of albuminuria in post-menopausal women with diabetes; further studies in a larger population are warranted.

Maternal history of type 2 diabetes is associated with diabetic nephropathy in type 1 diabetic patients.

AIMS: Insulin resistance is a key feature of type 2 diabetes. It is also involved in the development and progression of microvascular complications. We analysed the relationship between parental history of diabetes, insulin resistance and diabetic nephropathy (DN) and assessed the specific maternal and paternal influences of history of type 2 diabetes on DN in type 1 diabetic offspring. METHODS: We recorded information regarding family history of type 2 diabetes and of cardiovascular disease in 160 consecutive, unrelated type 1 diabetic patients. Insulin resistance was assessed using a validated estimation of the glucose disposal rate (eGDR). RESULTS: Type 1 diabetic patients with a maternal history of type 2 diabetes were more likely to be insulin-resistant (P=0.043) and to have renal complications (P=0.0041) than those from the reference group (without parental history of diabetes), while patients with a paternal history were not different from those from the reference group, regarding eGDR and DN. Time to development of abnormal albuminuria was significantly affected by maternal history of type 2 diabetes (log-rank=12.66; P=0.0004) and by familial history of premature cardiovascular disease (log-rank=5.48; P=0.0234). In multivariate analysis, a maternal history of type 2 diabetes was independently associated with nephropathy after adjustment for sex, diabetes duration and familial history of premature cardiovascular disease. CONCLUSION: Maternal history of type 2 diabetes is independently associated with DN in type 1 diabetic patients. This might suggest the transmission of a maternal trait related to microvascular complications, raising the hypothesis of imprinted genes predisposing to diabetic renal disease.

Protective effect of PEG 35,000 Da on renal cells: paradoxical activation of JNK signaling pathway during cold storage.

Polyethylene glycol (PEG), a high-molecular weight colloid, is added to preservation solutions in order to decrease cold- and ischemia-induced injuries of the grafted organ. We evaluated on LLC-PK1, a porcine proximal tubular epithelial cell line (1) the efficiency of several commercial preservation solutions (University of Wisconsin, Euro-Collins, Celsior, SCOT, IGL-1), and (2) whether adding PEG (400-35,000 Da) in a simple extracellular-type buffer modified cell integrity and mitogen-activated protein kinase (MAPK) signaling pathways. SCOT was the most efficient commercial solution. Moreover, only PEG 35,000 Da totally preserved cell viability, induced a decrease on reactive oxygen species production and a decrease on p38-MAPK activation. Furthermore PEG 35,000 Da stimulated c-Jun N-terminal kinase (JNK). However, the inhibition of JNK pathway, with the specific SP600125 inhibitor, in the presence of PEG 35,000 Da did not affect cell survival. We also confirmed on whole pig kidney the protective effect of PEG 35,000 Da on cold-induced tubular injuries. This study confirms PEG antioxidative properties, but we demonstrate that its effect on JNK signaling pathway had also a paradoxical effect on cell death. This sheds a new light on PEG effects during cell preservation, independently from the classical immuno-camouflaging hypothesis.

Protective role of selectin ligand inhibition in a large animal model of kidney ischemia-reperfusion injury.

Experiments in rodents have demonstrated an important role for selectins in kidney ischemia-reperfusion injury (IRI). However, the relevance of this in larger mammals, as well as the impact on long-term structure and function is unknown. We tested the hypothesis that small molecule selectin ligand inhibition attenuates IRI, cellular inflammation, and long-term effects on renal interstitial fibrosis. We used a porcine model of kidney IRI and used Texas Biotechnology Corporation (TBC)-1269, a selectin ligand inhibitor. Renal function, tissue inflammation, and tubulointerstitial fibrosis development were evaluated up to 16 weeks. Both warm and cold ischemia models were studied for relevance to native and transplant kidney injury. Pigs treated with TBC-1269 during 45 min of warm ischemia (WI) showed significantly increased glomerular filtration rate compared to control animals. In pigs with severe IRI (WI for 60 min), TBC-1269 treatment during IRI significantly increased renal recovery. Cellular inflammation was strongly reduced, particularly influx of CD4 cells. Quantitative measurement of fibrosis by picrosirius red staining showed strong reduction in TBC-1269-treated groups. TBC-1269 also reduced cold IRI, inflammation, and fibrosis in kidneys preserved for 24 h at 4 degrees C and autotransplanted. The selectin ligand inhibitor TBC-1269 provides a novel and effective approach to attenuate IRI in both warm and cold ischemia in large mammals, in both short and long terms. Selectin ligand inhibition is an attractive strategy for evaluation in human kidney IRI.

Determination of HbA1c concentrations in patients with acute myocardial infarction: comparison of the DCA 2000 device with the HPLC method.

BACKGROUND: Recent studies suggest that HbA1c is an important predictor of the glycometabolic state of patients admitted for acute myocardial infarction (AMI). OBJECTIVE: We aimed at comparing the results of HbA1c concentrations obtained by 2 different methods in patients with AMI. RESEARCH DESIGN AND METHODS: In a first study, HbA1c was measured in all patients consecutively hospitalized for AMI, during a 6 month period using the HPLC method and the DCA 2000 device in the biochemistry laboratory. In a second study, HbA1c measured by the DCA 2000 device in the intensive care unit was compared with HbA1c determined by HPLC in the biochemistry laboratory in a similar sample of patients. In patients without personal history of diabetes, those patients with HbA1c > 6.5% (HPLC method), were classified as possible diabetes. RESULTS: A total of 146 patients were included (119 males, 27 females; mean age: 63 +/- 15 years). Twenty-seven of the patients had a personal history of diabetes. HbA1c determined by 2 techniques were highly correlated (r = 0.939, P < 0.0001). The mean of the differences (Bland and Altman analysis) was 0.4 +/- 0.3%. Compared with the HPLC method, the sensitivity of DCA 2000 device for the detection of possible diabetes was 81.8 +/- 11.6 and the specificity was 99.1 +/- 0.9%. The diagnostic accuracy of DCA method was 97.5 +/- 1.4%. In the second study, the HbA1c concentrations of 21 additional subjects, determined in an intensive care unit, were not different from the first 21 patients of the first study. CONCLUSIONS: HbA1c can be effectively determined using the DCA 2000 device. This method is reliable and easy to be implemented in an intensive care unit.

[Value of systematic biological markers of inflammation for the prognosis at 12 months of patients undergoing programmed coronary angioplasty]. / Intérêt du dosage de marqueurs systémiques de l'inflammation dans l'évalualion pronostique à 12 mois de patients traités par angioplastie coronaire programmée.

Value of systematic dosage of biological markers of inflammation for the prognosis at 12 months of patients undergoing programmed coronary angioplasty Systematic dosage of proteins of inflammation has been suggested for assessing the prognosis of athero-thrombotic diseases. The authors undertook a study of plasma C-reactive protein (CRP) and interleukin 6 (IL-6) for evaluating the prognosis of patients undergoing programmed coronary angioplasty. A prospective monocentric study of 117 patients (65 +/- 8 years) was divided into a control group of 28 patients undergoing coronary angiography (Group 1) and 89 patients undergoing programmed coronary angioplasty (Group 2). Serum IL-6 and CRP levels were measured before arterial puncture and at H12 and H24 after coronary catheterisation. The follow-up period was 12 months. The angioplasty did not significantly increase CRP and IL-6 concentrations compared with coronary angiography. Twenty patients (Group 2) (22%) suffered a cardiovascular event in the 12 months' follow-up. These patients had significantly higher CRP levels at H0, H12 and H24 after coronary angioplasty than those who had uncomplicated outcomes. This was not observed for IL-6 concentrations because of the wide dispersion of the results obtained. Increased CRP concentrations between H0 and H24 was also a good predictive factor independently of high basal CRP levels potentially due to other causes than atheroma. Coronary angioplasty is associated with increased CRP at H0, H12 and H24. These values are correlated with the risk of future events at 6 and 12 months. This information is easily obtained and should help management of these patients.

Prognostic value of admission plasma glucose and HbA in acute myocardial infarction.

OBJECTIVE: Stress hyperglycaemia increases the risk of mortality after acute myocardial infarction in diabetic and in non-diabetic patients. We aimed to determine the contribution of admission plasma glucose and HbA(1c) on post-acute myocardial infarction prognosis. PATIENTS AND METHODS: Admission plasma glucose and HbA(1c) were simultaneously measured in all patients consecutively hospitalized for acute myocardial infarction. Patient survival was measured on 5 and 28 days after admission. Patients were defined as having 'previously diagnosed diabetes' (personal history of diabetes defined using ADA 1997 criteria), 'no diabetes', those without previously diagnosed diabetes and HbA(1c) below 6.5%, or 'possible diabetes', i.e. those without previously diagnosed diabetes and HbA(1c) above 6.5%. RESULTS: Of the 146 patients included, four had died by day 5 and 14 by day 28. Admission plasma glucose was higher in patients who had died by day 28 (11.7 +/- 5.8 vs. 8.0 +/- 3.3 mmol/l, P = 0.002), whereas HbA(1c) was not (6.4 +/- 1.9 vs. 6.1 +/- 0.8%, NS). Admission plasma glucose was significantly higher in those who had died by day 28 after adjustment on HbA(1c). A multivariate analysis, including sex, age and heart failure prior to acute myocardial infarction, showed that admission plasma glucose concentration was an independent predictor of survival after acute myocardial infarction. Twenty-seven of the patients had previously diagnosed diabetes and 119 had no history of diabetes. Eleven were found to have possible diabetes. Admission plasma glucose was significantly higher in previously diagnosed diabetes (11.1 +/- 5.6) than in the other groups: 7.7 +/- 2.9 in non-diabetes, 8.2 +/- 2.1 in possible diabetes (P < 0.0001). The relationship between HbA(1c)-adjusted admission plasma glucose and mortality after acute myocardial infarction was also found in the non-diabetes group. CONCLUSIONS: Admission plasma glucose, even after adjustment on HbA(1c), is a prognostic factor associated with mortality after acute myocardial infarction. Acute rather than the chronic pre-existing glycometabolic state accounts for the prognosis after acute myocardial infarction.

Serum triglycerides are a predictive factor for the development and the progression of renal and retinal complications in patients with type 1 diabetes.

OBJECTIVES: It is controversial that serum lipIds affect the development and progression of microvascular complications in patients with type 1 diabetes. METHODS: We prospectively followed 297 patients with type 1 diabetes without end-stage renal disease for 7 Years (range: 2-10). Serum lipIds were measured at baseline (total and HDL-cholesterol, triglycerIdes and calculated LDL-cholesterol, Lipoprotein (a)). The primary end-point was the occurrence of a renal event and the secondary end-point was the occurrence of a retinal event, defined as the progression to a higher stage of diabetic nephropathy or retinopathy, respectively. RESULTS: Serum triglycerIde (TG) levels were higher in patients who progressed in nephropathy than in those who dId not [median 1.21 (range 0.41-2.96) vs 0.91 (0.31-11.07) mmol/l; p=0.0037] and in those who developed retinal events than in those who dId not [1.05 (0.46-8.27) vs 0.87 mmol/l (0.31-11.07); p=0.0302], both in the whole cohort and in patients with normoalbuminuria at baseline. After adjustment for systolic blood pressure (SBP), diabetes duration, gender, stage of complications at baseline and glycohemoglobin (HbA1c), the relative risk for progression was 2.01 (95% CI: 1.07-3.77) for nephropathy and 2.30 (95% CI: 1.03-5.12) for retinopathy for patients having serum TG in the highest tertile, compared to the others. This result persisted when only patients with normoalbuminuria were consIdered. CONCLUSION: High triglycerIde levels are an independent predictive factor of both renal and retinal complications in patients with type 1 diabetes.

Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate.

BACKGROUND: Platelet activation plays an important role in arterial thrombosis and the widespread use of aspirin has reduced major events by 25% in the secondary prevention of cardiovascular diseases. However, it appears that aspirin antiplatelet effect is not uniform and 8-45% of the population are, in vitro, aspirin resistant, and it is well recognized that platelets can be activated by pathways that are not blocked by aspirin, such as adenosine diphosphate (ADP). OBJECTIVES: To investigate whether aspirin-resistant patients have a modified sensitivity to ADP-induced platelet activation MATERIALS AND METHODS: Seventy-two patients were enrolled. Platelet function was measured by the PFA-100(R) analyser; platelet GP IIb-IIIa activation by ADP 10 micro M was assessed by flow cytometry using PAC-1 MoAb. RESULTS: Using a collagen/epinephrine coated cartridge on the PFA-100(R), the prevalence of aspirin resistance was 29.2% (n=21). For aspirin-resistant patients, the collagen/ADP coated cartridge showed a closure time significantly shorter (p=0.004) compared to the sensitive and control groups. Platelets from aspirin-resistant patients bound PAC-1 significantly more (p=0.03) than the aspirin-sensitive patients and controls when activated with 10 micro M ADP. CONCLUSIONS: Platelets from aspirin-resistant patients appear to be more sensitive and activable by ADP. This hypersensitivity could provide a possible explanation for the so-called aspirin resistance, and this could justify therapeutic improvement with alternative antiplatelet agents.

Resistance to aspirin in vitro at rest and during exercise in patients with angiographically proven coronary artery disease.

BACKGROUND: Acetylsalicylic acid, or aspirin, is widely used in secondary prevention of coronary artery diseases, but the inhibition of platelet aggregation is not uniform in all individuals. OBJECTIVE: To investigate the prevalence of aspirin resistance at rest and during exercise in coronary artery disease patients. MATERIALS AND METHODS: Fifty patients with stable coronary artery disease were prospectively studied. All patients received aspirin (75-300 mg/day for >1 month) and no other antiplatelet therapy. Aspirin resistance was studied, at rest and immediately after a stress test, using the standardized platelet function analyzer (PFA-100(R), Dade-Behring). Aspirin resistance was defined as a normal collagen/epinephrine closure time (<186 s). RESULTS: Ten patients (20%) were aspirin-resistant at rest. Out of the 40 patients who were aspirin-sensitive at rest, 9 (22%) were aspirin-resistant immediately after the exercise stress test. There were no differences in aspirin sensitivity regarding gender, age, diabetes, hypertension, dyslipidemia, platelet count, medical treatment or number of the coronary arteries involved. CONCLUSIONS: Aspirin resistance is detected, at rest, in 20% of our patients with stable coronary artery disease. Aspirin treatment does not seem to protect against exercise-induced platelet activation in 22% of such patients, despite aspirin sensitivity at rest.

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes.

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.

Bright light during nighttime: effects on the circadian regulation of alertness and performance.

The present studies evaluated to what extent duration (all-night or 4-hour exposures) and timing of nocturnal bright light (BL) (beginning or end of the night) modulate effects on vigilance. The results showed that all-night BL exposure is able to alleviate the nocturnal decrements in alertness and performance. However, under certain circumstances, this continuous BL exposure may induce adverse effects on mood and finally reveal to be counterproductive. Shorter BL exposure (4 h) during nighttime helps improve mood and performance, although the effects of short BL pulses were less efficacious than all-night BL exposure. The latter part of the night appears the best time for using the alerting effect of BL. The immediate alerting effect of BL seems to be mediated by a global activation of the central nervous system.

The platelet cytoskeleton regulates the aggregation-dependent synthesis of phosphatidylinositol 3,4-bisphosphate induced by thrombin.

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.

Modifications of bone and connective tissue after orthostatic bedrest.

Eight male volunteers were submitted to a 6-week anti-orthostatic bedrest trial followed by a 1-month reambulation period. We prospectively monitored whole-body composition by dual-energy X-ray absorptiometry, bone and connective tissue metabolism by biochemical markers and calcium regulating hormones by 1-84 parathyroid hormone and 1,25-dihydroxyvitamin D(3). Bone mineral density (BMD) did not vary significantly; however, a trend toward an increase in head BMD and a decrease in trunk, lumbar vertebrae and lower limb BMD was observed. A decrease in the lower limb lean content occurred by day 27 and was maximum by day 42 after the beginning of bedrest; it normalized by day 30 after bedrest. The serum levels of both osteocalcin and C-terminal crosslinked telopeptide of type I collagen increased as a consequence of bedrest. A slight increase in the serum levels of the N-terminal propeptide of type III collagen, a marker of connective tissue metabolism, was observed during the bedrest period. Except for the C-terminal extension propeptide of type I collagen, all markers decreased to baseline pre-immobilization levels during the 1-month recovery phase. Serum PTH and 1,25-dihydroxyvitamin D(3) levels were low during the bedrest period and rose during the reambulation phase. These results seem to reflect early changes in bone and connective tissue metabolism as a result of bedrest unloading, but their order of magnitude remains moderate, thus emphasizing the necessity to perform longer-duration trials.