Direct observation of the anchoring process during the adsorption of fibrinogen on a solid surface by force-spectroscopy mode atomic force microscopy.
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Atomic force microscopy in a force-spectroscopy mode has been used to investigate the kinetics of the adsorption process of fibrinogen molecules on a silica surface. An original "approach/retraction" cycle of the tip/surface was used for this purpose. Fibrinogen molecules were adsorbed on the atomic force microscopy tip and were brought into contact with the silica surface for different interaction times varying from 5 to 2,000 ms. Multiple consecutive ruptures were observed. The mean number of ruptures nr per cycle increases steadily with the interaction time as well as the mean strength fr which varies from 300 pN for 5 ms to 1,400 pN for 2,000 ms. The minimal interaction time for a fibrinogen molecule to bind strongly to a silica surface during an adsorption process appears to lie between 50 and 200 ms. The histograms of the distances between two consecutive ruptures in one cycle exhibit maxima around 20-25 nm. This length is comparable to the characteristic distance between D and E globules of one fibrinogen molecule and suggests that fibrinogen molecules mainly adsorb through their D and E globules. (+info)
Measuring plasma fibrinogen to predict stroke and myocardial infarction: an update.
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Plasma fibrinogen is a major determinant of platelet aggregation and blood viscosity. The decrease in plasma fibrinogen by bezafibrate is associated with a decrease in the risk of reinfarctions. To strengthen the predictive value of plasma fibrinogen with respect to cardiovascular risk, we performed a meta-analysis of studies conducted between 1984 and 1998. Emphasis has been put on the relationship between high levels of plasma fibrinogen and fatal and/or nonfatal cardiovascular events in both the general population and in patients with previous cardiovascular events. Twenty-two studies (13 prospective, 5 cross-sectional, and 4 case-control) addressing the association between fibrinogen plasma concentrations and cardiovascular disease were analyzed. The overall estimate of risk of cardiovascular event in subjects with plasma fibrinogen levels in the higher tertile, was twice as high as that of subjects in the lower one (odds ratio, 1.99; 95% confidence interval, 1.85 to 2.13). High plasma fibrinogen levels were associated with an increased risk of cardiovascular disease in healthy as much as in high-risk individuals. A metaregression showed no confounding effects attributable to selected characteristics of retrieved studies. A subgroup analysis (study design, follow up, mean fibrinogen levels, percentage of smokers, and mean age) allowed us to conclude that fibrinogen is an independent risk factor for cardiovascular disease; that it interacts with major determinants of myocardial and cerebrovascular ischemia; and that, in secondary prevention studies, it enhances by 8% the prediction of future events by established risk factors. Thus, fibrinogen measurements should be encouraged to refine the overall risk profiles of individuals and to better tailor preventive interventions. (+info)
Activation of haemostasis by exercise, mental stress and adrenaline: effects on platelet sensitivity to thrombin and thrombin generation.
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Stress-induced activation of haemostasis may be involved in the triggering of acute coronary syndromes. We compared the effects of mental stress, dynamic exercise and adrenaline infusion on platelet sensitivity to thrombin using flow-cytometric analysis of platelet fibrinogen binding in whole blood, and platelet aggregability using filtragometry ex vivo, in healthy volunteers. Furthermore, we assessed thrombin generation [prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes in plasma] and thrombin activity (fibrinopeptide A in plasma). Exercise (bicycle ergometry) enhanced thrombin-induced platelet fibrinogen binding (P<0.05) and platelet aggregability (P<0.01), and elevated F1+2, thrombin-antithrombin complexes and fibrinopeptide A (P<0.05 for all three). Adrenaline infusion enhanced thrombin-induced platelet fibrinogen binding and platelet aggregability (P<0.05), and elevated thrombin-antithrombin complexes (P<0.05), whereas F1+2 and fibrinopeptide A levels were not significantly affected. Mental stress increased platelet sensitivity to high concentrations of thrombin only, and produced small increases in levels of thrombin-antithrombin complexes. Time control experiments showed no important changes with repeated measurements during rest. Platelet responses to exercise and adrenaline were reversible, with recovery 60 min later. Thus, heavy exercise and high levels of adrenaline reversibly increased platelet aggregability and platelet sensitivity to thrombin, and enhanced thrombin formation; the effects were most pronounced during exercise. Mental stress only weakly affected these parameters. (+info)
Hemorheology and walking of peripheral arterial occlusive diseases patients during treatment with Ginkgo biloba extract.
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AIM: To study the effects of Ginkgo biloba extract 761 (GbE) from the points of view of hemorheology for patients of peripheral arterial occlusive diseases (PAOD). METHODS: The treatment with GbE (240 mg.d-1, po) and the pain-free walking distance (PFWD) were carried out for 24 PAOD patients (12 nondiabetic, ND and 12 diabetic, D) over 48 wk. The parameters erythrocyte stiffness (ES) and relaxation time (RT), the blood plasma viscosity (eta), the plasma fibrinogen concentration (Cf) and the blood sedimentation rate (BSR), the PFWD, and maximal walking distance (MWD) were determined at 6 wk before treatment (-6), at the beginning of the treatment (0), and after 6, 11, 16, and 48 wk of treatment. RESULTS: At wk -6, ES and RT of both the ND- and D-group were not significantly different from a healthy control group. At wk 0, stiffness and RT were significantly higher than healthy control, and the mean PFWD was only 111 m. The eta value was significantly elevated and Cf and BSR were enhanced. Throughout 11 wk of treatment ES, RT, eta, and Cf decreased gradually and PFWD improved. Between 16 and 48 wk, ES, and RT were no longer significantly different from the controls, whereas eta and Cf decreased gradually but remained higher than normal, BSR decreased, and the PFWD improved by a factor of 3.8 times (D) and 3.3 times (ND). CONCLUSION: GbE gives therapeutic effects in PAOD patients. (+info)
Contribution of distinct adhesive interactions to platelet aggregation in flowing blood.
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Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ibalpha and von Willebrand factor (vWF) acted in synergy with alphaIIbbeta3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes. (+info)
Altered flow properties of blood and increased plasma fibrinogen in cyclosporin-treated renal allograft recipients.
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BACKGROUND: Abnormalities in blood rheology may be factors contributing to cardiovascular complications and the progression of renal failure in kidney allograft recipients. The haemorheological variables haematocrit, fibrinogen, whole blood viscosity, plasma viscosity, erythrocyte aggregation tendency and fluidity were measured in 27 cyclosporin A (CyA)-treated patients who had received a renal graft at least 6 months previously. Their creatinine clearance was in the range of 12-92 ml/min/1.73 m2 (mean 55+/-19). The values were compared with those obtained from a control group comprising 20 healthy subjects matched according to age, sex and smoking habits. RESULTS: The haematocrit, plasma fibrinogen, whole blood viscosity, plasma viscosity, erythrocyte aggregation tendency, body mass index (BMI), mean arterial pressure (MAP) and serum triglycerides were increased in the transplanted patients, and the serum high density lipoprotein (HDL)-cholesterol and erythrocyte fluidity decreased. The haemorheological variables were used as dependent variables in a stepwise regression analysis with age, MAP, BMI, urinary albumin excretion rate, blood CyA concentration, creatinine clearance, and serum triglycerides, cholesterol and HDL-cholesterol as independent variables. Plasma fibrinogen was positively correlated with BMI and blood CyA. The whole blood viscosity was positively correlated with blood CyA and negatively with serum HDL-cholesterol. Only serum triglycerides remained correlated with erythrocyte aggregation tendency. CONCLUSIONS: All variables with a known impact on blood viscosity were altered in the present group of renal transplant recipients. Inappropriate regulation of erythrocyte formation, overweight, the use of CyA, high triglycerides and low HDL-cholesterol levels may be factors contributing to this. The importance of impaired flow properties of blood for the development of cardiovascular diseases and transplant glomerulosclerosis needs to be examined. (+info)
Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing.
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We have developed biodegradable fibrin-derived microbeads as potent cell carriers. The fibrin-derived microbeads, 50-200 microm in diameter, were tested for their attachment to a wide range of cell types. Fibrin-derived microbeads were shown to be greatly haptotactic to cells (such as endothelial cells, smooth muscle cells and fibroblasts), which respond to fibrinogen in contrast to keratinocytes and different cell lines derived from leukocytic lineage. The cells on fibrin-derived microbeads could be maintained for more than 10 d and achieved a high density. 31P-nuclear magnetic resonance was employed to monitor phosphate metabolism in cells, with densities on the order of 100 million cells per g of fibrin-derived microbeads. The 31P-nuclear magnetic resonance adenosine triphosphate and phosphocreatine signals, equivalent to the signal obtained with perfused normal skin, indicated that metabolism of cells on fibrin-derived microbeads was responsive to oxygenation and nutrients. Light, fluorescent, and confocal laser microscopy revealed that the porous fibrin-derived microbeads accommodate up to 200-300 cells due to their high surface area which minimized contact inhibition. Cells could degrade the fibrin-derived microbeads and be transferred to seed culture flasks without trypsinization. In a pig skin wound healing model, fibrin-derived microbeads + fibroblasts were transplanted into full thickness punch wounds. This procedure was compared with other treatment modalities, such as the addition of human platelet-derived growth factor BB or fibrin-derived microbeads alone. By the third day after wounding, only the wounds in which fibroblasts on fibrin-derived microbeads were added showed significant formation of granulation tissue. Based on the above, we project many uses of our novel fibrin-derived microbead technology for cell culturing, wound healing and tissue engineering. (+info)
PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells.
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To better understand the means by which cells such as human platelets regulate the binding of the integrin alphaIIbbeta3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses alphaIIbbeta3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an alphaIIbbeta3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated alphaIIbbeta3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42MAPK, phospholipase A2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent ('outside-in') signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support alphaIIbbeta3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of alphaIIbbeta3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells. (+info)