Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.

Impedance biosensor for real-time monitoring and prediction of thrombotic individual profile in flowing blood.

A new biosensor for the real-time analysis of thrombus formation is reported. The fast and accurate monitoring of the individual thrombotic risk represents a challenge in cardiovascular diagnostics and in treatment of hemostatic diseases. Thrombus volume, as representative index of the related thrombotic status, is usually estimated with confocal microscope at the end of each in vitro experiment, without providing a useful behavioral information of the biological sample such as platelets adhesion and aggregation in flowing blood. Our device has been developed to work either independently or integrated with the microscopy system; thus, images of the fluorescently labeled platelets are acquired in real-time during the whole blood perfusion, while the global electrical impedance of the blood sample is simultaneously monitored between a pair of specifically designed gold microelectrodes. Fusing optical and electrical data with a novel technique, the dynamic of thrombus formation events in flowing blood can be reconstructed in real-time, allowing an accurate extrapolation of the three-dimensional shape and the spatial distribution of platelet thrombi forming and growing within artificial capillaries. This biosensor is accurate and it has been used to discriminate different hemostatic conditions and to identify weakening and detaching platelet aggregates. The results obtained appear compatible with those quantified with the traditional optical method. With advantages in terms of small size, user-friendliness and promptness of response, it is a promising device for the fast and automatic individual health monitoring at the Point of Care (POC).

Visualization of nitric oxide production by individual platelets during adhesion in flowing blood.

Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of l-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca(++) elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca(++) elevation. Simultaneous measurement of NO and Ca(++) indicated that NO production in individual platelets is preceded by Ca(++) elevations, with a lag phase of 33 ± 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca(++) elevation elicits the production of NO which, in turn, modulates thrombus size.