Computational protocol: Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

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[…] Forty-five microliters of normal citrated plasma diluted 1/2 with TBS were clotted upon the addition of 5 ul activation mix containing CaCl2 (final concentration 5 mM) and either thrombin (final concentration 0.88 U/ml (24 nM)) or different concentrations of MASP-1 (final concentrations 1 µg/ml (22 nM), 5 µg/ml (110 nM) or 10 µg/ml (220 nM)). As described earlier , clots were made in specially devised small perforated plastic vessels, incubated in a moist chamber for 2 hours followed by washing with sodium cacodylate buffer and subsequently fixed for 30 mins in 2% glutaraldehyde. Clots were recovered and further processed by a stepwise dehydration with acetone gradient and sputter coated with platinum palladium. Samples were viewed and photographed using a field emission gun, environmental scanning electron microscope (FEI, Quanta 200F, Hillsboro, USA). Ten images from different areas at three different magnifications (5000×, 10000×, 30000×) were taken per clot and visually examined by several investigators blinded to the clotting agent. Fiber diameters were measured with image analysis software package ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, http://imagej.nih.gov/ij). Ten fibers per image, corresponding to 100 fibers per clot, were measured. Fiber diameter data were normally distributed and compared between the four groups of clots (thrombin, MASP-1 10 µg/ml, MASP-1 5 µg/ml, MASP-1 1 µg/ml) by Oneway ANOVA with post-hoc Bonferroni analysis (SPSS Statistics software, version 17.0.0). […]

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