A blood sample from each mouse was divided into two 100 ml aliquots; one was diluted with 100 ml saline (control), the other with 100 ml saline containing LPS (500 ng/ml final concentration). Samples were incubated at 25uC. At time-points 0 (prior to LPS or vehicle), 30 and 60 min, a 20 ml aliquot was removed from each sample, diluted in 20 ml 2% paraformaldehyde and incubated for an additional 30 min. Each sample was then diluted in 250 ml PBS (0.1 mm pore membrane-filtered) and centrifuged at 456g for 12 min. The supernate, which contained platelets, was removed, placed into a new vial and used for imaging analysis. An aliquot of each platelet suspension was diluted 1:4 in PBS, and a 25 ml drop was placed on a glass slide, cover-slipped and sealed with glue. Platelets were then imaged in dark-field mode using a light microscope (Olympus BX41 with a 1006 oil-immersion lens) coupled to a CytoViva illumination system (CytoViva, Inc., Auburn, AL). Scanning from a corner of each cover-slip, platelets in each field were counted until 100 were totaled. Platelets were categorized according to shape morphology (discoid, irregular, flattened, pseudopodia) or whether exhibiting membrane granules or in aggregates (each aggregate was counted as 1). The number of platelets in each category was expressed as percentage of total 100 platelets counted.