Blood was collected from the retro-orbital sinus plexus (250-300 mL/mouse) of wild type and dTLR4 mice through siliconized capillary tubes coated with hirudin (thrombin inhibitor) and soybean trypsin inhibitor (STI, Factor Xa inhibitor) into 1.5 mL polypropylene tubes containing 20 mL of 100 mM hirudin and 1 mM STI [13] . For in vitro experiments, anticoagulated blood was aliquoted into pairs of tubes within 30 min after collection so that measurements from a vehicle-treated, control tube and LPStreated tube could be analyzed from each mouse at each time point. Vehicle (saline) or LPS (500 ng/mL) was added to one of each paired aliquots of blood. At designated time points (5, 30, and 60 minutes) after addition of vehicle or LPS, 10 mL whole blood from each aliquot was diluted into 990 mL (1:100) HEPES/ HANKS' buffer (130 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2 , 0.8 mM MgSO 4 , 0.44 mM Na 2 HPO 4 , 20 mM HEPES, pH 7.4) with 1 mg/ml albumin and 1 mM STI for staining of platelets positive for leukocyte antigen and leukocyte positive for platelet antigen. The remaining blood was used to prepare platelet free plasma (PFP) for MV analysis.