Analysis of Clot Lysis Time

We used two methods to assess clot lysis time (CLT) to mimic two distinct in-vivo physiological conditions. First, to represent CLT in stagnant (zero shear) conditions, we measured turbidity using light transmittance (SpectraMax, Molecular Devices; Sunnyvale, CA). Second, to measure CLT under shear conditions, we assessed loss of mechanical stiffness, using TEG (Haemoscope 5000; Braintree, MA). For both assays, plasma comprised 50% of the total mixture volume. Turbidity was measured at 405nm at 37°C [18]. Coagulation was initiated by inducing clotting with calcium chloride at a final concentration of 15 mM, human tissue factor at a final concentration of 0.6 pM (Dade Innovin; Siemens, USA), and phospholipids, DOPC:DOPS (7:3, w/w), at a final concentration of 12 μM (Avanti Polar Lipids; Alabaster, Al) [18–20]. To induce fibrinolysis, tissue plasminogen activator (tPA) (Alteplase, Genentech; San Francisco, CA) was immediately added to the plasma prior to clot formation at a final concentration of 60 ng/mL. Tris-buffered saline (50 mM Tris-HCl, 0.1 M NaCl, pH 7.4) was used as a buffering agent. Calcium chloride, tissue factor, phospholipid mixture, tPA, and buffer were mixed in disposable TEG cups containing heparinase (Haemonetics Corporation; Braintree, MA) prior to addition of plasma. Following mixing of reagents, human plasma was added to start the reaction. 100 μL of volume was transferred from the TEG cup to a 96 well plate in duplicate, and allowed to run on the spectrophotometer at 23°C. The remaining reaction volume was run using TEG. The CLT is derived from a clot-lysis profile and defined as the time between the midpoint of the rise to maximum turbidity or shear stress during coagulation, to the midpoint of the return back to baseline turbidity or shear stress during clot lysis (S1A–S1D Fig).