Blood, Vol. 113, Issue 5, 1139-1148, January 29, 2009

Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins
Blood Yun et al. 113: 1139

Supplementary materials for: Yun et al

Measurement of plasminogen and FVII activation rates
For determining plasminogen activation rates, bacteria (4 × 10⁸ cfu/mL to 1 × 10⁷ cfu/mL) were combined with human Glu-plasminogen (1 µM) and the chromogenic substrate, Spectrozyme PL (0.5 mM) in 200 µL MES buffer (50 mM 2-(N-morpholino) ethanesulfonic acid, 100 mM Tris, 0.1% Tween 80, pH 7.4). Plasmin formation was monitored for 2 hours at 37°C in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA) by continuously measuring the plasmin-catalyzed change in A₄₀₅ caused by p-nitroaniline released from Spectrozyme PL. The resulting plot of A₄₀₅ versus time was parabolic with the slope of the curve at any point proportional to the instantaneous plasmin activity (Fig. S1A). Rates of plasminogen activation were calculated by fitting the parabolic tracings to a second order polynomial of the form A₄₀₅= i + mt +Ct² where i is the Y-intercept (initial A₄₀₅), m is the initial slope of the curve, C is the rate of change of slope (quadratic constant), and t is time (minutes). Plasminogen activation rates were derived from C values via reference to the amidolytic activity of purified plasmin toward Spectrozyme PL. Bacteria exhibited negligible ability to hydrolyze this substrate. The assay is validated by the linear dependence of the rate of plasminogen activation on cell concentration (Fig. S1B). A similar procedure was used to quantify FVII activation rates, except that bacteria (2 × 10⁸ cfu/mL) were combined with purified FVII (200nM), recombinant soluble human tissue factor (1 µM), and the chromogenic substrate, Spectrozyme VIIa (0.5 mM), in MES buffer containing 0.1% PEG₈₀₀₀. FVII activation rates were determined via reference to the amidolytic activity of purified FVIIa.

Files in this Data Supplement:

• Figure S1. Plasminogen activation rates (JPG, 56.7 KB) -
  Rates of plasminogen activation were determined for various bacterial strains by incubating bacteria (10⁸ cfu/mL) with human Glu-plasminogen (1 µM) at 37°C and analyzing plots of plasmin activity generated by bacteria over time. (A) Representative progress curves of plasmin formation obtained by incubating either Y. pestis KIM5 (closed circles) or Y. pestis Δpla (open circles) with plasminogen. (B) Plot of initial rates of plasminogen activation versus concentration of Y. pestis KIM5 cells, to which a line was fitted using Sigmaplot (r² > 0.999). Data are mean ± SEM (n=3).