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• Figure I - (EPS) (575 KB) A PAR1-antibody completely blocks both the direct PAR1 contribution and its helper effect on PAR4 during thrombin-dependent platelet aggregation. Human platelets were gel-purified and aggregation measured as described in Figure 1. Platelets were pre-incubated with buffer (untreated), 1 μM RWJ-56110, 1 μM RWJ-56110, and 74 μg/mL PAR1-Ab, or 74 μg/mL PAR1-Ab before the addition of various concentrations of thrombin.
• Figure II - (EPS) (932 KB) Bivalirudin exhibits mixed non-competitive inhibition with the PAR4 cleavage peptide, N-Ac-PAPR-pNA. The PAR4 chromogenic substrate N-Ac-PAPR-pNA was dissolved in 20 mM Tris-HCl, pH 8.3 and 150 mM NaCl (TBS). Cleavage assays comprised 10-100 μM PAR4 peptide in TBS plus 0-30 nM bivalirudin and were initiated by the addition of thrombin freshly diluted in ice-cold TBS/0.1% PEG-8000 (final thrombin concentration = 208 pM) at 37 °C in a 96-well format. Initial rates were determined from the initial slopes of the progress curves using a SPECTRAmax 340 microplate spectrophotometer and SOFTmax PRO version 2.1. Quantification of p-nitroanilide product was determined using the extinction coefficient (ε₄₀₅ = 9890 M^-¹cm^-¹) and data were fit to the Michaelis-Menten equation. Inhibition data were tested against 12 inhibition models (linear, hyperbolic and parabolic models of competitive, noncompetitive, uncompetitive and full noncompetitive inhibition) by non-linear least-squares regression analysis as described in detail.¹¹ Inhibition of thrombin cleavage of N-Ac-PAPR-pNA by bivalirudin fit best (P<0.008) to a mixed non-competitive (NC) model (K_i = 3 nM) using Akaike's Information Criterion. The mechanism of inhibition is apparent from the replots: non-competitive gives increasing y-intercept and slope with increasing [I], whereas slope is unchanged for uncompetitive and y-intercept is unchanged for competitive inhibition. The replots are linear, therefore there is no partial inhibition (hyperbolic replots) and mixed behavior was from the alpha value (=3) from the nonlinear fit and from the intersection above the x-axis in the Lineweaver-Burke plot.
• Figure III - (EPS) (351 KB) The fluorescence resonance energy transfer (FRET) interaction between PAR1-CFP (donor) and PAR4-YFP (acceptor) is saturable and emanates primarily from the plasma membrane of COS7 cells. FRET was measured between PAR1Δ377-CFP and PAR4Δ348-YFP using COS7 cells co-expressing both receptors. A, Fluorescence measurements were conducted with transiently-transfected cells expressing differing amounts of PAR4Δ348-YFP at a concentration of 0.5 x 10⁶ cells/mL with excitation at 425 nm, emission at 527 nm, and 10-nm slit widths. All samples were analyzed for CFP and YFP content by fluorescence to control for receptor expression. B, Confocal FRET was performed on a Leica TCS SP2 instrument, with excitation using Ar (458/5mW, 514/20mW) lasers. PAR1-CFP emission was collected at 460-500 nm and PAR4-YFP was collected at 520-550 nm. Spectral bleed-through of the donor emission (average 36% of the CFP signal) into the acceptor channel and cross excitation of the acceptor by donor excitation (average 53% of the YFP signal) were determined and subtracted from the FRET scan (458 nm excitation, 520-550 nm emission) using ImageJ software to give the corrected FRET signal.