Skeletal muscle myosin promotes thrombin generation in PRP and PPP. Freshly prepared PRP and PPP (30 μL) from the same donor was incubated with various indicated concentrations of myosin for 10 min at 37°C. Then, fluorogenic thrombin substrate solution (I-1140) either with TF (Innovin; final, 0.5 pM) and CaCl2 (final, 11 mM) or with CaCl2 alone (final, 11 mM) was added to the plasma/myosin mixture (total final volume, 110 μL) to initiate thrombin generation at 37°C. For TF-induced thrombin generation assays using PRP and PPP, corn trypsin inhibitor (CTI; final, 50 µg/mL) was added to freshly prepared PRP and PPP immediately after blood was processed to obtain PRP and PPP. Thrombin generation was followed continuously using a SPECTRAmax GEMINI XS fluorometer (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths set at 360 nm and 460 nm, respectively. The first derivative of fluorescence versus time was used to produce thrombin generation curves with the correction for substrate consumption and inner filter effect.20 Thrombin generation is shown for PRP plus 0.5 pM TF-Ca++ and CTI (A) or for only Ca++ addition (C), or for PPP plus 0.5 pM TF-Ca++ and CTI (B) or for only Ca++ addition (D). The same sets of experiments were done for 4 different adult healthy blood donors, and data for 1 donor’s PRP and PPP are shown.
