Mechanism of SIPAct.
(A) Platelets from PPACK anticoagulated blood were diluted in HEPES containing 1.5 mM Ca2+ and 100 μM PPACK to achieve concentrations of 10 × 106cells/mL. This suspension was either incubated at 37°C under static conditions (○) or was presheared at 9600/s for 1 minute (■). In both cases, at t = 0, 15 μg/mL purified VWF was added and shear at 9600/s was applied. In control runs (●), diluted PRP with platelets at 10 × 106cells/mL was sheared in the viscometer at 9600/s in the absence of exogenous VWF for the duration of the experiment. (B) Citrated PPP was placed on the 37°C plate of the viscometer under static conditions (○) or was presheared in the viscometer at either 9600/s (⋄) or 6000/s (■) for 2 minutes. At t = 0, PRP was added such that the final platelet concentration was 10 × 106cells/mL, and all samples were sheared at 9600/s. (C) Citrated PRP at 300 × 106platelets/mL was placed in a cone-plate viscometer at t = −15 seconds, and shear was applied at 9600/s for 15 seconds. At t = 0, the suspension was diluted 30-fold in HEPES containing 10 μg/mL anti-GpIb monoclonal antibody (mAb) AN-51. Following dilution, shear was maintained at 9600/s for 3 minutes (solid line) or was reduced to 1891/s (dashed lines). (D) PRP at 300 × 106platelets/mL was sheared in the viscometer at 3251/s for 45 seconds (solid line) or was maintained under static conditions (dashed line) starting at t = −45 seconds. At t = 0, the suspension was diluted 30-fold as described in panel C, and the shear rate was increased to 9600/s. *Both the increase in platelet activation following dilution, and the absolute level of platelet activation were significantly higher than all other treatments in panels C and D. Data are mean ± SEM for n = 3-5.
