NPP4 promotes platelet aggregation in the presence of AP3A. Light transmission aggregometry was used to assess aggregation in response to agonists in platelet-rich plasma. Data are shown graphically as a percentage of light transmittance (y-axis) over time (x-axis). (A) Increasing concentrations of Ap3A demonstrate only a primary wave of aggregation followed by rapid platelet disaggregation. (B) Dose-dependent response of Ap3A in the presence of 100nM NPP4 demonstrates that low micromolar amounts of Ap3A are sufficient to induce robust platelet aggregation. (C-D) Dose dependence of NPP4 in the presence of 80μM Ap3A. Both the primary and secondary waves of platelet aggregation are dependent on NPP4 concentration, without evidence for disaggregation over the 10-minute time course of the experiments. In the absence of NPP4, 80μM AP3A elicits only a primary wave of aggregation followed by rapid disaggregation (blue curves, C-D). An inactive mutant form of NPP4 in which the catalytic threonine has been mutated to an alanine (T70A, green curve in panel D) fails to induce aggregation at the 100nM concentration.