The mean age was 31 years, and all subjects had vital signs and hematologic measurements within normal limits.

Mean 1 mM AA-induced maximum PA was ~ 76% by LTA at pre-dose, and it reached a 20% PA threshold at 11 ± 11 min with 162 mg and 7 ± 3 min with 650 mg ASA (p = NS) (Fig. 1a). With VN Aspirin test, the mean ARU levels were > 630 at pre-dose in both groups indicating that all subjects were ASA naïve prior to dosing, which reached below the 550 threshold by 20 ± 7 min with 162 mg and 13 ± 7 min with 650 mg ASA (p = 0.07) (Fig. 1b).

a 1 mM Maximum AA-Induced Platelet Aggregation. b Aspirin Reaction Units. c Serum Thromboxane B2. d Inhibition of Serum Thromboxane B2. e Plasma Acetylsalicylic Acid. f Plasma Salicylic Acid

Pre-dose TxB2 levels were 350 and 261 ng/ml and reduced to 1.1 and 0.4 ng/ml at 60 min with 162 and 650 mg ASA, respectively (p < 0.001). 95% inhibition of serum TxB2 level (therapeutic ASA response) was achieved at 38 ± 22 min and 22 ± 8 min with the 162 and 650 mg ASA doses, respectively (p = NS) (Fig. 1c, d).

There was a gradual increase in plasma exposure to ASA and SA after ASA administration reaching maximum plasma ASA and SA levels of ~ 10,000 and ~ 27,000 ng/ml within 60 min with 162 mg and 650 mg ASA, respectively (Fig. 1e, f).

A good correlation was observed between VN ARU and 1 mM AA-induced maximum PA (r = 0.69, p < 0.001), serum TxB2 levels (r = 0.74, p < 0.001) and inhibition of serum TxB2 (r = 0.79, p < 0.001) (Fig. 2a–c). A strong correlation was observed between 1 mM AA-induced PA and serum TxB2 levels (r = 0.82, p < 0.001) and inhibition of serum TxB2 (r = 0.90, p < 0.001 (Fig. 2d, e).

a Correlation between Aspirin Reaction Units and Arachidonic Acid-induced Platelet Aggregation. b Correlation between Aspirin Reaction Units and Serum Thromboxane B2. c Correlation between Aspirin Reaction Units and Inhibition of Serum Thromboxane B2. d Correlation between Arachidonic Acid-induced PA and Serum Thromboxane B2. e Correlation between Arachidonic Acid-induced PA and Inhibition of Serum Thromboxane B2

Receiver Operator Characteristic (ROC) curve analysis indicated that ARU 550 was associated with ≥ 81 ng/ml serum TxB2 with an AUC 0.940 (specificity = 89.66, sensitivity = 90.91, p < 0.001) and a cut point of ≤ 558 ARU was associated with > 95% inhibition of serum TxB2 with an AUC = 0.912 (specificity = 74.47, sensitivity = 95.65, p < 0.001). ROC curve analysis indicated that a cut point of ≤ 7% maximum 1mM AA-induced PA was associated with > 95% inhibition of serum TxB2 with an AUC 0.728 (specificity = 54.35, sensitivity = 100, p < 0.001).

The correlation between PK and PD measurements is shown in the Supplementary Figures 1a-d). Regression analyses demonstrated a correlation between ASA levels and serum TxB2 (r = 0.59, p < 0.001), inhibition of serum TxB2 (r = 0.75, p < 0.001) and ARU (r = 0.68, p < 0.001). The cutoff value of 686 ng/ml of ASA was correlated well with 1 mM-AA induced PA and ARU levels.

ROC curve analysis indicated that a cut point of > 686 ng/ml ASA was associated with > 95% inhibition of serum TxB2 (change from pre-dose) with an AUC 0.887 (specificity = 64.58%, sensitivity = 100%, p < 0.001) and a cut point of > 4907 ng/ml SA was associated with > 95% inhibition of serum TxB2 with an AUC 0.976 (specificity = 89.58%, sensitivity = 100%, p < 0.001).

In addition, the 686 ng/ml ASA cut off point was associated with ≤ 7% 1 mM AA-induced PA by LTA (AUC = 0.773, specificity = 76.19 and sensitivity = 95.00%, p = 0.001) and ≤ 585 ARU (AUC = 0.936, specificity = 90.48%, sensitivity = 92.68%, p < 0.001). Similarly, the 4907 ng/ml SA cut off point was associated with ≤ 7% 1 mM AA-induced PA by LTA (AUC = 0.716, specificity = 54.55 and sensitivity = 100%, p = 0.0015) and ≤ 558 ARU (AUC = 0.955, specificity = 81.82%, sensitivity = 96.55%, p < 0.001).