Fig. 4

N-terminal aspartic acids (D5 and D13) of apoA-IV are required for its inhibitory effect on platelets. a Schematic map of human apoA-IV. b Deletion of both the N-terminus and C-terminus of human apoA-IV abolished the inhibition of platelet aggregation induced by TRAP (250 μM) (39–335 vs Control: NS; ApoA-IV vs Control: P < 0.01). c, d Recombinant apoA-IV lacking the N-terminus (Δ1–38) abolished the inhibition of platelet aggregation (Δ1–38 vs Control: NS), while only deletion of the C-terminus (Δ336–376) enhanced the inhibition induced by (c) thrombin (0.5 U) and (d) ADP (2.5 μM) (Δ336–376 vs ApoA-IV: P < 0.05). e Point mutation of aspartic acids (D5E or D13E) at the apoA-IV N-terminal attenuated the inhibition of platelet aggregation (D5E and D13E vs ApoA-IV: P < 0.05). f–i Double D (D5 and D13) mutant human apoA-IV (f, g) and mouse apoA-IV (h, i) abrogated the inhibition of platelet aggregation (DM vs Control: NS; ApoA-IV vs Control: P < 0.01). Area under the curve of each group was compared. j Synthetic peptides of N-terminal 20 amino acids (N-20) of apoA-IV inhibited human platelet aggregation, while D5E, D13E or DM mutations on the N-20 synthetic peptides (D5E N-20, D13E N-20 and DM N-20, respectively) attenuated or abrogated its inhibition. For each group, the maximum platelet aggregation was compared to control. Aggregation traces are representative of four independent experiments. rH-DM double D mutant human apoA-IV, rM-DM double D mutant mouse apoA-IV. n = 4. Unpaired, two-tailed Student’s t-test or non-parametric Kruskal-Wallis one-way analysis of variance for multiple paired comparisons. Mean ± SEM. NS not significant, *P < 0.05 and **P < 0.01. Scale bars: 2 minute (b-j)