Fig. 4. The K2 domain of tPA interacts with the N terminus of apoB.

(A) A solid-phase binding assay was used to measure the interaction between immobilized LDL and recombinant human WT tPA (tPA-WT) or enzymatically inactive tPA-S513A. The binding of tPA-WT or tPA-S513A to wells without LDL is also measured under the same conditions as the control. OD, optical density. (B) A solid-phase binding assay was used to measure the interaction between recombinant human tPA and immobilized purified human MTP complex. (C) A solid-phase binding assay was used to measure the ability of tPA to inhibit the binding of MTP to immobilized LDL. (D) SPR was used to measure the interaction between human recombinant tPA and LDL. (E) A solid-phase binding assay was used to test whether anti-apoB N-terminal antibody (1D1) versus control IgG (raised against the β3 domain of apoB) blocks the binding between human recombinant tPA and immobilized LDL. (F) Human primary hepatocytes were transduced with the plasmid encoding WT tPA (tPA-WT), a tPA mutant without the K2 domain (tPA-Δ-K2), or tPA mutated in the K2 domain lysine binding site (tPA-D236, 238N). apoB secretion was measured by [³H]-labeling, as in Fig. 2. (G) A solid-phase binding assay was used to test whether an antibody against tPA-K2 domain interferes with the interaction between human recombinant tPA and immobilized purified LDL. (H) A solid-phase binding assay was used to measure whether tranexamic acid (TXA) interferes with the interaction between recombinant human tPA and immobilized purified LDL. (I) A schematic diagram depicting the interaction between the N terminus of apoB and the K2 domain of tPA. The diagram was generated using biorender.com. Data are shown as means ± SEMs; *P < 0.05 by one-way ANOVA followed by Dunnett’s test. n.s., not significant (P ≥ 0.05).