Targeting the RT loop of Src SH3 in Platelets Prevents Thrombosis without Compromising Hemostasis

Figures

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  Figure 1. Identification of antithromobotic targets based on the interaction of Src SH3 with integrin β3 that do not compromise hemostasis. a) The comparison of binding modes results of β3 heptapeptide (NITYRGT) and a class II peptide APPIPPPR with SH3 domain and the schematic representation of the structure of Src SH3 domain. The full length Src structure (PDB code: 2H8H)[ 52 ] was shown as cartoon with grayish‐white color while its SH3 domain was colored in salmon. RT loop and n‐Src loop of SH3 were colored in red and magenta respectively. Peptides and the interested residues in the top panel were shown as sticks with different colors. The carbon atoms in heptapeptide and APPIPPPR peptide were colored in light blue and yellow, respectively. The binding mode of heptapeptide with SH3 were obtained by molecular docking using Glide program (Schrödinger, LLC, New York, NY, 2015), and the binding mode of APPIPPPR with SH3 were obtained from X‐ray crystal structure with PDB code 4QT7.[ 19 ] Down panel is the schematic representation of the structure of Src SH3 domain and the positions of the mutant amino acids. b) Co‐IP results show the interaction of integrin β3 with WT or mutant (R95A, E97A,…

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  Figure 2. Small molecule DCDBS84 specifically binds to the RT loop of Src SH3. a) The workflow of screening for small molecules targeting SH3. b) Structural formula of DCDBS84. The dissociation constant (Kd) of DCDBS84 binding to Src SH3 was calculated by SPR. c) Chemical shift perturbation (CSP) analysis of Src SH3 upon the binding of DCDBS84. Mean (solid line) and mean + SD (standard deviation) (dashed line) values were indicated. Residues with CSP values over the dashed line were labeled. d) Src/DCDBS84 interaction characterized by HDX‐MS. Percentage change of deuterium uptake at 60 min was mapped onto the crystal models of Src (PDB code: 2SRC),[ 56 ] deuterium uptake was labeled as the color bar indicated. e) The Kd of the DCDBS84 binding to purified mutant (R95A, E97A, G116A, W118A, and Y131A) Src SH3. f) The proposed binding mode of DCDBS84 with SH3 domain. The SH3 domain and DCDBS84 were shown as cartoon and sticks, respectively. The interacting residues were also shown as sticks. DCDBS84 was colored cyan. The RT loop and n‐Src loop of SH3 were colored red and blue respectively. The yellow dash lines indicated H‐bond interaction.

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  Figure 3. Small molecule DCDBS84 disrupts the interaction of integrin β3 with Src SH3, affecting the phosphorylation of β3 and Src. a) Combination between purified GST‐integrin β3 cytoplasmic tail fusion protein and purified His‐Src‐SH3 in the presence of DCDBS84 at different concentrations (20, 40, 80 µmol L−1), dimethylsulfoxide (DMSO) or control (without treatment of DCDBS84 or DMSO), was detected by a pull‐down assay. b) Platelet lysates were incubated with 80 µmol L−1 DCDBS84, DMSO or control (without treatment of DCDBS84 or DMSO), immunoprecipitated by anti‐integrin β3 antibody and immunoblotted with anti‐Src antibody. c) Platelets from volunteers were treated with or without thrombin (0.1 U µL−1) and incubated with 80 µmol L−1 DCDBS84, 25 µmol L−1 integrilin (positive control), DMSO or control (without treatment of DCDBS84, integrilin or DMSO), protein lysates were immunoblotted with antibodies specific for β3, phosphorylated β3 (β3747 and β3759), Src and phosphorylated Src (Src416 and Src527).

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  Figure 4. DCDBS84 selectively inhibits platelet functions mediated by outside‐in signaling. a) Effects of DCDBS84 on platelet spreading on immobilized fibrinogen at different concentrations (20, 40, 80 µmol L−1), DMSO, 1 mmol L−1 RGDS or control (without treatment of DCDBS84, RGDS, or DMSO). Scale bar is 20 µm. b) Effects of DCDBS84 on platelet stable adhesion on immobilized fibrinogen at different concentrations (20, 40, 80 µmol L−1), data shown in the pattern (mean ± SD) were derived from the results of three separate experiments (**p < 0.01). Student's t‐test with two‐tailed analysis was performed by using GraphPad Prism. c) Effects of DCDBS84 at different concentrations (20, 40, 80 µmol L−1), DMSO, 1 mmol L−1 RGDS or control (without treatment of DCDBS84, RGDS, or DMSO) on platelet aggregation stimulated by ADP (2 µmol L−1). d) Effects of DCDBS84 on fibrin clot retraction at different concentrations (20, 40, 80 µmol L−1), DMSO, 1 mmol L−1 RGDS or control (without treatment of DCDBS84, RGDS or DMSO). The left panel displayed photographs of the fibrin clots at every 15 min after initiated by 1 U mL−1 thrombin. The right panel analyzed the clot size rate (compared to the primary clot…

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  Figure 5. DCDBS84 effectively suppresses occlusive thrombus formation without affecting bleeding time. a) Quantitative analysis of FeCl3‐induced occlusive carotid artery thrombosis in mice when intravenously injected with DCDBS84 (dissolved in DMSO) at 2.5, 5, and 10 mg kg−1, integrilin (dissolved in saline) at 0.18, 4.2, and 16 mg kg−1), DMSO or saline. Intragastric administration of aspirin (5.4, 125 mg kg−1), clopidogrel (1.25, 10, 200 mg kg−1) or saline was performed at 24 and 2 h prior to the initiation of the carotid artery injury procedure. Data were shown as mean ± SD. Student's t‐test with two‐tailed analysis was performed by using GraphPad Prism. Sample size (n) was showed in each group, from 8 to 11, *p < 0.05 and **p < 0.01. To correlate the in vivo doses (mg kg−1) with the in vitro concentrations (molar concentration), the doses in mg kg−1 could be converted to molar concentrations based on the estimation of mouse blood volume, i.e., 72 mL kg−1 for a 6‐ to 8‐week mouse[ 60 ] (70 mL kg−1 for a human adult).[ 61 ] The estimated molar concentration is thus 68.1 µmol L−1 for 2.5 mg kg−1 DCDBS84 (509.21 Da) and 3 µmol L−1 for 0.18 mg kg−1 integrilin (831.96 Da), and so on. b) Tail…

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  Figure 6. Working model to show the effect of DCDBS84 on thrombosis without compromising hemostasis.