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

Abstract Conventional antiplatelet agents indiscriminately inhibit both thrombosis and hemostasis, and the increased bleeding risk thus hampers their use at more aggressive dosages to achieve adequate effect. Blocking integrin αIIbβ3 outside‐in signaling by separating the β3/Src interaction, yet to be proven in vivo, may nonetheless resolve this dilemma. Identification of a specific druggable target for this strategy remains a fundamental challenge as Src SH3 is known to be responsible for binding to not only integrin β3 but also the proteins containing the PXXP motif. In vitro and in vivo mutational analyses show that the residues, especially E97, in the RT loop of Src SH3 are critical for interacting with β3. DCDBS84, a small molecule resulting from structure‐based virtual screening, is structurally validated to be directed toward the projected target. It specifically disrupts β3/Src interaction without affecting canonical PXXP binding and thus inhibits the outside‐in signaling‐regulated platelet functions. Treatment of mice with DCDBS84 causes a profound inhibition of thrombosis, equivalent to that induced by extremely high doses of αIIbβ3 antagonist, but does not compromise primary hemostasis. Specific targets are revealed for a preferential inhibition of thrombosis that may lead to new classes of potent antithrombotics without hemorrhagic side effects.


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The PDF file includes: Supporting Materials and Methods Figure S1. Screening small molecules by the inhibitory effect on platelet spreading on immobilized fibrinogen and binding affinity for SH3. Figure S2. Specification of DCDBS84 binding to Src revealed by HDX-MS and NMR spectrum.     Table S1. Small molecule screening process Legends for Movies S1 to S3 Legend for Data set S1 Legend for 124 compounds information

Other Supporting Materials for this manuscript includes the following:
Movie S1 (.mp4 format). Arteriolar thrombosis: DMSO control.
Intravital video-microscopy of the cremaster muscle arteriolar circulation and platelet thrombus formation (green) after laser injury.
Intravital videomicroscopy of the cremaster muscle arteriolar circulation and platelet thrombus formation (green) after laser injury.
Intravital videomicroscopy of the cremaster muscle arteriolar circulation and platelet thrombus formation (green) after laser injury.
Data set S1 (Microsoft Excel format). Raw data.
Information of 124 compounds (Microsoft Excel format).

Supporting Materials and Methods
Molecular docking and virtual screening: Molecular docking simulation was used to construct the binding mode of β3 heptapeptide (NITYRGT) with SH3. The full length Src structure (PDB code: 2H8H) [52] was used to compare the binding mode of β3 heptapeptide (NITYRGT) and a class II peptide APPIPPPR with SH3 domain. GLIDE program [53,54] (Schrödinger, LLC, New York, NY, 2015) was employed to perform the molecular docking study. The coordinates of chain A and RGT in the complex crystal structure of SH3: RGT (PDB entry: 4HXJ) [35] were used to construct the docking model. First, NITYRGT peptide was prepared with LigPrep [62] panel to produce multiple output structures with default settings. Then the Protein Preparation Wizard Workflow was used to prepare the protein structures (coordinates of chain A and RGT). And residues located within 15 Å centered on RGT tripeptide in SH3 were defined as NITYRGT binding sites in which the docking grids were created. Finally, the prepared peptide was docked into the binding site with extra precision (XP) mode without any constraint.
In the docking simulation of the SPECS database compounds against SH3, a similar procedure was used as above. Specifically, all of the compounds in SPECS database were prepared with LigPrep panel. Then the prepared compounds were docked into the defined binding sites of SH3 using extra precision (XP) mode without any constraint and were ranked by Glide-gscore. The top-ranked compounds were selected and then subjected to structure cluster. After carefully analysis and comparison, 124 compounds were purchased from SPECS supplier.
Animals and reagents: Src E97A transgenic mouse model was generated using gene targeting and blastocyst injection technologies in C57BL/6 mice. The gene ID is 20779 for the Src Rous sarcoma oncogene sequence. Genotypic identification was performed using PCR and sequencing. Mouse monoclonal antibody SZ-21 to the integrin β3 was a generous gift from C. Ruan (Jiangsu Institute of Hematology, Suzhou, China). Rabbit monoclonal antibody to Src, phospho-Src-Tyr416 (Src 416 ), phospho-Src-Tyr527 (Src 527 ) and horseradish peroxidase-or FITC-conjugated secondary antibodies were purchased from Cell Signaling Technology, anti-His was from Tiangen (Beijing, China), and anti-glutathione-S-transferase (GST), phospho-β3-Tyr747 (β3 747 ) and phospho-β3-Tyr759 (β3 759 ) were from Abcam. Alex Flour 488-conjugated human fibrinogen was from Sigma-Aldrich. Human α-thrombin was obtained from Enzyme Research Laboratories. Human adenosine diphosphate was purchased from Chronolog. Protein A/G agarose was purchased from Beyotime Biotechnology. All other reagents were obtained from Sigma-Aldrich.
Then, the wells were incubated sequentially with the purified Src SH3 WT or mutants (R95A, E97A, G116A, W118A and Y131A) with Flag tag, biotin-RLP1 peptide and HRP-conjugated streptavidin. The binding intensity of the biotin-RLP1 was then measured using a spectrophotometer (NanoQuant, Infinite M200, TECAN, Switzerland) at 450 nm. To detect the effects of DCDBS84 on the interaction of RLP1 peptide with Src SH3, DCDBS84 (100 μmol/L) was added into the wells when biotin-RLP1 peptide incubated with Src SH3.
Src kinase activity: The Src kinase activity was measured using the CycLex c-Src Kinase Assay/Inhibitor Screening Kit (MBL, Japan) according to the protocol provided by the company, detailed as follows. In an assay plate with wells pre-coated with "Tyrosine kinase-substrate-1", 10 μL of Src positive control (0.1 U/μL) or serial dilution of Src positive control were added on ice. Kinase reaction was initiated by adding 90 μL of kinase reaction buffer per well. The plate was then covered with plate sealer and incubated at 30 °C for 30 minutes. After washing the wells five time with wash buffer, residual wash buffer was removed by gentle tapping or aspiration.
100 μL of HRP conjugated detection antibody was then added into each well, followed by sealed incubation at room temperature for 60 minutes and then washing 5 times. After removing the residual wash buffer, 100 μL of substrate reagent was added to each well and then incubated at room temperature for 5-15 minutes. Next, 100 μL of stop solution was added to each well. Absorbance in each well was measured using a spectrophotometric plate reader at 450 nm. To detect the effects of His-Src SH3 and GST-β3 cytoplasmic tail fusion proteins were expressed in E coli and were grown in LB broth at 37 °C. All these plasmids were verified by DNA sequencing. 200 μmol/L IPTG was added into the cultural medium to induce expression. When the OD (600) value was approximately 0.6-0.8, the fermentation was continued for 12 hours at 22 °C. Then, the bacteria were harvested by centrifugation and lysed with lysis solution. The His-Src-SH3 clear lysate was loaded onto a His-trap column, then elution by buffer containing imidazole. The eluant was cleaved by thrombin overnight at 22 °C, followed by purification using Q column.
Finally, the purified Src SH3 protein was obtained. When the His-Src-SH3 was used for pull-down assay, thrombin cleavage was omitted. And GST-β3 cytoplasmic tail fusion protein could be eluted by the buffer containing glutathione. Samples were injected at 50 μL/min across an immobilized VIII / Pepsin Cartridge (2.1 mm × 30 mm, NovaBio Assays) and digested. Peptide fragments were subsequently collected on a Acclaim PepMap300 C18 column (5 μm, 1.0 mm × 15 mm; Thermo Fisher, USA) for desalting with 0.1% formic acid in H 2 O and then isolated by liquid chromatography using an ACQUITY UPLC Peptide CSH C18 column (130 Å, 1.7 μm, 1 mm × 50 mm; Waters, USA) at a flow rate of 45 μL/min with an acetonitrile gradient starting with 1% and increasing to 35% over 10 min.
Mass spectrometric data were acquired using a Fusion Orbitrap mass spectrometer (Thermo Fisher, USA) with a measured resolving power of 60,000 at 350-1500 m/z.
The deuterium uptake data of the peptides was analyzed using HDExaminer (version 2.4) software. A total set of 173 peptides with 84.3% of Src sequence coverage were employed to track deuterium exchange. Based on three experiments of repeated determinations, 5% of the deuteration difference was selected as the threshold to indicate that the peptides had significant deuteration behavior differences in the experiment. Percentage change of deuterium uptake at 60 min was mapped onto the crystal models of Src (PDB code: 2SRC). [56] Surface plasmon resonance (SPR): SPR assay was performed on Biacore T200 instrument (GE Healthcare, USA) with HBS-EP running buffer (10 mmol/L HEPES, PH 7.4, 3 mmol/L EDTA, 150 mmol/L NaCl, 0.05% surfactant P20). Protein Src SH3 was covalently immobilized on a CM5 chip. Small molecules were serially diluted and injected at a flow rate of 30 μL/min for 120 seconds of association, subsequently 120 seconds of dissociation The Kd value of small molecules were determined using Biacore T200 evaluation software (GE Healthcare, USA). cryoprobe. Complete backbone resonance assignments were obtained from 15 N-HSQC, HNCACB and HN(CO)CACB spectra using uniformly 15  Platelet preparation: Whole blood was acquired from healthy volunteers with informed consent. Anticoagulated with 1/10 volume of 3.8% (w/v) trisodium citrate, the blood was centrifuged at 300 g for 10 minutes at 22 °C to obtain platelet-rich plasma (PRP). The platelet concentration in PRP was adjusted to 1-3×10 8 /mL by adding platelet poor plasma. Washed platelets were prepared with blood anticoagulated with 1/4 volume of acid citrate dextrose (ACD, 85 mmol/L trisodium citrate, 21 mmol/L citric acid, and 83 mmol/L dextrose). The PRP was recentrifuged and the pellets were washed with CGS (13 mmol/L trisodium citrate, 120 mmol/L NaCl, 30 mmol/L glucose, pH 6.5) twice, and were resuspended in HEPES-Tyrode's buffer (137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO 3 , 0.3 mmol/L NaH 2 PO 4 , 5.5 mmol/L glucose, 5 mmol/L N-2-hydroxyethylpiperazine -N-2-ethane sulfonic acid (HEPES), 1 mmol/L CaCl 2 , 1 mmol/L MgCl 2 , and 0.1% bovine serum albumin (BSA), pH 7.4) at a final concentration of 1-3×10 8 /mL platelets. The platelet suspensions were rested for 60 minutes before use.

Co-immunoprecipitation and Western blot:
Platelets at a concentration of 2×10 8 /mL were incubated with DMSO or DCDBS84 (80 μmol/L) for 60 minutes at 37 °C, then lysed with NP-40 buffer (0.5% NP-40, 50 mmol/L Tris-HCl, pH 7.2, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L sodium vanadate, 1 mmol/L of phenylmethysulfonyl fluoride containing the protease inhibitor. Platelet lysis (500 μg protein) were incubated with anti-integrin β3 antibody or normal mouse IgG as control overnight at 4 °C and subsequently incubated with pre-clear protein A/G beads (20 μL) for 2 hours at 4 °C. Complexes were washed by NP-40 buffer 3 times, followed by WB assay to identify the interaction of integrin β3 with Src, Gα13 or Kindlin3. In addition, the platelets were treated with DMSO, DCDBS84 (80 μmol/L) or integrilin (25 μmol/L) for 60 minutes at 37 °C. After that, the platelets were separated into two parts, one as inactive state, and the other as the activated state when thrombin was added into the platelets on platelet aggregation apparatus (APACT4004, Germany). Platelets were lysed by RIPA (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail and phosphatase inhibitor cocktail. Then the WB was performed using β3 747 , β3 759 , Src 416 , and Src 527 antibodies.

Platelet spreading on immobilized fibrinogen:
Washed platelets were resuspended at a concentration of 1×10 8 /mL in Tyrode's buffer, subsequently pre-incubated with DCDBS84 (20 μmol/L, 40 μmol/L, 80 μmol/L), RGDS (1 mmol/L) and DMSO for 60 minutes and then allowed to adhere and spread on fibrinogen-coated (20 μg/mL) coverslips in the wells of 12-well plate at 37 °C for 60 minutes. After washing with PBS 3 times, the adherent platelets were fixed, and stained with TRIFC-labeled phalloidin as previously described. Finally, the spreading shapes of these platelets were observed by a conventional fluorescence microscope (Leica, Germany) with a 60× objective in small molecule screening, and a laser confocal microscope (Leica TCS SP8, Germany) with a 63× objective in DCDBS84 spreading assay. This assay was performed as described previously. [12,57] Platelet stable adhesion on immobilized fibrinogen: Platelets were washed and incubated with DCDBS84 (20 μmol/L, 40 μmol/L, 80 μmol/L), RGDS (1 mmol/L) and DMSO for 60 minutes. 100 μL platelets were added to the fibrinogen-coated (20 μg/mL) wells of 96-well plate in triplicate and incubated for 60 minutes at 37 °C.
Then the wells were washed vigorously with PBS 3 times to remove unstable adherent platelets. Then, stable adherent platelets were mixed with p-nitrophenyl phosphatase (PNPP) substrate solution (100 mmol/L of sodium acetate, 1% Triton X-100, 3 mg/mL of PNPP) for 60 minutes at 37 °C. The enzyme reaction was stopped with 1 mol/L of NaOH, and the optical density was measured at 405 nm with a microplate reader. [12,57] Platelet aggregation: Platelet aggregation was performed as previously described. [12,57]  Soluble fibrinogen binding: Soluble fibrinogen binding assay was performed as described previously. [12,57] Washed platelets were suspended in Tyrode's buffer at Thrombus formation under flow: Ex vivo flow-based platelet adhesion assay was performed according to previous studies. [58] In brief, microfluidic channels (Fluxion Biosciences Inc. USA) were coated with 20 μg/mL collagen incubated at 4 °C overnight. The next morning, the coated channels were blocked with 2% BSA.
Whole blood from healthy volunteers was collected with hirudin as an anticoagulant and incubated with integrilin (100 μmol/L), DMSO and DCDBS84 (200 μmol/L) for 60 minutes at 37 °C. Calcein AM (4 μmol/L) was used to label platelets in the whole blood in the dark. The labelled blood was perfused through the micro-channels at wall shear rates of 500, 1500, or 5000 s -1 for 5 minutes. Adherent platelet aggregates were monitored using an inverted fluorescent microscope and CCD camera (Nikon Eclipse Ti-s, Japan). The data were analyzed using the Bioflux 200 software (Labtech, USA).
FeCl 3 -induced thrombosis: FeCl 3 -induced thrombosis assay was performed as described previously. [13] 6-to 8-week C57BL/6 mice were anaesthetized by intraperitoneal injection of phenobarbital. Intravenous injection of different concentrations of DCDBS84 (2.5 mg/kg, 5 mg/kg or 10 mg/kg), integrilin (0.18 mg/kg, 4.2 mg/kg or 16 mg/kg), DMSO, saline or Dasatinib (5 mg/kg) was performed 15 minutes before assay. Intragastric administration of aspirin (5.4 mg/kg, 125 mg/kg), clopidogrel (1.25 mg/kg, 10 mg/kg, 200 mg/kg) or saline was carried out by using a gavage needle and syringed at 24 h and 2 h prior to the initiation of the carotid artery injury procedure. Carotid arterial thrombosis was induced with a filter paper disc (diameter = 2 mm) that was soaked with 1.2 μL of 7.5% FeCl 3 . Filter paper was removed 3 minutes later then blood flow was monitored with a laser doppler system (Model MA-0.5VB). Then tails of mice were cut 5 mm from the tip with a sharp blade, and bleeding was monitored by blotting with filter paper every 15 seconds. Bleeding time was defined as no evidence of rebleeding for 60 seconds. Bleeding exceeding 15 minutes was immediately stopped by applying pressure.
Data set S1. Raw data (provided as separate excel file).
Information of 124 compounds (provided as separate excel file).