Study Design
Patients who developed STEMI and underwent PPCI between August 2016 and November 2019 at the Shanghai Ninth People’s Hospital, affiliated with the Shanghai Jiao Tong University School of Medicine, were recruited in this study. Patients were excluded if they had a history of coronary heart disease, late presentation (> 12 h), infectious or inflammatory disease, severe liver or renal disease or thrombolytic therapy. The STEMI diagnosis was confirmed by coronary angiography, which was followed by the administration of 300 mg aspirin, 180 mg ticagrelor, and 0.15 g·kg-1·min-1 tirofiban infusion. On the basis of the thrombolysis in myocardial infarction (TIMI) flow grade, patients who underwent PPCI were divided into two subgroups: the SR group and non-SR (NSR) group. The observational clinical study complied with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations [13]. All patients provided informed written consent prior to sample collection, and the study protocol was approved by the Shanghai Ninth People’s Hospital Institutional Ethics Committee (HKDL2017300) (No. 2016-256-T191) and performed in accordance with the ethical standards outlined in the 1964 World Medical Association Declaration of Helsinki.
Plasma Preparation and ANGPTL2 Quantification
After informed consent was obtained, blood was collected from the patients into plasma sodium citrate anticoagulant tube prior to medication. Plasma fraction was obtained by differential centrifugation at 3000 rpm for 10 min at 26 ℃. ANGPTL2 levels were measured using an enzyme-linked immunosorbent assay kit (E1919Hu, USCN Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using an Infinite M200PRO microplate reader (Tecan, Switzerland, Germany).
Platelet Preparation and Measurement of Platelet ANGPTL2 Levels
Blood was collected from patients into empty syringes moistened with sodium citrate and transferred into polypropylene centrifuge tubes containing 100 µL/mL Whites anticoagulant (2.94% sodium citrate, 136 mM glucose, pH 6.4), 0.1 g/mL PGE1, 1 U/mL apyrase and gently mixed. Platelet-rich plasma (PRP) was fractionated using differential centrifugation (290 g for 10 min). Supernatant was aspirated and the platelets were resuspended with PRP containing 5 mM EDTA and subjected to second differential centrifugation (850 g for 10 min). Washed platelets were resuspended in the modified Tyrode’s buffer as previously described [14].
Platelet ANGPTL2 levels were evaluated immunoblotting as previously described with goat anti-ANGPTL2 antibody (1:1,000, AF2084, R&D Systems, USA) and the secondary antibodies (1:3,000, #L3042, Signalway Antibody, China) [9]. Image Pro Plus v6.0 (Media Cybernetics, USA) was used to quantify specific protein bands and to determine ANGPTL2 expression levels in platelets. Then data were normalized not only according to GAPDH but also the NSR group mean to represent the relative expression of ANGPTL2.
Generation of Angptl2 –/– Mice
All animal experiments were approved by the Shanghai Ninth People’s Hospital institutional ethics committee (HKDL2017300). Global Angptl2 deficient (Angptl2–/–) mice were obtained from Jackson Laboratory, while wild-type (WT) C57BL/6 mice genetic background control mice were obtained from Shanghai SLAC Experimental Animal Co., Ltd. Angptl2 deficiency in platelets was confirmed by comparing western blots of platelet extracts of Angptl2−/− and wild-type (WT) C57BL/6J mice. All mice were maintained in the Division of Laboratory Animal Resources under specific pathogen free conditions. After the mice were bred, animals homozygous for the null allele were screened and identified. Mice were euthanized with an overdose of isoflurane anesthesia.
In Vivo Thrombosis Models
Male WT and Angptl2−/−mice with a C57BL/6J background (age, 4–5 weeks old) were used to establish the ferric-chloride (FeCl3)-induced mesenteric arteriole injury model. The mice were anesthetized with a ketamine/xylazine mixture (200:10 mg·kg− 1) via jugular vein cannulation. Rhodamine dye to label platelets was also delivered via this catheter. After the mesentery was exteriorized through a midline abdominal incision, arterioles with diameters of 80–100 µm were treated with filter paper pre-saturated with 250 mM FeCl3 for 5 min. The mesenteric arterioles and thrombus formation were visualized using an AxioPlan 2 microscope equipped with an AxioCam MRm camera (Carl Zeiss, Göttingen, Germany). Z-STACK slices of the vessel were captured. Thrombus volumes were determined from the area and height of the clot after the rendered z-stack images were deconvolved with AxioVision Rel 4.6 software (Carl Zeiss, Göttingen, Germany) to produce a three-dimensional image. Thrombus stability was determined by calculating the percentage of the vessel occupied by the thrombus within 2 min and quantified using a score from 1 to 10, with 1 representing 0–10% occupancy and 10 representing 91–100% occupancy [15–16]. In these experiments, three mice per group, with 10 thrombi per mouse, were studied.
Platelet Secretion Assay
Fresh blood was collected from the abdominal aorta of mice anesthetized via isoflurane inhalation, with syringes containing 100 mL/L sodium citrate anticoagulant (2.94% sodium citrate, 136 mM glucose [pH 6.4]), 0.1 g/mL prostaglandin E1, and 1 U/mL apyrase. Washed platelets were prepared following the above method. The count was read using an automatic blood cell analyzer (BM860, BWLIN MAN, China) and adjusted to 1 × 106 platelets/µL with a modified Tyrode’s buffer.
Platelet secretion was analyzed by measuring the surface expression of α (P-selectin)- and dense-granule, markers of granule release. For α-granule analysis, platelets from WT and Angptl2−/− mice were stained with a fluorescein isothiocyanate-conjugated monoclonal antibody targeting P-selectin (Biolegend, #148305). For dense-granule exocytosis analysis [17], 100 µL of washed platelets (2 × 107/mL) were incubated with 4 µM fluorescent quinacrine (Q3251, Sigma-Aldrich) at 37 ℃ for 10 min. After stimulated by collagen (#385, Chrono-Log), thrombin (T4648, Sigma-Aldrich, USA) or adenosine diphosphate (ADP; A5258, Sigma-Aldrich), platelet secretion was analyzed by flow cytometry using CytoFLEX (Beckman Coulter, USA).
Platelet Aggregation
Then, the washed platelets (300 µL) were used for an aggregation assay in a Lumi-Aggregometer (Chrono-Log, Havertown, PA, USA) [9]. The role played by Angptl2 in agonist-induced platelet aggregation was investigated by stimulating platelets from WT and Angptl2−/− mice with collagen, thrombin or ADP. For further analysis, purified ANGPTL2 was added at concentrations of 0.1, 0.5, and 1 µg/mL to washed WT platelets stimulated with 1 µg/mL collagen, 0.067 U/mL thrombin, or 1 µM/mL ADP. The effect of ANGPTL2 was determined on the basis of decrease in platelet aggregation.
Platelet Spreading on Immobilized Fibrinogen
Chamber slides with microtiter wells (Nalge Nunc, Rochester, NY, USA) were coated with 50 µg/mL human fibrinogen (Sigma-Aldrich) and incubated at 4°C overnight. Washed mouse platelets (2 × 107/mL) were stored in Tyrode’s buffer containing 1 mM CaCl2 and 1 mM MgCl2, and incubated at 37°C for 90 min. Subsequently, the washed platelets were stimulated with 0.01 U/mL thrombin, and spread on immobilized fibrinogen for 60 min. Then, they were fixed, permeabilized, stained with fluorescein-labeled phalloidin (Invitrogen, Carlsbad, CA, USA), and visualized using an upright fluorescence Axio Scope.A1 microscope (Carl Zeiss, Göttingen, Germany) equipped with a 100×/1.30 oil objective lens, an X-Cite 120Q light source (EXFO, Mississauga, CA, USA), and a digital camera. At least six images were randomly chosen for each experiment and analyzed under blinded conditions. The platelet surface area was analyzed using Image J software v1.8.0 (National Institutes of Health, Bethesda, MD, USA) [9].
Platelet-Mediated Clot Retraction
Washed mouse platelets were mixed with citrated normal human platelet-depleted plasma to a concentration of 3 × 108/mL. Subsequently, coagulation was induced using 0.5 U/mL thrombin. The clots were allowed to retract at 37°C and then photographed at 60 min. Two-dimensional clot size was quantified from photographs by using Image J software v1.8.0, and clot retraction was expressed in terms of the retraction ratio (1 – [final clot size/initial clot size]) [9].
Western Blot Analysis
Western blot was performed as previously described [9]. Platelets (3 × 108/mL) from WT and Angptl2–/– mice were stimulated with 1 µg/mL collagen at 37°C. After the platelets were treated with collagen for 1, 2, 3, 4, or 5 min, they were lysed with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) on ice for 30 min. Subsequently, they were centrifuged at 13,000 × g for 10 min at 4°C to remove cell debris, following which the protein supernatant was stored in a new tube for western blot analysis. The target protein levels were determined using anti-Angptl2 polyclonal antibody (1:1000, #AF 1444, R&D, USA), the secondary antibodies (1:3,000, #L3042, Signalway Antibody, China), anti-phospho-Shp1 Y564 (1:1,000, #8849, Cell Signaling Technology,USA), anti-phospho-Shp2 Y580 (1:1,000, #5431, Cell Signaling Technology, USA), anti-phospho-Src Y416 (1:1,000, #6943, Cell Signaling Technology, USA), anti-phospho Syk Y525 (1:1000, #PA5-104904, Invitrogen, USA), anti-phospho-PLCγ2 Y1217 (1:1,000, #3871, Cell Signaling Technology, USA), anti-GAPDH antibody (1:1,000, #5174, Cell Signaling Technology, USA) and the secondary antibodies (1:3,000, #L3012, Signalway Antibody, China). GAPDH was used as a loading control for total protein.
Statistical Analysis
Statistical analysis was performed using SPSS (v.21.0; IBM Corp., Armonk, NY, USA). Quantitative data were presented as the mean ± standard deviation (SD). Continuous variables were tested for normality by using the Kolmogorov-Smirnov test. The chi-square test was used for categorical variables. Multivariate logistic regression analysis was used to determine the independent predictors of SR. Statistical significance was determined via an independent sample t-test between two groups or ANOVA among multiple groups using SPSS (v.21.0). Statistical significance was set at P < 0.05.