Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis

<p>Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5′-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.</p

S14161 was unable to disrupt platelet aggregation induced by higher doses of agonists.

<p>Gel-filtered platelets (2.5×10⁸/ml) were pre-incubated for 10 min with S14161 (2.5 µM or 5 µM). Platelet aggregation was initiated with different concentration of collagen (1 µg/ml, 2 µg/ml, and 10 µg/ml) (A) or with thrombin (0.05 U/ml, 0.1 U/ml, and 0.2 U/ml) (B). The data are representatives of at least three individual experiments.</p

S14161 suppresses agonist-stimulated platelet PI3K signaling phosphorylation.

<p>Gel-filtered human platelets (2.5×10⁸/mL) were preincubated with or without S14161 (2.5 µM, 5 µM and 10 µM) or LY294002 (10 µM). Then, platelets were stimulated with collagen (2 µg/mL), thrombin (0.1 U/mL) or U46619 (1 µM) for 3 min with stirring at 1000 r.p.m. in an aggregometer at 37°C. Platelets were lysed, and immunoblotted using the corresponding antibodies recognizing total or phosphorylated AKT (Ser473 or Thr308) and phosphorylated GSK3β (Ser9). Densitometric band scanning was performed using an Odyssey Infrared Imaging System (LI-COR Biosciences). Data are representatives of at least 3 independent experiments.</p

S14161 inhibited platelet adhesion on collagen-coated surfaces under flow ex vivo and thrombus formation in vivo, but not hemostasis.

<p>(A) Human whole blood was labeled with Calcein-AM, treated with (lower channels) or without (upper channels) S14161 (10 µM), and perfused over collagen-coated surface at a shear rate of 40 dyn/cm² (1000 s⁻¹). Platelet adhesion and aggregates were photographed. Data plotted are means ± standard errors of fluorescence intensity of three individual experiments (**P<0.01, ***P<0.001). Arrow indicates the flow direction. (B) S14161 (2 mg/kg) (n = 9) or vehicle (n = 8) was administered intraperitoneally to mice and time to occlusion of the carotid artery after the application of 7.5% FeCl₃ for two minutes was measured using Visual Sonics View 2100. Time to loss of blood flow was recorded. (C) S14161 (2 mg/kg) (n = 12) or vehicle (n = 10) was administered intraperitoneally to mice. The bleeding end was immersed in saline at 37°C, and the time to bleeding cessation was recorded. *P<0.05 vs. vehicle control. N.S. means no significance.</p

Influence of S14161 on platelet alpha-granule secretion and integrin inside-out signaling.

<p>Gel-filtered human platelets (2×10⁷/mL) were preincubated with S14161 (10 µM) at 37°C for 10 minutes in the presence of fluorescent-conjugated antibodies against P-selectin or fibrinogen. Samples were then challenged with convulxin (0.5 nM) or thrombin (0.1 U/mL), and incubated for an additional 15 minutes. P-selectin expression (A, C) and fibrinogen binding (B, D) of single platelet were monitored by Flow cytometry. Data plotted are means ± standard errors of the mean; n = 3; *P<0.05, **P<0.01 and ***P<0.001 with vs. without S14161 treatment.</p

S14161 inhibited platelet aggregation.

<p>Gel-filtered human platelets (2.5×10⁸/mL) were preincubated for 10 minutes with different concentrations of S14161 (2.5 µM, 5 µM and 10 µM) or vehicle (DMSO). Platelet aggregation was initiated with collagen (2 µg/mL), thrombin (0.1 U/mL), U46619 (1 µM), or ADP (10 µM). The aggregation curves are the representatives of at least three individual experiments. Means ± standard errors of the mean of platelet aggregation percentage from three experiments are plotted in the bar charts; *P<0.05, **P<0.01 and ***P<0.001 as compared with control. N.S. means no significance (n = 3).</p

S14161 inhibited platelet aggregation more potently than LY249002.

<p>Gel-filtered human platelets (2.5×10⁸/mL) were preincubated for 10 minutes with different concentrations of LY249002 (10 µM, 20 µM and 40 µM) (A, B) or equal concentration of S14161 or LY249002. Platelet aggregation was initiated with collagen (2 µg/mL), thrombin (0.1 U/mL). The aggregation curves are the representatives of at least three individual experiments. Means ± standard errors of the mean of platelet aggregation percentage from three experiments are plotted in the bar charts; *P<0.05, **P<0.01 and ***P<0.001 as compared with control. N.S. means no significance (n = 3).</p

Effects of S14161 on platelet spreading on immobilized fibrinogen and fibrin clot retraction.

<p>(A) Gel filtered human platelets (2×10⁷/mL) were preincubated with S14161 (2.5 µM, 5 µM and 10 µM) or vehicle for 30 min at 37°C and allowed to spread on fibrinogen-coated slides. Images were obtained with an Olympus fluorescence microscope. Statistical data were derived from quantitative results (means ± SEM) calculated from the mean of the average surface area of individual platelets from 3 separate experiments. ***P<0.001 vs. vehicle control. (B) Platelet rich plasma was incubated with S14161 (10 µM) or vehicle. Fibrinogen (2 mg/mL) was added and fibrin clot formation was initiated by adding 1 U/mL of thrombin. Clot retraction was monitored over time, and photographs of the clots were taken at different time points. The histograms of the clot size were generated from the photographs by calculating the ratio of the surface area of the retracted clot versus that of the initial clot. *P<0.05 and **P<0.01 compared with vehicle control (n = 3).</p

Evaluation of the toxic and apoptotic effect of S14161 on platelets.

<p>(A) Gel-filtered platelets (2.5×10⁸/ml) were preincubated with S14161 (2.5 µM, 5 µM and 10 µM) or vehicle and then incubated with Alamar Blue reagent for 4 hours at 37°C. The fluorescence was acquired with the excitation and emission wavelengths of 570 nm and 585 nm, respectively, and the Tyrode buffer control-subtracted fluorescence was plotted in the bar charts (mean ± standard error, n = 3). (B) Gel-filtered platelets (2×10⁷/ml) were preincubated with S14161 (2.5, 5, and 10 µM), vehicle, or dibucaine (500 µmol/L) at 37°C for 15 min, and then mixed with Annexin V binding buffer and Annexin V-APC. Samples were analyzed by flow cytometry and the percentage of annexin V positive platelets was calculated (mean ± standard error, n = 3). ***P<0.001. N.S., not significant compared to vehicle control.</p