Colchicine inhibits ROS generation in response to glycoprotein VI stimulation

Abstract

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y₁₂ receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H₂DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO⁺/CD62P⁺) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.

Figure 1

Colchicine decreases platelet aggregation response to collagen in whole blood and platelet rich plasma (PRP). Hirudinised whole blood and PRP from healthy controls (n = 10) was assessed for platelet aggregation and the rate of aggregation (velocity) after incubation with collagen (3 µg/mL) in the presence or absence of 20 nM colchicine and 2 mM colchicine (PRP only). Significant decrease in aggregation and velocity was observed in whole blood (p = 0.006 and p = 0.03; a and b) and in PRP after incubation with 20 nM colchicine (p = 0.005 and p = 0.019; c and d) and 2 mM colchicine (p = 0.0001 and 0.0019; e and f). Statistical significance was determined by paired t test to vehicle control. Data presented as mean ± SD.

Figure 2

Colchicine decreases ADP-induced platelet aggregation in platelet rich plasma (PRP). Hirudinised whole blood and PRP from healthy controls (n = 9–10) was assessed for platelet aggregation and the rate of aggregation (velocity) after incubation with ADP (6.7 µM) in the presence or absence of 20 nM colchicine and 2 mM colchicine (PRP only). No significant decrease in aggregation or velocity was observed in whole blood (a,b) or in PRP after incubation with 20 nM colchicine (c,d). Significant decrease in aggregation and velocity was observed in PRP after incubation with 2 mM colchicine (p = 0.009 and 0.004; e,f). Statistical significance was determined by paired t test to vehicle control. Data presented as mean ± SD.

Figure 3

Colchicine inhibits ROS generation in response to GPVI stimulation with CRP-XL. Citrated PRP was used to assess the effect of colchicine on ROS generation, by flow cytometry with H₂DCF-DA, in response to stimulation of the GPVI receptor with CRP-XL (2 µg/mL) or no agonist control. (a) ROS generation was reduced in platelets pre-incubated with colchicine in a concentration dependent manner in response to CRP-XL, no difference was observed between the no agonist controls. (b) ROS generation was significantly decreased by 20 nM (p = 0.045, n = 5) and 2 mM colchicine (p = 0.0003)—area under the curve from (a) corrected for the non-stimulated response. Statistical significance determined by Students t test; data presented as mean ± SD.

Figure 4

Colchicine does not cause GPVI shedding. Colchicine does not change platelet GPVI expression in PRP as assessed by %GPVI expression (a) or GPVI mean fluorescence intensity (b). GPVI was shed in response to the positive control (NEM, p = 0.009 for % and p = 0.01 for MFI) and rescued with GM6001 (a broad-spectrum metalloproteinase inhibitor, p = 0.008 for % and p = 0.01 for MFI). Statistical significance was determined by Students paired t test; data presented as mean ± SD; n = 4.

Figure 5

Colchicine does not alter ADP-induced calcium flux. Citrated PRP was used to assess the effect of colchicine on ADP-induced calcium flux by flow cytometry with Fluo-4 AM. (a) Calcium flux was not significantly altered in platelets pre-incubated with colchicine and stimulated with ADP (5 µM). Curves are corrected for the non-stimulated response. (b) Calcium flux was not significantly altered by 20 nM (p = 0.26) and 2 mM colchicine (p = 0.24)—area under the curve from (a). Statistical significance determined by Students t test; data presented as mean ± SD, n = 4 donors. The average of duplicate curves was used for each donor.

Figure 6

Effect of colchicine on P-selectin, CD63 expression and conformational change of GPIIb/IIIa. Flow cytometry was performed to assess the cell surface expression of P-selectin/CD62P, CD63 and conformational change of GPIIb/IIIa in response to stimulation with 0.2 µg/mL CRP-XL, 0.5 µM ADP or 2 µM ADP after preincubation with vehicle or colchicine (20 nM and 2 mM). A significant reduction in CD62P expression was only observed in those samples pre-incubated with high concentrations of colchicine [(a) CRP-XL, p = 0.0005; (b) 0.5 µM ADP, p = 0.014 and (c) 2 µM ADP, p = 0.005]. There was no significant difference in CD63 expression in response to colchicine under any of the conditions examined [(d) CRP-XL, (e) 0.5 µM ADP and (f) 2 µM ADP]. A significant reduction was only observed in GPIIb/IIIa conformational change (PAC-1 binding) in those samples pre-incubated with high concentrations of colchicine and stimulated with ADP, no change was observed in platelets stimulated with CRP-XL (g) [(h) 0.5 µM ADP, p = 0.02 and (i) 2 µM ADP, p = 0.002]. Statistical significance was determined by comparing conditions with the vehicle control using either paired t test or Wilcoxon matched pair rank test as appropriate. Data presented as mean ± SD, n = 3–4.

Figure 7

Colchicine inhibits the formation of procoagulant platelets. Washed platelets were subjected to dual stimulation with thrombin and collagen in the presence of vehicle, 20 nM or 2 mM colchicine, and the formation of procoagulant (GSAO⁺/CD62P⁺) platelets determined with flow cytometry. (a) Representative zebra plots showing procoagulant platelets (GSAO⁺/CD62P⁺, top right quadrant) at rest or after dual stimulation with thrombin and collagen with vehicle, 20 nM or 2 mM colchicine pre-incubation. (b) Percentage procoagulant platelets at rest, and after stimulation in the vehicle control, 20 nM and 2 mM colchicine. Mean ± SD, n = 6, Wilcoxon test vs vehicle control.