MMP‐13 binds to platelet receptors αIIbβ3 and GPVI and impairs aggregation and thrombus formation

Figures

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  Figure 1. Washed platelet adhesion assays. (A) Platelets adherent to 10 μg/ml coated MMP‐13 variants in the presence of 2 mmol L−1 Mg2+ (red bars) or 2 mmol L−1 EDTA (orange bars) where stated. Where appropriate, platelets were pre‐incubated with anti‐GPVI, αIIbβ3 or α2β1 antagonists. BSA and GPP 10 were used as Mg2+‐independent negative controls. The platelet α2β1 binding‐peptide GFOGER was included as an Mg2+ dependent positive control. *P < .05; **P < .01; †(one‐way anova and Holm multiple comparison test) relative to untreated platelets in either the presence of Mg2+ or EDTA, as appropriate. (B) Inhibition of platelet adhesion to CRP by anti‐GPVI scFvs as described above (C) Platelets adherent to MMP‐13(E204A) and MMP‐13 CAT and HPX domains. CRP‐XL was used as a positive control. **P < .01 (one‐way anova and Holm multiple comparison test) relative to adhesion to MMP‐13(E204A). Data represent mean A405 ± SE of three experiments

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  Figure 2. Competition, GPVI and Glanzmann platelet binding assays. (A) MMP‐13(E204A) and either 10B12 or GR144053 were used to obtain IC 50 values for the inhibition of washed platelet adhesion to 10 μg/ml coated fibrinogen (i, iii) or CRP (ii, iv), respectively. (v) Adhesion of MMP‐13(E204A) to recombinant human GPVI monomer and dimer. Plates were coated with 10 μg/ml GPVI or BSA as a negative control. MMP‐13(E204A) at a concentration of 83 nM was allowed to adhere for 1 h at room temperature, then detected using an antibody directed at the MMP‐13 linker region, as described in Methods. Data represent mean A450 ± SE of three experiments. (B) Platelets from a healthy donor (red bars) and from a Glanzmann thombasthenic individual (orange bars) were allowed to adhere to MMP‐13, fibrinogen and CRP‐XL coated plates. Where appropriate, platelets were pre‐incubated with anti‐GPVI (1C3), or αIIbβ3 antagonists as described for Figure 1. Data represent mean A405 ± SE of duplicate readings for one experiment due to the rarity of the Glanzmann donor

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  Figure 3. Platelet receptor cleavage and shedding assays. (A) Degradation analysis of recombinant platelet receptors by active and MMP‐13(E204A). Recombinant human (rh)GPVI and purified αIIbβ3 and fibrinogen type I were incubated with MMP‐13 or MMP‐13(E204A) for 2 h at 37°C. An equal volume of Tris buffer was used as a negative control. Samples were subjected to electrophoresis under reducing conditions and Coomassie stained. Images are representative of three experiments. (B) Shedding analysis of platelet receptors. Washed platelets were incubated with the calcium ionophore A23187, a thrombin and collagen type I mixture or MMP‐13 for 1 h at 37°C. The platelets were then pelleted, the supernatant isolated and subjected to SDS‐PAGE under reducing conditions and Western blotted. Platelet GPVI and the integrin β3 chain were detected using the appropriate antibodies as described in materials and methods. Recombinant human GPVI or αIIbβ3 were loaded onto the gels where appropriate as positive controls. Images are representative of three experiments

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  Figure 4. Inhibition of platelet aggregation by MMP‐13(E204A). Different agonists were added to washed platelets following pre‐incubation with 80 nmol L−1 MMP‐13(E204A). The equivalent volume of 0.01 mol L−1 acetic acid was used as a negative control. Mean donor responses performed in duplicate and repeated three times with different donors are shown in (A), and representative individual traces in response to MMP‐13 only, A23187, thrombin, cross‐linked collagen related peptide (CRP‐XL), HORM ® and type I collagen shown in (B)

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  Figure 5. Activation of platelets in whole blood. Whole blood was mixed with anti P‐Selectin and the agonists 2 mmol L−1 proMMP‐13(E204A)/MMP‐13, 100 μg/ml CRP‐XL, 100 μg/ml HORM® equine collagen I fibers, thrombin activating peptide (TRAP; 500 μmol L−1) calcium ionophore A23187 (100 μmol L−1) or HBS (negative control) added. Alexa 488 conjugated anti‐mouse was then added and after 10 minutes at 24°C the volume was made up to 500 μl with isotonic solution. After 30 minutes fluorescence was measured using an Accuri C6 flow cytometer (BD Biosciences, Oxford, UK). Data represent mean A450 ± SE of three separate donors. **P < .005; (one‐way anova and Holm multiple comparison test)

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  Figure 6. Platelet adhesion and thrombus deposition on fibrillar type I collagen. Untreated whole blood and blood pre‐incubated with 80 nmol L−1 MMP‐13 or negative control where stated was drawn through a flow chamber for 5 minutes over (A) collagen type I fibers or (B) collagen type I fibers co‐coated with MMP‐13 using a syringe pump to generate a wall shear rate of 1000 s−1, corresponding to arteriolar conditions. Surface coverage (i) mean height (ii) and (iii) ZV50 are the mean taken from a minimum of three different donors as measured using confocal microscopy. *P < .05; **P < .01; (one‐way anova and Holm multiple comparison test) relative to MMP‐untreated platelets