Differential Proteoglycan Expression in Atherosclerosis Alters Platelet Adhesion and Activation

Figures

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  Figure 1. Platelet adhesion and spreading on different extracellular matrices. (a) Platelets fluorescently labelled with DIOC6 were incubated in 96-well plates coated with either 20 µg/mL collagen I (COL1); collagen III (COL3); Biglycan (BGN); Decorin (DCN); Versican (VCAN); high-molecular-weight hyaluronan (HMW-HA); low-molecular-weight hyaluronan (LMW-HA) or goat serum blocking buffer (CONTROL) for 45 min at 37 °C. Following washing, (b) the number of platelets remaining and (c) the average platelet area was quantified as a measure of platelet adhesion and spreading, respectively. Data are presented as mean ± SEM, (n = 5), * p < 0.05, ** p < 0.01.

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  Figure 2. Platelet activation and fibrinogen binding. Platelets were stimulated with 20 µg/mL collagen I (COL1); collagen III (COL3); Biglycan (BGN); Decorin (DCN); Versican (VCAN); high-molecular-weight hyaluronan (HMW-HA); low-molecular-weight hyaluronan (LMW-HA) or vehicle control and (a) surface P-selectin levels (n = 3) and (b) fibrinogen binding (n = 4) were measured using flow cytometry. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01.

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  Figure 3. Thrombus formation on individual extracellular matrix proteins under arterial and elevated shear stress. Whole blood labelled with DIOC6 was perfused through ibidi µ-slides coated with 100 µg/mL type 1 collagen (COL1); type III collagen (COL3); versican (VCAN); biglycan (BGN); decorin (DCN); high-molecular-weight hyaluronan (HMW) or low-molecular-weight hyaluronan; (LMW) at (a–c) arterial shear stress (15 dynes/cm2) or (d–f) elevated shear stress (75 dynes/cm2). Platelets were visualised using fluorescence microscopy, and thrombi size at (b) arterial and (e) elevated shear stress and platelet area coverage at (c) arterial and (f) elevated shear stress were quantified using Fiji/ImageJ. Data represent mean ± SEM; n = 5; * p < 0.05; ** p < 0.01.

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  Figure 4. Platelet adhesion and spreading on type I and type III collagen compared to ‘rupture’ and ‘erosion’ composite matrices. Washed platelets were incubated for 45 min at 37 °C in wells coated with type I collagen (COL1; 20 µg/mL), type III collagen (COL3; 20 µg/mL), a ‘rupture’ composite (RUP) containing type I collagen, biglycan and decorin (20 µg/mL of each protein) or erosion composites (ERO) consisting of type III collagen, versican and high-molecular-weight hyaluronan (H) or low-molecular-weight hyaluronan (L) (20 µg/mL each). (a) Platelets were visualised using fluorescence microscopy, and (a) platelet number and (b) platelet area were quantified using Fiji/ImageJ. Data represent mean ± SEM. n = 5; * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. Thrombus formation on disease-relevant matrix composites using arterial and elevated shear stress. Whole blood labelled with DIOC6 was perfused through Ibidi µ-slides coated with 100 µg/mL type I collagen (COL1), type III collagen (COL3), a ‘rupture’ composite (type I collagen, biglycan and decorin; RUP) or erosion composites (ERO) consisting of type III collagen, versican and high-molecular-weight hyaluronan (H) or low-molecular-weight hyaluronan (L) at (a–c) arterial (15 dynes/cm2) and (d–f) elevated (75 dynes/cm2) shear stress. Chambers were imaged. Platelets were visualised using fluorescence microscopy, and thrombi size at (b) arterial and (e) elevated shear stress and platelet area coverage at (c) arterial and (f) elevated shear stress were quantified using Fiji/ImageJ. Data represent mean ± SEM; n = 5; * p < 0.05; ** p < 0.01, **** p <0.0001.

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  Figure 6. Thrombus formation in the presence of aspirin. Whole blood labelled with DIOC6 was perfused through ibidi µ-slides coated with 100 µg/mL type I collagen (COL1), a ‘rupture’ composite (type I collagen, biglycan and decorin; RUP) or erosion composites (ERO) consisting of type III collagen, versican and high-molecular-weight hyaluronan (H) or low-molecular-weight hyaluronan (L) at arterial (15 dynes/cm2) shear stress. Chambers were imaged. Platelets were visualised using fluorescence microscopy, and (a) thrombi size and (b) platelet area coverage were quantified using Fiji/ImageJ. Data represent mean ± SEM; n = 5; * p < 0.05; ** p < 0.01.

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  Figure 7. Thrombus formation on type 1 collagen with the addition of soluble decorin (DCN) or biglycan (BGN). Whole blood labelled with DIOC6 was perfused through ibidi µ-slides coated with 100 µg/mL type I collagen (COL1) at arterial shear stress (15 dynes/cm2) following the addition of recombinant (a–c) decorin (DCN) or (d–f) biglycan (BGN) (20 µg/mL) to the whole blood. Platelets were visualised using fluorescence microscopy, and (b,e) thrombi size and (c,f) platelet area coverage were quantified using Fiji/ImageJ. Data represent mean ± SEM; n = 4; * p < 0.05; ** p < 0.01.