Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα

Abstract

Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

Graphical abstract

Figure 1.

Potency of synthetic glycopolymer–induced platelet aggregation is chain length dependent. (A) Isolated human and mouse platelet aggregation was measured in response to increasing molar concentrations of synthetic glycopolymers C329 and C13. Maximal increase in light transmission is presented as mean ± SEM (n = 3 experiments). Original traces of C329-induced (Bi) and C13-induced (Bii) human platelet aggregation and C329-induced (Ci) and C13-induced (Cii) mouse platelet aggregation. (D) Comparison of equal weight doses (30 µg/mL) of fucoidan from F vesiculosus (FV) and dextran sulfate (DS).

Figure 2.

Differences in human platelet tyrosine phosphorylation patterns comparing synthetic glycopolymers with rhodocytin, the snake venom agonist for CLEC-2. (A) Isolated platelets were stimulated using the synthetic glycopolymer C329 (0.2 µM) or the CLEC-2 agonist rhodocytin (R; 50 nM) for 0.3, 1, 2, or 5 minutes. Control (Ctrl) sample was treated with an equal volume of H₂O. Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by western blot. Membranes were stained for tyrosine phosphorylation (4G10), p-Syk Tyr^525/526, p-Syk Tyr³⁴⁸, and LAT Tyr¹⁹¹. (Bi) Phosphorylation patterns of p-Syk Tyr^525/526, p-Syk Tyr³⁴⁸, and LAT Tyr¹⁹¹ were also compared for fucoidan from F vesiculosus (FV; 30 µg/mL). Quantification of Syk (Bii) and LAT (Biii) phosphorylation. Graphs represent data from 3 or 4 experiments, and densitometry quantification is presented in arbitrary units (a.u.) as mean ± SEM. Control = 1 a.u. *P < .05 vs 0.3-minute control.

Figure 3.

Functional consequence of Syk and Src inhibition in synthetic glycopolymer– and fucoidan-stimulated human platelets. Isolated platelets were incubated for 5 minutes with the Syk inhibitor BAY 61-3606 (1 µM) or the Src inhibitor PP2 (10 µM). Samples were stimulated using the CLEC-2 agonist rhodocytin (R; 50 nM), dextran sulfate (DS; 30 µg/mL), fucoidan from F vesiculosus (FV; 30 µg/mL) or the glycopolymers C329 (0.2 µM) or C13 (4.1 µM) and analyzed by aggregometry. The GPVI agonist convulxin (CVX; 10 ng/mL) and thrombin (Thr; 0.1 U/mL) were used as controls. (A) Original traces. (B) Quantification of maximal aggregation. The inert analog PP3 (10 µM) was used as a control against PP2. Aggregation was measured as increases in light transmission. All samples contained <0.05% dimethyl sulfoxide. Representation of n = 3 experiments. *P < .05, treatment vs control.

Figure 4.

The PI3K/Akt signaling pathway is essential for human platelet activation by synthetic glycopolymers and natural fucoidan. (Ai) Original traces of platelet aggregation by C329 (0.2 µM), C13 (4.1 µM), fucoidan from F vesiculosus (FV; 30 µg/mL), and dextran sulfate (DS; 30 µg/mL) in the presence or absence of wortmannin (1 µM) or LY294002 (50 µM). (Aii) Quantification of human platelet aggregation. PAR1 activating peptide SFLLRN (10 µM) and GPVI agonist convulxin (CVX; 10 ng/mL) were used as controls. All samples contained <0.05% dimethyl sulfoxide. Maximal increase in light transmission is presented as mean ± SEM (n = 3 or 4 experiments). *P < .05, PI3K inhibitors vs control. (B) Isolated platelets were stimulated using synthetic glycopolymer C329 (0.2 µM), FV (30 µg/mL), DS (30 µg/mL), or the CLEC-2 agonist rhodocytin (R; 50 nM). Control (Ctrl) sample was treated with an equal volume of H₂O. Reactions were stopped at 0.3, 1, or 2 minutes, and samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by western blot. Graph represents n = 3 experiments; densitometry quantification is presented in arbitrary units (a.u.) as mean ± SEM. Control = 1 a.u. *P < .05, treatment vs 0.3-minute control.

Figure 5.

Synthetic glycopolymers and sulfated polysaccharides cause human platelet aggregation via PEAR1 by direct binding to EGF-like repeats 13 and 12 to 14 of PEAR1. (A) Aliquots of platelet suspension were stimulated with convulxin (CVX; 10 ng/mL), rhodocytin (R; 50 nM), dextran sulfate (DS; 30 µg/mL), fucoidan from F vesiculosus (FV; 30 µg/mL), C329 (0.2 µM), or C13 (4.1 µM) for 20 seconds, after which immunoprecipitation for PEAR1 was performed. Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by western blot. Membranes were probed with the pan tyrosine phosphorylation antibody 4G10. Control sample was treated with an equal volume of H₂O instead of agonist. *P < .05, treatment vs control. (B) Aggregation was measured as the increase in light transmission using isolated platelets. Isolated platelets were incubated for 2 minutes with anti-PEAR1 antibody (0.3 µg/mL) or matching isotype control (0.3 µg/mL) and subsequently stimulated with C329, C13, FV, or DS. R was used as control. All samples contain <0.0002% sodium azide. (C) Isolated platelets were preincubated with anti-PEAR1 antibody (0.3 µg/mL) or corresponding isotype control (0.3 µg/mL) for 2 minutes. Samples were stimulated with DS, FV, C329, or C13 for 1 minute. Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by western blot. Representation of n = 3 experiments. *P < .05, isotype vs PEAR1 antibody. (Di) Illustration of PEAR1 and schematic overview of the EGF-like repeats that were analyzed by AVEXIS. The red EGF-like repeat is number 13, which is sufficient to bind sulfated synthetic glycopolymers. (Dii) Detection of PEAR1–glycopolymer interaction by AVEXIS. SPs and NSPs were plate immobilized, acting as a bait for EGF-like repeats 1 to 12, 13, and 12 to 14. Absorbance for β-lactamase activity was measured at 485 nm. Data are mean ± SEM (n = 3 experiments). *P < .05, SPs vs NSPs.

Figure 6.

Mouse platelets deficient in PEAR1 (Pear1^−/−) still aggregate to high concentrations of synthetic glycopolymers, whereas CLEC-2–deficient platelets (Clec1b^{fl/fl;PF4-Cre}) do not. Murine platelets were analyzed for platelet aggregation upon stimulation by C329 and C13. (Ai) Original traces of PEAR1-deficient platelets compared with wild-type platelets stimulated with C329 (0.03 µM) and C13 (0.7 µM). (Aii) Quantification of maximal aggregation. Representation of 3 to 5 experiments. *P < .05, wild-type vs Pear1^−/− mice. (Bi) Original traces of CLEC-2–deficient platelets compared with wild-type platelets stimulated by C329 (0.2 µM) and C13 (4.1 µM). (Bii) Quantification of maximal aggregation within 10 minutes. Collagen (10 µg/mL) was used as a control for Clec1b^{fl/fl;PF4-Cre} platelet reactivity. Representation of 3 experiments. *P < .05, wild-type vs Clec1b^{fl/fl;PF4-Cre} mice.

Figure 7.

Synthetic glycopolymers require GPIbα to induce full aggregation in murine and human platelets. (A) Detection of GPIbα–glycopolymer interaction by AVEXIS. SPs and NSPs were plate immobilized, acting as a bait for the full-length extracellular domain of GPIbα. CD200 was used as a negative control protein. Absorbance for β-lactamase activity was measured at 485 nm, presented as mean ± SEM (n = 3 experiments). *P < .05. (Bi) Human platelet aggregation. Platelet aliquots were treated with anti-GPIbα antibody SZ2 (0.3 µg/mL), HIP2 (0.3 µg/mL), or isotype control (0.3 µg/mL) for 2 minutes before stimulation by C329 or C13. (Bii) Quantification of maximal aggregation within 10 minutes. Thrombin was used as a control at 0.3 and 0.2 U/mL. Increase in light transmission is presented as mean ± SEM (n = 3 experiments). *P < .05. (Ci) Mouse platelet aggregation. Murine wild-type platelets and transgenic mouse platelets deficient in GPIbα extracellular domain (hIL4-Rα/GPIbα-Tg) were stimulated using synthetic glycopolymers C329 (0.2 µM) and C13 (4.1 µM). (Cii) Quantification of maximal increase in light transmission within 10 minutes. Collagen (10 µg/ml) was used as a control for platelet viability. *P < .05, wild-type vs hIL4-Rα/GPIbα-Tg mice.