Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2)

Abstract

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.

FIGURE 1.

Fucoidan-induced platelet responses. A, washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37 °C under stirring conditions. Platelet aggregation was measured using a Lumi aggregometer. The traces are representative of data from at least three independent experiments. B, fucoidan-induced αIIbβ3 activation was measured by activating washed, aspirin-treated human platelets with fucoidan (50 μg/ml) and analyzed with FITC-PAC1 antibody by flow cytometry. C, dense granule secretion was assessed by measuring fucoidan-induced (50 μg/ml) ATP release from human platelets. D, α granule expression was measured by CD62P (P-selectin) expression in response to fucoidan (50 μg/ml) and was analyzed using flow cytometry. Graphs represent mean ± S.E. of % positive cells from at least three different experiments (***, p ≤ 0.05). E, washed human platelets without aspirin treatment were stimulated in a platelet aggregometer, and levels of TXB₂ were determined according to established protocol (4).

FIGURE 2.

Role of Src family tyrosine kinase (SFK)-dependent pathways in fucoidan-induced platelet activation. A, washed, aspirin-treated human platelets were incubated with YM-254890 (50 nm), a selective Gq inhibitor, for 5 min and stimulated with fucoidan (50 μg/ml) for 3 min at 37 °C under stirred conditions. Platelet aggregation was measured using lumi-aggregometer. The traces are representative of data from at least three independent experiments. B, washed, aspirin-treated human platelets were pretreated with SFK inhibitor PP2 (10 μm) or PP3 (control) at 37 °C for 5 min followed by stimulation with fucoidan (50 μg/ml) for 3 min under stirred conditions. Platelet aggregation was measured by aggregometry. The tracings are representative of data from at least three independent experiments. DMSO, dimethyl sulfoxide.

FIGURE 3.

Effect of SFK inhibition on fucoidan-induced platelet activation. A, fucoidan-induced phosphorylation of Syk and Lat. Washed, aspirin-treated human platelets were stimulated with fucoidan (50 μg/ml) for different time periods. Samples were subjected to SDS-PAGE and analyzed for phosphorylation of Syk and Lat. The results are representative of data from platelets at least three independent experiments. B, washed, aspirin-treated human platelets were stimulated with fucoidan (50 mg/ml) in the presence of the SFK inhibitor PP2 for 30 s, and the effect on Syk (Y525/26) and LAT (Y191) phosphorylation were analyzed. C, washed, aspirin-treated human platelets loaded with 5 μm FURA-2 AM were pretreated with PP3 or PP2 (10 μm) and activated with fucoidan (50 μg/ml) to measure intracellular Ca²⁺ mobilization.

FIGURE 4.

The role of Syk in fucoidan-induced aggregation, calcium mobilization, and αIIbβ3 activation. A, washed, aspirin-treated human platelets were pretreated with vehicle (dimethyl sulfoxide), OXSI-2 (2 μm), or Go 6976 (1 μm) for 5 min at 37 °C prior to activation with convulxin (CVX; 100 ng/ml) or fucoidan (50 μg/ml) (US-unstimulated). αIIbβ3 expression was analyzed with PAC-1-FITC antibody. Graphs are represented mean ± S.E. of % positive cells from three different experiments (***, p < 0.01). B, the effect of Syk inhibitors (OXSI-2 or Go 6976) on SFK phosphorylation induced by fucoidan (50 μg/ml) in human platelets was analyzed. C, time course analysis of PLCγ2 tyrosine phosphorylation from aspirin-treated human platelets stimulated with fucoidan (50 μg/ml). D, platelets were stimulated with 50 μg/ml of fucoidan for 1 min. PLCγ2 tyrosine phosphorylation was measured in the presence of OXSI2, Go6976, or dimethyl sulfoxide. E, washed, aspirin-treated human platelets loaded with 5 μm FURA-2 AM were pretreated with OXSI-2 (2 μm) or Go 6976 (1 μm) and activated with fucoidan (50 μg/ml) to measure the effect of Syk inhibition in Ca²⁺ mobilization.

FIGURE 5.

Fucoidan does not activate GPVI or FcγRIIA. A, fucoidan-induced platelet aggregation on FcRγ chain-null murine platelets. Wild-type or FcRγ chain-null murine platelets were stimulated with fucoidan (50 μg/ml) and CRP (Collagen Related Peptide) allowed to aggregate for 3 min at 37 °C under stirred conditions in a Lumi-aggregometer. The traces are representative of data from at least three independent experiments. B, human platelets were stimulated with fucoidan (50 μg/ml) in the absence and presence of IV.3 monoclonal antibody (10 μg/ml) for 3 min. The traces are representative of data from at least three independent experiments.

FIGURE 6.

Fucoidan-induced platelet responses are mediated by CLEC-2 receptor. A, wild-type or CLEC-2 knock-out platelets (2 × 10⁸/ml) were stimulated with fucoidan (50 μg/ml) and allowed to aggregate at 37 °C under stirred conditions in a Lumi-aggregometer (n = 10). B, washed human platelets (1 × 10⁹/ml) were stimulated with 100 nm rhodocytin or 50 μg/ml fucoidan. Reaction was terminated by addition of an equal volume of 2× lysis buffer. Platelet lysates were precleared, and detergent-insoluble debris were removed using centrifuge. Antibodies against CLEC-2 were added to the resultant supernatant and incubated overnight with protein A Sepharose. Precipitated proteins were separated by SDS-PAGE and Western blotted (WB) with the phosphotyrosine antibody. The data are a representation of three experiments.

FIGURE 7.

Model depicting tyrosine kinase-dependent signaling pathway in platelets activated with fucoidan. Fucoidan activates platelets through an SFK-dependent pathway through the CLEC-2 receptor. YP represents tyrosine phosphorylation sites on the cytoplasmic domain of CLEC-2 receptor.