Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets.
BACKGROUND
P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate.


METHODS
Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators.


RESULTS
The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and <25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction.


CONCLUSIONS
Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.
Bachelet
Laure
L
Inserm, U698, Cardiovascular Bioengineering, CHU X. Bichat, University Paris 7, Paris, F-75877, France.
Bertholon
Isabelle
I
Lavigne
Damien
D
Vassy
Roger
R
Jandrot-Perrus
Martine
M
Chaubet
Frédéric
F
Letourneur
Didier
D
D000328:Adult; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D016264:Dextran Sulfate / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D000080506:Sialyl Lewis X Antigen; D013379:Substrate Specificity
D000925:Anticoagulants; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D006493:Heparin; D016264:Dextran Sulfate; C007789:fucoidan