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Role of chondroitin 4‐sulphate as a receptor for polycation induced human platelet aggregation
Author(s) -
Donato José L.,
Marcondes Sisi,
Antunes Edson,
Nogueira Marie D.,
Nader Helena B.,
Dietrich Carl P.,
Rendu Francine,
Nucci Gilberto
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb16057.x
Subject(s) - chemistry , platelet , polylysine , proteoglycan , biochemistry , chondroitin , platelet rich plasma , glycosaminoglycan , biology , extracellular matrix , immunology
1 Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4‐sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2 Platelets were desensitized with phorbol 12‐myristate 13‐acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet‐rich plasma (PRP). Changes in the intracellular Ca 2+ concentration were measured in fura2‐am (2 μ m ) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [ 32 P]‐phosphate labelled platelets. The release of dense granule contents was measured in [ 14 C]‐5‐hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA 2 ) was assessed by radioimmunoassay. Surface chondroitin 4‐sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37°C). The amount of chondroitin 4‐sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3 The addition of PMA to PRP before polylysine inhibited the aggregation by 88 ± 18% ( n = 3). Staurosporine (1 μ m , 5 min) prevented the PMA‐induced inhibition. Chondroitinase AC (4 pu ml −1 to 400 μu ml −1 , 3 min) abolished the polylysine‐induced aggregation in PRP but caused only a discrete inhibition of ADP‐induced aggregation. The concentration of chrondroitin 4‐sulphate in PRP (0.96 ± 0.2 μg/10 8 platelets, n = 3) and in washed platelets (WP; 0.35 ± 0.1 μg/10 8 platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 ± 0.1 μg/10 8 platelets and WP = 0.08 ± 0.06 μg/10 8 platelets). 4 Washed platelets had a significantly lower concentration of chondroitin 4‐sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA 2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 μ m , 10 min). The hydrolysis of platelet surface chondroitin 4‐sulphate strongly reduced the polylysine‐induced phosphorylation. 5 Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4‐sulphate present on the platelet membrane.