A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation.
In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5. Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10-100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose-response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.
Bani
Daniele
D
Department of Anatomy, Histology and Forensic Medicine, University of Florence, viale G.Pieraccini 6, I-50139 Florence, Italy. daniele.bani@unifi.it
Nistri
Silvia
S
Cinci
Lorenzo
L
Giannini
Lucia
L
Princivalle
Marc
M
Elliott
Lucy
L
Bigazzi
Mario
M
Masini
Emanuela
E
D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000378:metabolism / Q000494:pharmacology*
D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C033395:relaxin receptors; D012065:Relaxin; D006152:Cyclic GMP