Elsevier

Regulatory Peptides

Volume 144, Issues 1–3, 1 December 2007, Pages 10-16
Regulatory Peptides

A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation

https://doi.org/10.1016/j.regpep.2007.05.004Get rights and content

Abstract

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.

Introduction

Relaxin (RLX) belongs to a newly defined hormone family, termed the relaxin peptide family, that includes three different RLXs, H1, H2 and H3, as well as insulin-like peptide (INSL)3, INSL4, INSL5 and INSL6 [1], [2]. In humans, 3 separate genes have been found and designated RLN1, RLN2 and RLN3 [1], [2]. The peptide encoded by the RLN2 gene, H2 RLX, is the major circulating form and is produced mainly in the corpus luteum [3]. Circulating RLX accounts for most of the known biological effects of the hormone in humans and experimental animals. They act on the female reproductive system as well as on non-reproductive targets, including the cardiovascular system and the connective tissues [2], [4], [5], [6], [7].

RLX is the ligand for two leucine-rich repeat-containing G-protein coupled receptors (LGRs), LGR7 and LGR8 [8], now classified as relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively [9]. RXFP1 is the main and most specific H2 RLX receptor, but it is also able to bind to H1 and H3 RLX, although with less affinity. On the other hand, RXFP2 chiefly binds INSL3 and also H1 and H2 RLX, but with less affinity [8], [10]. These receptors have been found on most if not all RLX target tissues and cells and are abundantly expressed in the reproductive, nervous, renal and cardiovascular systems [8]. More recently other G-protein coupled receptors for peptides of the relaxin family have been discovered and termed RXFP3 (formerly GPCR135) and RXFP4 (formerly GPCR142) [9]. RXFP3 binds with high affinity H3 RLX [11] while RXFP4 binds H3 RLX [12] and also INSL5 [13].

Many of the milestone studies on the biological effects of RLX had been carried out using highly purified, natural RLX, extracted mainly from sow ovaries [14]. Characterisation of the RLN2 gene sequence has also allowed the production of synthetic human RLX by recombinant DNA technology [15]. Availability of recombinant H2 RLX, besides giving a further momentum to RLX research, has made clinical trials possible of RLX in promoting cervical ripening in women with delayed delivery [16] and in the treatment of scleroderma [17].

In view of the ever-increasing interest in RLX as a potential new drug, for instance in fibrosis, reproductive dysfunctions and cardiovascular diseases [2], [6], [7], [17], [18], [19], [20], it is extremely important to develop reliable methods to compare the biological activity of different RLXs. In 1995 we described a specific effect of RLX on the inhibition of human and rabbit platelet aggregation [21]. The current study shows that inhibition of platelet aggregation may be the basis for a simple, rapid and cheap bioassay for RLX with several advantages over the currently used bioassays.

Section snippets

Relaxins and related peptides

The following peptides of the RLX family were used for bioactivity comparison: i) highly purified luteal porcine RLX (pRLX, 2500–3000 U/mg) used as reference standard, prepared according to Sherwood and O'Byrne [14] and kindly donated by Dr O.D. Sherwood, University of Illinois at Urbana-Champaign (Urbana, IL, USA); ii) recombinant human H2 RLX from Phoenix (cat. #035-62, Phoenix Europe GmbH, Karlsruhe, Germany); iii) recombinant human H2 RLX (batch B917056/1/1), formerly used for clinical

Platelet aggregation assay

A 10-min incubation of platelet suspensions with pRLX or the H2 RLX preparations at concentrations of 10, 30 and 100 ng/ml caused a dose-dependent inhibition of platelet aggregation induced by ADP (3 μM). The lowest RLX dose of 3 ng/ml was ineffective, whereas the highest RLX dose of 300 ng/ml was substantially less effective than 100 ng/ml. As shown in Fig. 1, the efficacy of the tested RLX preparations was different: pRLX showed the highest and most specific effects, judging from nearly

Discussion

In the past, RLX bioassay had been based on in vivo methods on laboratory rodents, such as the interpubic ligament elongation bioassay, which are expensive, cumbersome and require a lot of relaxin and a large number of animals to obtain a dose–response relationship [reviewed in [24]]. To override these drawbacks, ex vivo bioassays were set up, such as the uterine contractility inhibition bioassay [25], the mouse brain RLX receptor binding assay [26] or the isolated atrial bioassay [27], [28],

Acknowledgments

We are grateful to Dr O.D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign (Urbana, IL, USA) for the generous gift of purified porcine RLX.

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