A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation
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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Cited by (24)
Relaxin as an anti-fibrotic treatment: Perspectives, challenges and future directions
2022, Biochemical PharmacologyCitation Excerpt :RLX can also potentially regulate the innate immune response to tissue injury, and thrombosis-induced cardiovascular disease, through its suppression of platelet aggregation, given that platelets have been shown to influence innate immune cells such as neutrophils, dendritic cells and macrophages [88]. The RLX (or porcine relaxin)-induced suppression of isolated platelets in vitro (at 3–300 ng/ml) was mediated via the promotion of cyclic guanosine monophosphate (cGMP) and inhibition of calcium [89,90], while RLX suppressed platelet aggregation and activation in rodent models of AAD [70], I/R-injury [71] and liver transplantation [80,91]. Although circulating relaxin levels (of 0.4–380 pg/ml) appeared to correlate more with serum fibrinogen levels rather than platelet numbers in diabetic patients associated with aberrant coagulation [92], platelet activity was not determined in these diabetic subjects.
Recombinant human H2 relaxin (serelaxin) as a cardiovascular drug: aiming at the right target
2020, Drug Discovery TodayCitation Excerpt :Pioneer experiments on isolated and perfused rodent hearts demonstrated that purified porcine RLX (1–10 nmol/l) induced a marked and dose-related vasodilation and blood flow enhancement, even greater than that induced by equivalent doses of known vasodilators, such as acetylcholine and sodium nitroprusside [36]. Of note, porcine RLX and serelaxin were demonstrated to have nearly similar affinity to human RXFP1 and biological activity [37,38]. Later on, several studies from different laboratories provided compelling evidence that RLX is a potent vasodilator, effective on the arteries and the microcirculation of the heart, uterus, mammary gland, kidney, liver, small bowel, and brain [31–33].
Understanding relaxin signalling at the cellular level
2019, Molecular and Cellular EndocrinologyCitation Excerpt :cGMP activates protein kinase G (PKG) and cyclic nucleotide-gated ion channels, and is broken down by PDEs (Lucas et al., 2000). Relaxin stimulation produces acute increases in cGMP in human platelets (Bani et al., 2007), human hepatic stellate cells (Fallowfield et al., 2014), rat renal myofibroblasts (Wang et al., 2016), HUVECs, HUVSMCs, HUASMCs, and HCFs (Sarwar et al., 2015). The relaxin-induced cGMP concentration-response curves in HUVECs, HUVSMCs, and HCFs cells were bell-shaped.
Relaxin induces up-regulation of ADAM10 metalloprotease in RXFP1-expressing cells by PI3K/AKT signaling
2018, Molecular and Cellular EndocrinologyCitation Excerpt :This prevents binding of the modified hormone to RXFP1. To test the effective lack of bioactivity of iRLX, we measured cAMP generation in human THP-1 cells constitutively expressing RXFP1, as described (Bani et al., 2007). These findings confirm that, at variance with authentic RLX, iRLX did not induce any cAMP rise in THP-1 cells.
Heart Disease and Relaxin: New Actions for an Old Hormone
2018, Trends in Endocrinology and MetabolismCitation Excerpt :The benefits of NO signaling in the context of I/R injury have been reported comprehensively in literature [47]. Relaxin has also been shown to reduce human and rabbit platelet aggregation in vitro in a NO-dependent manner [48]. A recent study investigated the cardioprotective effect of serelaxin in adult mice at the onset of reperfusion.
Relaxin family peptide receptors - from orphans to therapeutic targets
2008, Drug Discovery Today