Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.

Hematophagous leeches express a broad variety of secretory factors in their salivary glands; among them are hirudins, inhibitors of blood coagulation, and decorsins/ornatins, inhibitors of platelet aggregation. Here, we describe the identification and molecular and functional characterization of putative hirudins and decorsins/ornatins in two leech species of American origin, Limnobdella mexicana and Haementeria vizottoi. The leech species represent two orders of leeches, the proboscis-bearing Rhynchobdellida and the non-proboscis-bearing Arhynchobdellida. Members of the hirudin superfamily, such as hirudins or decorsins/ornatins, are described for the first time in the genus Haementeria. Both species expressed very potent inhibitors of platelet aggregation, but only the putative hirudins of L. mexicana displayed high thrombin-inhibitory potency, whereas the putative hirudin of H. vizottoi turned out to be a hirudin-like factor. The results of our study provide new insights into the evolutionary background of the blood-sucking lifestyle in leeches.

Haematophagous leeches rely on a broad variety of bioactive compounds to secure a sufficient blood meal from their vertebrate prey. Both the primary (platelet aggregation) and secondary (blood coagulation) haemostasis are the main targets of action. The platelet aggregation inhibitor decorsin was first described in the North American leech, Macrobdella decora Say, 1824, whereas the bivalent thrombin inhibitor hirudin was originally identified in the European medicinal leech, Hirudo medicinalis Linnaeus, 1758. Hirudin blocks both the catalytic site and the fibrinogen-binding site (exosite I) of thrombin. Haemadin of the Indian land leech Haemadipsa sylvestris Blanchard, 1894, is also a highly efficient bivalent thrombin inhibitor but blocks exosite II of thrombin. So far, only the archetypal form of haemadin from H. sylvestris has been purified and functionally characterized, and two putative haemadins have been identified in the salivary transcriptome of Haemadipsa interrupta Moore, 1835, a terrestrial leech inhabiting mainly the Malayan peninsula. Here, we describe the identification of ten additional putative haemadins in the transcriptomic data set of H. interrupta, first generated by another study. Furthermore, we identified a putative oligomeric decorsin, which represents the first finding of this anticoagulant in a haemadipsid leech. Both the putative decorsin and a selection of haemadins were expressed, purified, and functionally characterized. The putative haemadins displayed a broad spectrum of thrombin-inhibitory potencies, whereas the putative oligomeric decorsin was indeed a weak inhibitor of platelet aggregation.

Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells.

Hematophagous leeches rely on a broad diversity of bioactive factors that interfere with the host’s defense systems to secure a successful blood meal. The most prominent examples of such factors are the antithrombotics that address either the primary hemostasis (the platelet aggregation) or the secondary hemostasis (the blood coagulation). Whereas the inhibitors of platelet aggregation mainly cause continuous blood flow, coagulation inhibitors mainly keep the blood fluid within the stomach of the leech. The critical dependency of hematophagous leeches on the accurate action of all antithrombotic factors may explain the presence of multiple genes for each type of these factors that is regularly observed in leech genomes. The genome of Hirudinaria manillensis Lesson, 1842, the Asian buffalo leech, contains five individual genes that encode variants of the coagulation inhibitor hirudin. However, no genes that encode putative decorsins, the archetype of leech-derived inhibitors of platelet aggregation, have been described to date. Here, we report the identification of one monomeric and four multimeric decorsin encoding genes in the genome of H. manillensis. A selection of the putative decorsins was expressed as recombinant proteins, purified and functionally characterized. All but one of these putative decorsins displayed platelet aggregation-inhibitory potencies. Furthermore, we provide a mathematical calculation based on the mechanism of alternative pre-mRNA splicing that illustrates the potential to generate an enormous variety of different factors from one single multimeric ornatin gene.

African hematophagous leeches are largely underinvestigated both in terms of their biodiversity and the characterization of the repertoire of bioactive components of their salivary gland secretions. Asiaticobdella (formerly Aliolimnatis ) fenestrata Moore, 1939, also referred to as the African medicinal leech, is hence a promising candidate for the identification and functional characterization of putative anticoagulants that may serve as inspirations for the development of new or the enhancement of existing drugs for medical purposes. A. fenestrata inhabits flooded plains of southern African regions and mainly feeds on mammals like black lechwe ( Kobus leche Gray, 1850). Whereas several anticoagulants, including two putative hirudins of A. fenestrata , have already been identified in previous investigations, functional characterizations of these components are yet missing. We conducted an in-depth re-analysis of the salivary gland transcriptomic data set of A. fenestrata and identified two additional hirudin-like factors that exhibit a remarkable structural feature—the presence of an RGD motif at or near the N-terminal end of the molecules. The presence of such an RGD motif is a key feature of leech-derived platelet aggregation inhibitors such as ornatins and decorsins. However, both in ornatins and in decorsins, the respective RGD motif is located within the molecule between the 5th and the 6th cysteine residues, but not at or near the N-terminus. We named the two putative platelet aggregation inhibitors “fenestrins.” Both the two putative hirudins and the two fenestrins of A. fenestrata were expressed as recombinant proteins, purified, and functionally characterized in either coagulation or platelet aggregation assays.