Project 1: Studies on the role of CLEC-2 and Syk in lymphatic development and response to radiation & Project 2: A role for atrial natiuretic peptide in platelet inhibition and evidence for compartmentation of cGMP signalling.

Abstract

Project 1: The platelet C type lectin-like type II (CLEC-2) transmembrane receptor has been shown to interact with its endogenous ligand, podoplanin, on lymphatic endothelium and mediate separation from blood vessels. Here, conditional deletion of CLEC-2 or a mutation in signalling molecule Syk, specifically in the megakaryocyte/platelet lineage, was seen to cause a severe blood-lymphatic mixing phenotype. As constitutive loss of CLEC-2 in mice is lethal at birth, radiation chimeras have been pivotal to investigating the role of CLEC-2 in platelet function. This pilot study was conducted to investigate the role of platelets in the recovery from radiation injury. Mice were subjected to irradiation and reconstituted with CLEC-2+/+ or CLEC-2-/- foetal liver cells. Immunofluorescence in the intestinal mesentery at 9 days showed disrupted vasculature in CLEC-2-/- mice. The only visible difference between the two sets of chimeras was blood in the intestine of CLEC-2-/- mice at 28 days. Terminal deoxynucleotidyl end labeling (TUNEL) showed no difference in the percentage of apoptotic cells in the livers of CLEC-2+/+ and CLEC-2-/- mice but an increase in the spleen. These results support the idea of a progressive intestinal phenotype and support a role for CLEC-2 and Syk in the separation and organization of blood and lymphatic vessels.

Project 2: Nitric oxide (NO) has long been accepted as a potent and powerful inhibitor of platelet function acting through soluble guanylyl cyclase (sGC) to elevate levels of cyclic guanosine 3’,5’-monophosphate (cGMP). In cells such as cardiac myocytes, cGMP levels are also influenced by natriuretic peptides (NP) which bind to a particulate GC (pGC). Despite a number of studies indicating binding sites for atrial NP (ANP) on platelets, their influence on platelet function is unclear. Here it is demonstrated that NO and ANP mediate concentration-dependant inhibition of platelet aggregation to thrombin which is not overcome by high concentrations of the protease. ANP also stimulated weak phosphorylation of vasodilator stimulated phosphoprotein (VASP) in contrast to the robust response to NO and which was highly variable between donors. Further, sildenafil, a selective PDE5 inhibitor, was shown to enhance NO but not ANP mediated platelet inhibition and VASP phosphorylation indicating soluble and particulate cGMP pools are compartmentalised in platelets. Together these findings indicate a role for ANP in inhibiting platelet function which is distinct from that of NO.