thromboxane-a2 and sphingosine-kinase

Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function.. To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function.. We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo.. We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Topics: Animals; Arachidonic Acid; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Carotid Artery Injuries; Disease Models, Animal; Erythrocytes; Lysophospholipids; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thrombosis; Thromboxane A2; Vascular System Injuries

FTY720 (Fingolimod) is a recently approved orally administered drug for the treatment of multiple sclerosis. Phase II and III clinical trials have demonstrated that this drug modestly increases BP. We previously showed that inhibition of sphingosine kinase increases vascular tone and BP in hypertensive, but not normotensive rats. Since FTY720 is reported to have inhibitory effects on sphingosine kinase, we investigated whether FTY720 increases vascular tone and BP only in hypertensive rats via this mechanism.. The contractile and BP modulating effects of FTY720 were studied in vivo and ex vivo (wire myography) in age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs).. Oral administration of FTY720 induced an increase in mean arterial pressure in SHR, whereas a decrease in BP was observed in WKY rats, as measured 24 h after administration. Similar to the sphingosine kinase inhibitor dimethylsphingosine (DMS), FTY720 induced large contractions in isolated carotid arteries from SHR, but not in those from WKY. In contrast, the phosphorylated form of FTY720 did not induce contractions in isolated carotid arteries from SHR. FTY720-induced contractions were inhibited by endothelium denudation, COX and thromboxane synthase inhibitors, and by thromboxane receptor antagonism, indicating that (like DMS-induced contractions) they were endothelium-dependent and mediated by thromboxane A₂.. These data demonstrate that FTY720 increases vascular tone and BP only in hypertensive rats, most likely as a result of its inhibitory effect on sphingosine kinase.

Topics: Animals; Blood Pressure; Carotid Arteries; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Fingolimod Hydrochloride; Hypertension; Immunosuppressive Agents; Isoenzymes; Male; Muscle, Smooth, Vascular; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sphingosine; Thromboxane A2; Vascular Resistance; Vasoconstrictor Agents