The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa.
The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response.
Pearce
Andrew C
AC
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. andrew.pearce@spc.ox.ac.uk
Wonerow
Peter
P
Marshall
Stuart J
SJ
Frampton
Jon
J
Gartner
T Kent
TK
Watson
Steve P
SP
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009842:Oligopeptides / Q000494:pharmacology*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; C113734:leucyl-seryl-alanyl-arginyl-leucyl-alanyl-phenylalanine; D014443:Tyrosine; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium