Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins.
We have previously demonstrated that platelets in polycythaemia vera (PV) exhibit decreased aggregation after stimulation with platelet activating factor (PAF) and reduced expression of GPIIIa on both resting and stimulated platelets. In the present study, we investigated if these results were related to changes in the mobilization of intracellular calcium, activation of phospholipase D (PLD) or amounts of GPIIIa and the intracellular tyrosine kinases Fak, Syk, Grb2, Shc and rhoA. Intracellular calcium levels were not different in resting platelets from 14 PV patients and 15 healthy controls (median 43 nmol/L, range 10-114, vs. 36 nmol/L, range 10-119). After stimulation with PAF (1 micromol/L) an equal increase was seen (125 nmol/L for PV platelets, range 67-257, vs. 113 nmol/L for controls, range 60-250). Also formation of phosphatidyl ethanol (PEt) was similar after exposure to 0.5 U/ml thrombin (0.28% PEt of total phospholipid, range 0.16-1.10, vs. 0.24 for controls, range 0.11-2.3) and 1 micromol/L PMA (0.25, range 0.16-0.32, vs. 0.14, range 0.09-0.6). In contrast to the reduced amount of GPIIIa on the surface of PV platelets, immunoblotting on whole cell lysates showed no reduction in PV patients compared to controls, indicating the possibility of an impaired incorporation of GPIIIa to the cell membrane. Levels of Fak, Syk, Shc, Grb2 and rhoA appeared equal in patients and controls. Similar intracellular proteins were tyrosine phosphorylated after stimulation with thrombin, PAF and PMA. In summary, defective platelet aggregation after stimulation with PAF is caused by neither defective mobilization of intracellular calcium nor, in contrast to the situation in PV granulocytes, an impaired activation of PLD. Moreover, no apparent differences in the intracellular amounts of Fak, Syk Shc, Grb2 and rhoA could be detected between PV and control platelets.
Le Blanc
K
K
Department of Hematology, The Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden. Katarina.Leblanc@hematol.hs.sll.se
Berg
A
A
Palmblad
J
J
Samuelsson
J
J
D048868:Adaptor Proteins, Signal Transducing; D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D004792:Enzyme Precursors / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000737:chemistry / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D039661:Integrin beta3; D016023:Integrins / Q000097:blood; D047908:Intracellular Signaling Peptides and Proteins; D019751:Intramolecular Transferases / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010739:Phospholipase D / Q000097:blood / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000494:pharmacology; D011087:Polycythemia Vera / Q000097:blood* / Q000503:physiopathology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D004792:Enzyme Precursors; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D039661:Integrin beta3; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D010714:Phosphatidylethanolamines; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; D010739:Phospholipase D; D020742:rhoA GTP-Binding Protein; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; D002118:Calcium