Defective platelet aggregation in polycythaemia vera is not caused by impaired calcium signaling, phospholipase D activation or decreased amounts of focal adhesion proteins

Abstract

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.

Marked heterogeneity in protein levels and functional integrity of the thrombopoietin receptor c‐mpl in polycythaemia vera

“…Moliterno et al (1998 ) demonstrated that TPO stimulation of PV platelets conferred an impaired tyrosine phosphorylation of proteins with molecular masses of 125, 95 and 85 kDa. In contrast, thrombin induced similar phosphorylation patterns to those in controls, a finding that we have also made ( Le Blanc & Samuelsson, 1998). Furthermore, phosphorylation of JAK‐2 and its substrate STAT‐5 was also diminished in all patients that they investigated ( Moliterno et al , 1998 ).…”

“…Immunoblots showed a major band around 85 kDa, corresponding to c‐mpl, as well as weaker bands of lower molecular weight, a phenomenon of unknown significance and previously noted with the same antibody (Dr T. Kato, personal communication). In a parallel study of signal transduction in PV platelets, we examined the same samples for the expression of GPIIIa, which was the same in PV and healthy controls ( Le Blanc & Samuelsson, 1998), ensuring that similar amounts of platelet lysate protein was studied in both groups. Of the seven c‐mpl‐positive patients, only two were receiving bone marrow‐suppressive agents (one busulphan and one hydroxyurea), whereas six out of eight c‐mpl‐negative patients were treated with such agents (three hydroxyurea, two anagrelide and one α‐interferon).…”

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

“…Washed, Fg depleted platelets were prepared by repeated centrifugation using a method modified from LeBlanc et al (18). PGE1 (1 µM) and apyrase (1.0 U/ml) were added to the prepared PRP to minimize activation, followed by centrifugation at 1000 g for 10 minutes.…”

Induction of central signalling pathways and select functional effects in human platelets by β‐boswellic acid

“…Together, despite the pronounced elevation of [Ca 2 þ ] i , only select functional platelet responses were observed after stimulation with b-BA. Along these lines it was found that platelets in polycythaemia vera exhibit decreased aggregation after stimulation with PAF, although an equal increase in [Ca 2 þ ] i was seen as compared to platelets from healthy donors (Le Blanc et al, 2000). Also, a patient was described with defective platelet aggregation in response to ionophore A23187, despite normal increases in [Ca 2 þ ] i (Fuse et al, 1999).…”