Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.

Oxidation of membrane proteins and functional activity of band 3 in human red cell senescence.

The state of oxidation of membrane proteins was analyzed in red cell subpopulations of different age by quantifying the oxidation of methionine to its sulfoxide and by determining the amount of thiol groups in ghost membrane preparations and the reactivity of thiols of individual membrane proteins in intact cells. The results obtained show that oxidation of methionine occurs early during red cell life in the circulation, and can be detected in middle-aged and senescent cells. Thiol content of ghost membranes is kept constant in all the red cell subpopulations analyzed, but reactivity of thiol groups to the thiol reagent N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM) in intact cells decreased 30% in alpha-spectrin, band 3 (B3), 4.1 and 4.2 proteins, probably as a consequence of conformational changes of these molecules. Since the role played by band 3 in the exposure of senescence antigen has been described by many authors, the functional activity of the anion transporter has been analyzed by measuring the 4-4'-diisothiocyano-stilbene-2-2-'-disulfonate (DIDS) binding capacity in different red cell subpopulations. The results obtained are in agreement with the possibility that during senescence band 3 undergoes conformational changes involving the anion channel subsite being more exposed to the extracellular space and responsible for binding of DIDS.

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes.

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

Meningeal hematopoiesis causing exophthalmus and hemiparesis in myelofibrosis: effect of radiotherapy. A case report.

Meningeal myeloid metaplasia (MM) is very rarely observed in patients with myelofibrosis. We report the occurrence of meningeal MM causing exophthalmus and fever in a patient with myelofibrosis secondary to polycythemia vera. A computerized tomography (CT) scan showed multiple intracranial and intraorbital enhancing masses. A needle aspirate of retrobulbar space confirmed the diagnosis of extramedullary hematopoiesis. The patient subsequently developed a rapidly worsening tumor-like syndrome with hemiparesis, aphasia, and loss of sphinteric control. The administration of radiotherapy caused a complete and stable regression of clinical symptoms and a marked reduction of MM masses.