Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Abstract

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A(2). We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34(+) hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34(+) cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34(+) cells synthesized more PGE(2) than TXB(2) (214 +/- 50 vs. 30 +/- 10 pg/10(6) cells), whereas the reverse was true in mature megakaryocytes (TXB(2) 8,440 +/- 2,500 vs. PGE(2) 906 +/- 161 pg/10(6) cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE(2) and TXB(2) to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE(2) and TXA(2) may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Figure 1

(a and b) Bone marrow biopsies were reacted with anti-COX-1 (a) or anti-COX-2 (b) Ab, and peroxidase-labeled secondary Ab. Megakaryocytes were positive for both COX-1 (a) and COX-2 (b); original magnifications: ×400. (c and d) Cytospins of purified CD34⁺ cells were stained with anti-COX-1 Ab (c) or anti-COX-2 (d) and peroxidase-labeled secondary Ab. The purity of this preparation was 94%. Original magnifications: ×1,000.

Figure 2

(a and b) Double immunofluorescence of cytospins from CD34⁺ cells at day +6 of culture, stained for CD61 revealed by FITC-conjugated secondary Abs (a) and COX-1 revealed by phycoerythrin (PE)-conjugated secondary Abs (b). (c and d) Double immunofluorescence of cytospins from CD34⁺ cells at day +6 of culture, stained for CD61 revealed by PE-conjugated secondary Abs (c) and COX-2 revealed by FITC-conjugated secondary Abs (d). (e and f) double immunofluorescence of cytospins from CD34⁺ cells at day +14 of culture, stained for CD42b revealed by FITC-conjugated secondary Abs (e) and COX-2 revealed by PE-conjugated secondary Abs (f).

Figure 3

TPO-treated CD34⁺ cells were harvested at different days of culture, counted, and incubated with AA. PGE₂ or TXB₂ were measured in the supernatants. Results are means of two to six determinations for each time point.

Figure 4

Cells from day +14 of culture were incubated with selective COX inhibitors before AA incubation. PGE₂ (Right) and TXA₂ (Left) production were measured, and the dose-response curves for each compound are shown. The respective IC₅₀s are indicated on each plot. Results are means of two determinations for each concentration.

Figure 5

(a and b) Cytospins of washed platelets were reacted with anti-COX-2 Ab, and peroxidase-labeled secondary Ab. (a) Platelets from a control subject. Note that few platelets display a clear positivity (in brown color, one positive platelet is indicated by the arrowhead), the majority are negative (one example of negative platelet is indicated by the asterisk). (b) Platelets from a patient who underwent peripheral stem cell transplantation. Several positive platelets are present in the field (indicated by the arrowhead); a negative platelet is indicated by the asterisk. The increased platelet size of patient compared with control is typical of high platelet regeneration status. (c and d) Double immunofluorescence of platelets stained for COX-2 revealed by PE-conjugated secondary Abs (c) and iPGES revealed by FITC-conjugated secondary Abs (d). (e and f) Double immunofluorescence of cytospins from CD34⁺ cells at day +14 of culture stained for iPGES revealed by FITC-conjugated secondary Abs (e) and COX-2 revealed by PE-conjugated secondary Abs (f). Original magnifications ×1,000. (c and d Insets) Digital magnification ×10.

Figure 6

TXB₂ (Upper) and PGE₂ (Lower) production during whole blood clotting. Peripheral venous samples were obtained from patients with high platelet regeneration and healthy subjects and clotted in the presence of vehicle, the COX-2 inhibitor NS-398 (10 μM), or aspirin (ASA: 50 μM). Values are expressed as percentage of the respective vehicle-treated sample. *, P < 0.01 vs. vehicle-treated samples.