Constitutive Expression and Involvement of Cyclooxygenase-2 in Human Megakaryocytopoiesis

Abstract

Objective-Cyclooxygenase-1 (COX-1), but not COX-2, is expressed in human platelets, and thromboxane A 2 (TXA 2 ) produced via COX-1 induces platelet aggregation. The objectives of this study were to investigate the expression of COX-1 and COX-2 during platelet differentiation and to determine whether these enzymes are involved in the differentiation. Methods and Results-CD34ϩ progenitor cells isolated from human cord blood were cultured with thrombopoietin and c-kit ligand. The cells differentiated into megakaryocytes (CD34 Ϫ /CD41 ϩ ) after 8 days of culture and into platelets (CD41 ϩ /prodium iodide Ϫ ) after 14 days of culture. The CD34 ϩ cells expressed a trace of COX-1 gene and no COX-2 gene. On day 5, COX-2 gene expression was observed and continued throughout the remainder of the culture. COX-1 gene expression increased after 8 days of culture. The treatment of this liquid culture with indomethacin, a dual inhibitor of COX-1 and COX-2, and NS-398, a COX-2-specific inhibitor, suppressed megakaryocyte differentiation. In contrast, at a dose of 10 Ϫ7 M, mofezolac, which is a highly selective inhibitor of COX-1, did not affect differentiation. NS-398 -induced suppression of megakaryocyte differentiation was partly abrogated by stable analogues of TXA 2 . Conclusions-We report here that COX-2 and COX-1 are constitutively expressed in megakaryocytes, and TXA 2 produced by COX-2 plays an important role in megakaryocytopoiesis.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy

“…In light of previous evidence of COX-2 expression in mature megakaryocytes from normal [24][25][26][27][28][29] and ET 30 bone marrow specimens, and of in vitro data showing that platelet COX-2 might contribute to TXA 2 biosynthesis in hematopoietic hyperregenerative conditions (eg, recovery after bone marrow transplantation), 24 we sought to explore the contribution of platelet COX-2 to TXA 2 biosynthesis in ET. Etoricoxib, a highly selective COX-2 inhibitor, 42 was used as a pharmacologic tool to test this hypothesis in vivo, along with experiments adding a selective COX-2 inhibitor, NS-398, in vitro to blood samples from aspirin-treated ET patients.…”

“…22 Aspirin achieves its antithrombotic effect by permanently and selectively inactivating platelet cyclooxygenase 1 (COX-1), thus blocking TXA 2 biosynthesis. 23 It has been shown that COX-2 is up-regulated during megakaryopoiesis, 24 is expressed by normal megakaryocytes and circulating young platelets, [24][25][26][27][28][29] and it is overexpressed in bone marrow megakaryocytes of ET patients. 30 Under conditions of physiologic thrombopoiesis, platelet COX-2 does not appear to contribute to TXA 2 production.…”

“…32 In fact, only 23 of 104 serum TXB 2 measurements in ET patients were below this threshold. Although we could not obtain pre-aspirin measurements, the data of the present study are nevertheless consistent with the less-thancomplete inhibition (Ͻ 90%) of serum TXB 2 that we previously reported in a limited number of untreated ET patients who were given aspirin of 50 or 100 mg, 19 at a time when antiplatelet therapy was not uniformly prescribed.In light of previous evidence of COX-2 expression in mature megakaryocytes from normal [24][25][26][27][28][29] and ET 30 bone marrow specimens, and of in vitro data showing that platelet COX-2 might contribute to TXA 2 biosynthesis in hematopoietic hyperregenerative conditions (eg, recovery after bone marrow transplantation), 24 we sought to explore the contribution of platelet COX-2 to TXA 2 biosynthesis in ET. Etoricoxib, a highly selective COX-2 inhibitor, 42 was used as a pharmacologic tool to test this hypothesis in vivo, along with experiments adding a selective COX-2 inhibitor, NS-398, in vitro to blood samples from aspirin-treated ET patients.…”

A Novel Nuclear Signaling Pathway for Thromboxane A2 Receptors in Oligodendrocytes: Evidence for Signaling Compartmentalization during Differentiation

“…Furthermore, TPR activation is also known to Four milligrams of protein of isolated extranuclear (E) and nuclear (N) fractions from rat OPCs/OLGs was incubated with the TPR-immunoaffinity matrix. After washing, the bound proteins were eluted, subjected to SDS-PAGE, and probed using antibodies against TPR and various G␣ subunits: G q , G 13 (29,34,47), and is thought to be involved in apoptosis (11,49). As is the case with other GPCRs, TPRs have been shown to activate multiple G proteins, in particular G q and G 13 ( 10, 23, 35, 45).…”

Regulatory effects of TLR2 on megakaryocytic cell function

“…In addition, increases in MCP-1 secreted by the platelets could increase the immune response in the circulation. COX2 levels were shown to be up-regulated during megakaryocyte maturation [48][49] and have an effect on this process. 49 Increases in both TLR2 and NFB1 further enhance the megakaryocyte TLR2 cell-surface recognition of PAMPs and increase the number of signaling proteins to respond to any activation of this receptor.…”