Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.