Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines
Bibliographic record
Abstract
Abstract 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".