# Aspirin inhibition and recovery of cyclooxygenase activity and thromboxane biosynthesis in human megakaryocytes: a translational surrogate model

## TL;DR

This study shows that aspirin inhibits COX-1 in human megakaryocyte cell lines, leading to reduced thromboxane production and delayed recovery, similar to effects in platelets.

## Contribution

The study introduces a reliable megakaryocyte-based model to study aspirin's effects on COX-1 inhibition and recovery kinetics.

## Key findings

- A single aspirin exposure suppressed TXB2 production by over 85% in megakaryocytic cell lines.
- COX-1 inhibition by aspirin is time- and dose-dependent, with recovery taking 48–72 hours.
- Repeated aspirin exposure leads to sustained COX-1 inhibition and delayed recovery, mirroring platelet behavior.

## Abstract

Low-dose aspirin irreversibly acetylates cyclooxygenase (COX)-1 in anucleate platelets and progenitor megakaryocytes, permanently suppressing thromboxane (TX)A2-dependent platelet activation. Although aspirin pharmacodynamics is well characterized in platelets, the kinetics of COX inhibition and recovery in human megakaryocytes remains poorly defined, due to ethical issues associated with invasive, bone-marrow trephine sampling, and low megakaryocyte yield. We studied aspirin pharmacodynamics in human megakaryocytic cell lines as a reliable and feasible surrogate model. We characterized COX-1 and COX-2 expression and activity in MEG-01 and CHRF-288-11 megakaryocytic cell lines, treated with a range of aspirin concentrations and exposure duration. COX activity was quantified by the production of TXB2 from exogenous arachidonic acid. A single 10-μM aspirin exposure suppressed TXB2 by 90 ± 2% (MEG-01) and 85 ± 4% (CHRF-288-11), with full recovery within 48–72 hours. Both COX-isozymes were detected by western blot and immunohistochemistry; however, selective COX-1 inhibition by SC-560 reduced TXB2 by >75%, whereas COX-2 inhibition by NS-398 had minimal effect. Repeated aspirin exposure every 24 hours produced concentration- and time-dependent TXB2 suppression, achieving 89 ± 2% inhibition by day 2 at 1 μM and 73 ± 3% by day 4 at 0.1 μM. TXB2 biosynthesis recovered by 86 ± 2% and 99 ± 10% at days 2 and 3, respectively. These findings identify COX-1 as the principal source of TXA2 in megakaryocytes and demonstrate that aspirin inhibits megakaryocyte COX-1 time- and dose-dependently, with delayed recovery likely reflecting de novo synthesis of COX-1 protein, thereby providing mechanistic insight into the sustained antiplatelet effect of low-dose aspirin in humans.

In human megakaryocyte cell lines, once-daily aspirin treatment at low-concentration range time-dependently inhibits COX-1 with delayed recovery after aspirin withdrawal. This closely mimics the kinetics of platelets, supporting the translational utility of the megakaryocyte-based surrogate model.