Fifty years with aspirin and platelets

Abstract

In 2021, we reached the 50th anniversary of the publication of Sir John Vane's seminal paper in Nature New Biology describing the experiments supporting his mechanistic hypothesis that inhibition of prostaglandin synthesis might explain the main pharmacological effects of aspirin and aspirin-like drugs, that is, reduction in pain, fever and inflammation. Bengt Samuelsson's subsequent discoveries elucidating the cyclooxygenase pathway of platelet arachidonic acid metabolism motivated my research interest towards measuring platelet thromboxane A₂ biosynthesis as a tool to investigate the clinical pharmacology of cyclooxygenase inhibition by aspirin in health and disease. What followed was a long, winding road of clinical research leading to the characterization of low-dose aspirin as a life-saving antiplatelet drug that still represents the cornerstone of antithrombotic therapy. Having witnessed and participated in these 50 years of aspirin research, I thought of providing a personal testimony of how things developed and eventually led to a remarkable success story of independent research.

Keywords: aspirin; cardiac pharmacology; clinical pharmacology; cyclo-oxygenase; pharmacodynamics; platelets/thrombocytes; prostaglandins.

FIGURE 1

Assays of platelet biochemistry and function before and during aspirin intake in healthy volunteers. Forty‐eight subjects were randomized to one of eight groups, according to treatment duration, ranging from 1 to 8 weeks. Each participant received enteric‐coated aspirin 100 mg once daily. Maximal aggregation (Tmax) values of (a) ADP‐, (b) collagen‐ and (c) arachidonic acid‐induced aggregation; (d) aspirin response units of Verify‐Now‐Aspirin®; and absolute values of (e) serum thromboxane B₂ and (f) urinary 11‐dehydro‐thromboxane B₂ at baseline and during aspirin intake. Data shown are means ± SD; values were determined at baseline (Week 0, n = 48), Week 1 (n = 47), Week 2 (n = 42), Week 3 (n = 34), Week 4 (n = 28), Week 5 (n = 23), Week 6 (n = 17), Week 7 (n = 11) and Week 8 (n = 6). *P < 0.01, significantly different from baseline. ^# P < 0.001, significantly different from baseline. Reproduced from Santilli et al. (2009).

FIGURE 2

The three main steps in the understanding of the molecular mechanism of action of aspirin in inhibiting thromboxane‐dependent platelet function. See text for details of these discoveries.

FIGURE 3

Clinical pharmacology of platelet cyclooxygenase acetylation. Panel (a) depicts reappearance of active (unacetylated) cyclooxygenase in the circulation after a single 325‐mg aspirin dose. Values are presented as tritium incorporation (counts per minute) per milligram platelet particulate protein. Protein recovery did not vary significantly at the different time points. Data shown are individual values with means ± SD. Panel (b) depicts the effect of daily aspirin on platelet cyclooxygenase. Data shown are means ± SD. Open bars represent mean cyclooxygenase level before aspirin ingestion. Solid bars represent mean cyclooxygenase level 24 h after cessation of drug. Percentage figures above each solid bar indicate the percent inhibition attained by the respective aspirin dose. Number of subjects participating in the experiment was 6, 8 and 6 for the 20‐, 80‐ and 325‐mg doses, respectively. Reproduced from Burch, Stanford and Majerus (1978).

FIGURE 4

Clinical pharmacology of platelet thromboxane (TX) inhibition. Panel (a) depicts log‐linear inhibition of platelet cyclooxygenase activity by aspirin in healthy subjects. TXB₂ production during whole blood clotting was measured before and 24 h after oral aspirin ingestion. The results are expressed as percent inhibition, each subject serving as his or her own control. Data shown are means ± SD. Numbers in parentheses indicate the number of subjects for each dose of aspirin. Panel (b) shows selective cumulative inhibition of platelet TXA₂ production by low‐dose aspirin in healthy subjects. Serum TXB₂ concentrations and urinary excretion of 6‐keto‐PGF_1a, expressed as percentage of pre‐aspirin values, were measured in three healthy subjects before, during and after aspirin 30 mg daily. Data shown are means ± SEM. The arrows indicate duration of daily aspirin intake. Redrawn from Patrignani et al. (1982).

FIGURE 5

Acetylation of platelet cyclooxygenase (COX)‐1, inhibition of thromboxane (TX)B₂ production and reduction of vascular events by aspirin are saturable at low doses. The left panel is redrawn from Patrignani et al. (2014); the centre panel is redrawn from Patrignani et al. (1982); and the right panel is redrawn from Antithrombotic Trialists' Collaboration (2002).

FIGURE 6

Aspirin‐resistant thromboxane (TX) biosynthesis in essential thrombocythemia (ET) is explained by accelerated renewal of the drug target. Under conditions of normal megakaryopoiesis, low‐dose aspirin acetylates cyclooxygenase (COX)‐isozymes in both circulating platelets (COX‐1) and bone‐marrow megakaryocytes (COX‐1 and COX‐2), but negligible amounts of unacetylated enzymes are resynthesized within the 24‐h dosing interval. This pharmacodynamic pattern is associated with virtually complete suppression of platelet TXA₂ production in the peripheral blood throughout the dosing interval. Under conditions of abnormal megakaryopoiesis, such as in ET, an accelerated rate of COX‐isozyme resynthesis is biologically plausible in bone‐marrow megakaryocytes, accompanied by faster release of immature platelets with unacetylated enzyme(s) during the aspirin dosing interval, and in particular between 12 and 24 h after dosing. This pharmacodynamic pattern is associated with incomplete suppression of platelet TXA₂ production in the peripheral blood and time‐dependent recovery of TXA₂‐dependent platelet function during the 24‐h dosing interval. Immunohistochemistry panels depicting COX‐isozyme expression in bone‐marrow megakaryocytes (top and middle panels) and blood platelets (lower panel) are a courtesy of Prof. Bianca Rocca. Pharmacodynamic data are from Pascale et al. (2012).

FIGURE 7

Effect of aspirin on long‐term risk of colorectal cancer. The effect of aspirin (75–300 mg daily) assignment compared with control, on subsequent incidence of colorectal cancer in all randomized patients (n = 8073) in the Thrombosis Prevention Trial (TPT), the Swedish Aspirin Low Dose Trial (SALT) and the United Kingdom Transient Ischaemic Attack (UK‐TIA) Aspirin Trial (lower dose aspirin versus control) (upper panel); time to first colorectal cancer in all Colorectal Adenoma/Carcinoma Prevention Programme 2 (CAPP2) study participants (n = 861) followed up for 10 years and for 20 years in England, Finland and Wales (middle panel); cumulative incidence of colorectal cancer from time of randomization by randomized aspirin (100 mg every other day) assignment in 39,876 women aged 45 and over in the Women's Health Study (WHS), 33,682 of whom continued observational follow‐up, with P‐value from log‐rank test (lower panel). Reproduced from Rothwell et al. (2010), Burn et al. (2020) and Cook et al. (2013).

FIGURE 8

The potential role of platelet activation in the early stage of colorectal carcinogenesis. In the first stages of intestinal tumourigenesis, platelets may play a key role, because they are activated in response to intestinal mucosal injury and participate in tissue repair. However, when platelet activation is not controlled in time and space, the same mechanism may contribute to the induction of several signalling pathways through paracrine soluble mediators, such as thromboxane (TX)A₂ and prostaglandin (PG)E₂, growth factors and inflammatory cytokines, in turn inducing cyclooxygenase (COX)‐2 expression in adjacent nucleated cells, and an eicosanoid amplification loop promoting cell proliferation and angiogenesis. A sequential involvement of COX‐1 (in platelets) and COX‐2 (in various nucleated cells) in the early events leading to the transformation of an apparently normal intestinal mucosa into an adenomatous lesion would explain the similar protective effect of low‐dose aspirin and COX‐2 inhibitors in reducing the recurrence rate of a sporadic colorectal adenoma over the first 3 years of treatment and protecting against cancer development over 5–10 years. This working hypothesis was first articulated by Patrono et al. (2001) and further developed by Patrignani and Patrono (2016, 2018).