10.4081/btvb.2024.121
This historical and personal review mainly focuses on the contribution of our research group and other Italian Colleagues to the development of aspirin pharmacology and its clinical use as an antithrombotic drug, in the Sixties, Seventies and Eighties. The main lines of research that have been developed over the last three decades, both at the experimental and clinical level, are not the subject of the present review.
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Chiara Cerletti
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Bleeding, Thrombosis and Vascular Biology
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Blood platelets, prostaglandins and aspirin: a historical and personal rereading
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2024-01-22
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Bleed Thromb Vascul Biol
Chiara Cerletti
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10.4081/btvb.2024.121
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Blood platelets, prostaglandins and aspirin: a historical and personal rereading
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                                                                                                            NARRATIVE REVIEW

Blood platelets, prostaglandins and aspirin:
a historical and personal rereading
Giovanni de Gaetano, Chiara Cerletti

Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy



ABSTRACT
    This historical and personal review mainly focuses on the contribution of our research group and other Italian Colleagues to the de-
velopment of aspirin pharmacology and its clinical use as an antithrombotic drug, in the Sixties, Seventies and Eighties. The main lines
                                                                       of research that have been developed over the last three decades,
                                                                       both at the experimental and clinical level, are not the subject of
                                                                       the present review.

  Correspondence: Chiara Cerletti, Department of Epidemiology and
  Prevention, IRCCS Neuromed




                                                                                                ly
  Via dell’Elettronica, 86077 Pozzilli (IS), Italy.
  Tel.: +39.0865.915248.
                                                                             Platelets were born 15 years before aspirin




                                                                                        on
  E-mail: chiara.cerletti@moli-sani.org

  Key words: aspirin, platelets, prostaglandins.                                  Platelets were discovered by Giulio Bizzozero more than 140
                                                                             years ago and rediscovered in the early Sixties after many decades




                                                                                  e
  Contributions: the authors contributed equally.                            of oblivion.1 Overlooked for more than two centuries after the mi-

  Conflict of interest: the authors declare no potential conflict of in-
  terest.
                                                                            us
                                                                             croscope was made available to hematologists, considered as an
                                                                             artifact or a Cinderella, platelets became a prima ballerina in the
                                                                             early Sixties, when they were considered dangerous cells to be in-
                                                                            al
  Funding: none.
                                                                             hibited by aspirin and other more expensive drugs.
                                                                                  In the late Fifties and early Sixties, Baserga in Ferrara,2 Born
                                                         ci

  Conference presentation: the content of this review was presented          and Cross in London,3 and O’Brien in Portsmouth,4 described
                                                       er


  in part at the “Convegno ASA 2022: l’acido acetilsalicilico ha 125         an optical platelet test, roughly based on the decrease of optical
  anni”, held in Matera (MT), Italy, May 14/15, 2022.                        density of a platelet suspension corresponding to platelet dis-
                                                      m



                                                                             ruption by a hypotonic solution or by clump formation by ADP
  Acknowledgements: the authors wish to gratefully remember Pro-
                                                                             or other stimuli. Very soon, especially the aggregometer devel-
  fessors Bruno Bizzi, Raffaello Breda, Marc Verstraete, Melvin Sil-
                                          om




  ver, Bryan Smith and Alfredo Leonardi. The authors’ studies                oped by Born, appeared to be an easy and practical method and
  mentioned in this review could not have been performed without             platelet aggregation and function could be studied in dozens of
  the faithful collaboration of many Colleagues in Milano, Santa             laboratories all around the world. One of us, at the Laboratory
                                    -c




  Maria Imbaro, Campobasso and Pozzilli: it would be a very long             of Verstraete and Vermylen, in Leuven, Belgium, started to pre-
  list. Our profound gratitude and appreciation go to all of them, one       pare in 1968 his PhD thesis using an original aggregometer pro-
                           on




  by one. Let’s only mention here Professors Josef Vermylen, Silvio          vided by Born himself.
  Garattini and Giovanni Di Minno, Vittorio Bertelé, Elisabetta De-
  jana, Roberto Latini, Jaime Merino, Silvia Villa, Francesca Bucchi,
                     N




  Mary Gambino, Marilena Crescente and the late Manuela Livio
  and Grazyna Rajtar. The authors are grateful to Dr. Fabrizia Noro          Meeting of aspirin with platelets
  for figures editing.
                                                                                 Aspirin, acetylsalicylic acid, was born 15 years after platelets,
  Received: 22 January 2024.                                                 in 1897, but it was only after 70 years, in 1967, that platelets and
  Accepted: 4 March 2024.                                                    aspirin met each other officially for the first time and a never-end-
                                                                             ing story was begun.
  Publisher’s note: all claims expressed in this article are solely those
  of the authors and do not necessarily represent those of their affili-         In reality, already in the Fifties, French investigators had ob-
  ated organizations, or those of the publisher, the editors and the re-     served that aspirin,5,6 in relatively small doses, resulted in a pro-
  viewers. Any product that may be evaluated in this article or claim        longation of bleeding time. They had also noted that this effect
  that may be made by its manufacturer is not guaranteed or endorsed         was exaggerated in patients who had underlying bleeding disor-
  by the publisher.                                                          ders. These clinical observations were confirmed in the USA by
                                                                             Quick,7 the inventor of the prothrombin time, who also made the
  ©
   Copyright: the Author(s), 2024
                                                                             important observation that, unlike aspirin, sodium salicylate had
  Licensee PAGEPress, Italy
  Bleeding, Thrombosis and Vascular Biology 2024; 3:121                      no effect on bleeding time.
  doi:10.4081/btvb.2024.121                                                      Weiss and Aledort first showed that prolongation of the bleed-
                                                                             ing time by aspirin (3 grams/day for two days!) was associated
  This work is licensed under a Creative Commons Attribution-                with a marked impairment of collagen-induced platelet aggrega-
  NonCommercial 4.0 International License (CC BY-NC 4.0).                    tion.8 By contrast, aspirin ingestion did not inhibit primary ADP-
                                                                             induced aggregation. Other groups almost at the same time,9-11


                                             Bleeding, Thrombosis and Vascular Biology 2024; 3:121
2                                                        G. de Gaetano and C. Cerletti




confirmed and extended these original findings. Sodium salicylate          clinical trials of aspirin in ischemic arterial disease,19 that aspirin
failed to prevent platelet aggregation induced either by collagen          was effective in the secondary prevention of different ischemic
or ADP.10 The general provisional conclusion was that aspirin –            arterial diseases such as myocardial infarction and stroke, as con-
possibly by a poorly defined platelet membrane stabilizing effect          firmed some years later by a larger meta-analysis.20
– inhibited the platelet release reaction.12 The effects of aspirin
ingestion on platelet aggregation occurred very rapidly but were
of long duration (4 to 7 days),10 suggesting an irreversible damage        Development of platelet pharmacology
to platelet population, which persisted until the affected platelets
had been replaced by a sufficient number of new platelets. The                  On the basis of the evidence available in the early
possible critical role of the acetyl group in the aspirin effect was       Seventies,21,22 aspirin, dipyridamole and sulfinpyrazone, three
also rapidly singled out. Altogether, these findings reasonably ex-        drugs already in clinical use for other indications, had been
plained the mild hemostatic defect produced by aspirin and indi-           shown to possess antiplatelet effects but for many years no
cated that it should be avoided in patients in whom control of             «new» antiplatelet compound came to the stage of clinical in-
hemostasis (especially during and after surgical operations), could        vestigation. In 1971 a group of three articles reported that aspirin
be a problem.                                                              blocked the production of PGE2 and PGF2α in human platelets
     «The heretic – says William in The Name of the Rose – may             (as confirmed in other experimental systems) and John Vane pro-
be born from the saint and the possessed from the seer».13 No sur-         posed that prostaglandin inhibition might explain some or even
prise, therefore, that a more intriguing outcome of these studies          all pharmacologic properties and clinical effects of aspirin (and




                                                                                                       ly
on hemostasis, was the possibility that, though being a potential          of other non-steroidal anti-inflammatory drugs).23-25 However,




                                                                                               on
hemorrhagic drug by inhibiting platelet aggregation, aspirin might         neither PGE2 nor PGF2α appeared to play a role in platelet ag-
become a useful anti-thrombotic agent.14-16                                gregation, so that the mechanism by which aspirin could prevent
     If aspirin was capable of inhibiting collagen-induced platelet        platelet aggregation by interfering with prostaglandin synthesis




                                                                                         e
aggregation, might it also prevent arterial thrombus formation?            remained obscure for some time.26
     In the early Seventies, the case for testing aspirin in the pre-           In the early Seventies, de Gaetano et al. had reported the oc-
vention of myocardial infarction and other arterial occlusion dis-
eases became therefore quite strong, although, at that time, the
                                                                                 us
                                                                           currence of platelet aggregation and release reaction induced by
                                                                           a commercial mixture of essential fatty acids (Thrombofax, Ortho
role of platelet aggregation and even of thrombosis in myocardial          Diagnostics), possibly by a substance generated during a short-
                                                                         al
infarction was not a common knowledge.17                                   term incubation with platelets. Thrombofax-induced aggregation
                                                             ci

     This is possibly a reason why aspirin was first clinically tested     was fully prevented by aspirin.27,28 The team of Melvin Silver at
as a prophylactic measure in post-operative venous thromboem-              Cardeza Foundation in Philadelphia confirmed this observation
                                                           er


bolism; the results of the Medical Research Council of England’s           by using a purified arachidonic acid preparation.29 They also re-
trial were however negative.18 We had to wait until the late Eight-        ported the formation of an intermediate in platelet prostaglandin
                                                          m



ies to be informed, by a pioneering meta-analysis of the first six         biosynthesis and its association with platelet activation.30
                                                om
                                          -c
                                  on
                            N




Figure 1. Schematic metabolism of arachidonic acid.


                                          Bleeding, Thrombosis and Vascular Biology 2024; 3:121
                              Blood platelets, prostaglandins and aspirin: a historical and personal rereading                               3



     In 1975, Samuelsson and his associates at the Karolinska In-
stitutet in Stockholm elucidated platelet arachidonic acid me-            The biochemical selectivity of aspirin
tabolism first confirming and characterizing the generation of
                                                                               A biochemical selectivity of aspirin was achieved in rats in a
unstable biosynthetic intermediates, the cyclic endoperoxides
                                                                          rather unusual way: an animal made thrombocytopenic by an-
PGG2 and PGH2, then with the description of an extremely po-
                                                                          tiplatelet antibodies was exchange-transfused with blood from an-
tent but labile platelet-aggregating prostanoid named thrombox-
                                                                          other animal pretreated with aspirin a few hours before (in order
ane (Tx)A2 (Figure 1).31
                                                                          to allow complete elimination of the intact drug from the periph-
     The discovery of TxA2 clearly identified the biochemical
                                                                          eral circulation). The recipient rat had therefore aspirinated
link, still missing at that time, between inhibition of platelet
                                                                          platelets but non aspirinated vessel walls. Notwithstanding this
arachidonic acid metabolism and impaired platelet function.
                                                                          pharmacologic success, the bleeding time of the animals did not
     One year later, however, the discovery was announced by
                                                                          change significantly.38
Moncada et al.,32 in Vane’s laboratory in London, of prostacyclin              As summarized in the next paragraph (The development of
(PGI2), an unstable vasoactive and platelet-inhibiting                    the low-dose aspirin concept and its clinical application),39-48 bio-
prostaglandin produced by the vessel walls. The synthesis of              chemical selectivity in man was successfully demonstrated in
PGI2 was also inhibited by aspirin.                                       Rome, by Patrono’s team, by administration of repeated small
                                                                          doses of aspirin to normal volunteers.39 This was explained by the
                                                                          fact that platelet cyclo-oxygenase, once irreversibly acetylated by
The aspirin dilemma




                                                                                             ly
                                                                          aspirin, could not be replaced as long as the affected platelets re-
                                                                          mained in the circulation. As a consequence, the effects of single,




                                                                                     on
     This created the so-called aspirin dilemma, that is the clin-
                                                                          partially effective doses of aspirin could be expected to accumu-
ical relevance of a concomitant inhibition by this drug of two
                                                                          late – and this, in fact, occurred.
major modulators of platelet and vascular homeostasis with op-
                                                                               Concomitantly, the same repeated low doses of aspirin failed




                                                                               e
posing biological effects.26
                                                                          to affect vascular prostacyclin biosynthesis. However, cumulative
     Although faint experimental evidence was only available
that aspirin would be thrombogenic at high doses, it was thought
that inhibition of vascular PGI2 might limit the potential an-
                                                                        usinhibition of PGI2 synthesis measured on vascular segments was
                                                                          reported after administration of repeated low doses of aspirin to
                                                                          patients with atherosclerosis.49
                                                                   al
tithrombotic effects of aspirin resulting from inhibition of                   One point of debate was that suppression of platelet TxA2
platelet TxA2 production. Consequently, doubts were generated             biosynthesis might not necessarily result, by itself, in inhibition
                                                    ci

on the clinical usefulness of aspirin as a potential antithrombotic       of platelet function in vivo, if not accompanied by a simultaneous
drug.
                                                  er


                                                                          inhibition of the intermediate prostaglandin endoperoxides PGG2
     The simultaneous inhibition of TxA2 and PGI2 synthesis               and PGH2.50,51 The report that when pairs of agonists (such as PAF
could have been the reason for the disappointing results of early
                                                 m




clinical trials on the antithrombotic effect of relatively large
doses of aspirin.19 It was even shown that animals treated with
                                       om




high doses of aspirin, which inhibited PGI2 synthesis, might
have an increased thrombotic tendency.33 Moreover, humans tak-
ing high doses of aspirin exhibited a shortened bleeding time.34
                                 -c




The assumption was made, and popularized, that to achieve an-
                         on




tithrombotic efficacy, the inhibitory effect of aspirin on platelet
cyclo-oxygenase should be retained, while that on the vascular
enzyme should be minimized (Figure 2). Several experimental
                   N




approaches were therefore adopted to estimate the dose of as-
pirin which would suppress the synthesis of TxA2 but not that
of prostacyclin. Neri Serneri’s group in Florence reported in a
study on 25 volunteers that inhibition of platelet cyclo-oxyge-
nase occurs with smaller doses of aspirin and lasts longer than
inhibition of vessel-wall cyclo-oxygenase. They suggested that
3.5 mg/kg (corresponding to about 250 mg for a person of 70
kg b.w.) would be the best dose of aspirin to most likely produce
a consistent inhibition of platelet aggregation but only a slight
inhibition of prostacyclin production.35
     Another hypothesis was based on the assumption that the
platelet enzyme would be more sensitive to aspirin than the vas-
cular enzyme. Although studies in vitro comparing platelets with
cultured human endothelial cells, showed that aspirin exerted a
similar inhibitory profile,36 the search for a clinically appropriate
dose of aspirin continued to be intense; all attempts using single
oral doses of aspirin failed to significantly dissociate the drug’s
pharmacological effects on platelets and vascular cells, both in          Figure 2. Functional model of cyclo-oxygenase inhibition by
                                                                          aspirin and other non-steroidal anti-inflammatory drugs.
experimental animals and in man.35,37


                                          Bleeding, Thrombosis and Vascular Biology 2024; 3:121
4                                                        G. de Gaetano and C. Cerletti




and adrenaline) were used to induce platelet aggregation, repeated         clotting in a glass test tube at 37°C.40 This paper was accepted for
low doses of aspirin appeared to be no longer effective did not at-        publication in Thrombosis Research by the Editor Maria
tract any clinical attention.52,53                                         Benedetta Donati. At about the same time, the discovery of 2,3-
     The low-dose aspirin concept, even before being successively          dinor-TxB2 as a major enzymatic metabolite of TxB2 allowed in-
evaluated in controlled clinical trials, received an enthusiastic re-      vestigating TxA2 biosynthesis in vivo and its pharmacological
ception by many clinicians. They were fascinated not only by the           reduction by aspirin.41,42
apparent simplicity of this pharmacologic approach, but wel-                    Patrono’s group showed that it was possible to dissociate the
comed the foreseeable reduction, or even disappearance, of side            effect of low-dose aspirin (30 mg daily) on serum TxB2 (almost
effects (mainly gastrointestinal) related to the chronic intake of         exclusively a product of platelet cyclo-oxygenase (COX)-1) from
relatively high doses of aspirin, despite a pioneer meta-analysis,         the effect on urinary 6-keto-PGF1α (mainly a product of renal COX-
already mentioned, of the results of the first six controlled clinical     2) by exploiting the cumulative nature of platelet COX-1 inacti-
trials had shown a dose-unrelated beneficial effect of aspirin in          vation he had observed on repeated daily dosing. Platelet COX-1
the secondary prevention of mortality in patients with myocardial          activity was almost abolished after approximately one week of
infarction.19 The dose-unrelated beneficial effect of aspirin had          daily dosing with 30 mg aspirin, while furosemide-induced renal
been confirmed in patients with unstable angina.54-56 To better un-        PGI2 biosynthesis was not significantly diminished.39 The nonlin-
derstand the clinical problem of the lack of dose-response rela-           ear relationship between inhibition of the maximal biosynthetic
tionship of aspirin, de Gaetano et al.57 became interested in the          capacity of platelets (as reflected by serum TxB2 measurements)
possible effects of salicylate – this metabolite has a longer plasma




                                                                                                         ly
                                                                           and the inhibition of platelet activation in vivo (as reflected by uri-
half-life than the parent molecule and may accumulate during re-           nary TxM measurements), required a persistent inhibition of the




                                                                                                on
peated drug administration. The importance of plasma salicylate            former (>95%) to produce a measurable effect on the latter.43 The
levels in regulating the interaction between aspirin and cyclo-oxy-        ‘hit-and-run’ platelet inhibition by a very short half-life acetylsal-
genase suggested that a better knowledge of the pharmacokinetics           icylic acid molecule permanently inactivated a platelet protein that




                                                                                         e
of aspirin and salicylate might help resolve the aspirin dilemma,58        could not be re-synthesized within the 24-h dosing interval.44
as described in more detail in the paragraph Implications of the
salicylate-aspirin interaction.59-68
     In young healthy subjects, administration of high-dose aspirin
                                                                                 us
                                                                                Another indirect support to the use of low-dose aspirin de-
                                                                           rived from studies, debunking the (apparently logical) assumption
                                                                           that platelet TxA2 synthase, rather than COX-1 inhibition, should
                                                                         al
(650 mg × 2) or indobufen (200 mg × 2) – a cyclo-oxygenase in-             be preferred to obtain a clear dissociation between TxA2 and PGI2
hibitor unrelated to salicylate – significantly inhibited not only, as     synthesis blockade. Indeed, following pharmacological selective
                                                             ci

expected, serum TxB2 generation but also the rise in tissue plas-          platelet TxA2 synthase inhibition, a combination of endogenous
minogen activator induced by a provoked standard venous occlu-             PG endoperoxides and PGE2 resulted in normal aggregation, de-
                                                           er


sion, without affecting the pre-occlusion values. In contrast,             spite a full suppression of TxA2 production.45,46 It was concluded
salicylate (569 mg × 2, a dose equimolar to 650 mg × 2 of aspirin)         that inhibition of TxA2 synthase does not prevent platelet aggre-
                                                          m




did not affect either TxB2 generation or the fibrinolytic response.        gation as the functional result appears to be modulated by an in-
                                                om




Low-dose aspirin (20 mg × 7 days) while reducing serum TxB2                terplay of the endogenous aggregating PG-endoperoxides and
generation by about 90%, did not modify the increased fibrinolytic         PGE2, formed in excess, concomitantly to TxA2 selective suppres-
response to venous occlusion.69 The hypothesis that the rise in fib-       sion. Low-dose aspirin was in contrast able to prevent not only
rinolytic activity occurring during this hypoxemic challenge is
                                          -c




                                                                           the final generation of TxA2, but also that of compensatory en-
mediated by local generation of vascular PGI2 was clearly demon-           dogenous PGs.46
                                  on




strated both in humans and in experimental animals.70,71                        All these findings led Peto et al. to successfully test a 160 mg
     Thus, low-dose aspirin, by sparing vascular cyclo-oxygenase           daily dose of aspirin in the first large-scale, placebo-controlled
activity, would leave intact not only the antiaggregating (PGI2)           randomized trial for efficacy and safety in the short-term treatment
                            N




but also the fibrinolytic potential of the vessel wall. The solution       of patients with acute myocardial infarction.47 This marked the
of the aspirin dilemma could therefore have wider implications             transition from descriptive phenomenology and empirical trials
than simply the platelet-oriented TxA2-PGI2 balance.                       to molecular understanding of platelet pharmacology, and the de-
                                                                           sign of new randomized clinical trials (RCT).
The development of the low-dose aspirin concept                                 The first RCT performed in Italy on low-dose aspirin, the Pri-
and its clinical application                                               mary Prevention Project (PPP),48 reported a significant reduction
                                                                           of composite thrombotic endpoints in healthy individuals at car-
     Although by Born’s optical platelet aggregometry several in-          diovascular risk. This trial had also the rare characteristic of being
vestigators had already reported in the Sixties the ability of aspirin     performed in a diffuse national general practice context.
to inhibit platelet function at low-doses,10 it was not until the dis-          Both Patrono and FitzGerald were awarded by the Institut de
covery of TxA2 and the development of mechanism-based bio-                 France the prestigious recognition of the Lefoulon Delalande
chemical end points that the human pharmacology of platelet                Grand Prize, in 2013, for their studies on aspirin pharmacology.
inhibition by aspirin could be properly elucidated.
     Patrono et al. at the Catholic University in Rome, replaced           Implications of the salicylate-aspirin interaction
the smooth muscle strips that Vane had used to quantify the release
of unstable prostanoids (e.g. ‘rabbit aorta contracting substance’)             Cerletti et al. in 1982, suggested that to inhibit platelet activity,
with a soluble antibody against TxB2, the stable hydrolysis product        aspirin had first to bind, through its salicylate moiety, to a binding
of TxA2, to determine the synthesis and release of platelet TxA2           site on the enzyme, thus allowing to its acetyl group to interact
triggered by endogenously formed thrombin during whole blood               with a nearby enzyme active site,59 but the COX-1 channel was


                                          Bleeding, Thrombosis and Vascular Biology 2024; 3:121
                              Blood platelets, prostaglandins and aspirin: a historical and personal rereading                                 5



only described many years later.60 Platelet enzyme irreversible           atively large doses of oral aspirin, serum TxB2 generation ap-
acetylation explained the well-known long-lasting platelet func-          peared to be suppressed, even when there was no detectable as-
tion inhibition by aspirin.61 Other non-steroidal anti-inflammatory       pirin in the peripheral blood.58,72-76 It was suggested that
drugs (NSAID) such as indomethacin, shared with salicylate (thus          pre-systemic first-pass deacetylation of aspirin within the entero-
with aspirin) a common binding site, but were unable to perma-            hepatic circulation was responsible for the low (or absent) periph-
nently block the active site, thus explaining their short-term in-        eral drug levels. The sparing of vascular cyclo-oxygenase after
hibitory effect on platelet function.58,59,62                             oral (compared with intravenous) administration of the same dose
     The concept that low-dose might be preferred to high-dose            of aspirin was clearly shown in rats and in a patient with a porta-
aspirin in the long term prevention of ischemic cardiovascular            caval shunt (Figure 3).77-79
events was supported by a number of original reports on salicy-
late-aspirin interaction that is developed as salicylate accumulates
in blood following high dose aspirin.57,58,63,64 Salicylate was shown     Platelets and aspirin in diabetes 2 patients
to reduce platelet inhibition by aspirin, supporting the FitzGerald’s
observation, that the inhibitory effects of aspirin on platelet func-           Until the extensive work of Santilli et al.80 in the last decade,
tion ex vivo could be obscured during chronic high dose aspirin           little attention had been given to early observations on a possible
administration in man.44                                                  peculiar application of a low-dose aspirin concept in diabetes 2
     Subsequent studies on the interaction of aspirin with salicylate     patients. The readers will find in the paragraph below (Platelet
were extended, both in experimental animals and in humans, to             turnover and aspirin in type 2 diabetes) a brief review of the con-




                                                                                             ly
the interaction of aspirin with other NSAIDs, such as in-                 tributions to the beginning of this experimental and clinical re-




                                                                                     on
domethacin and ibuprofen.65-67 It was shown in man that previous          search topic by North American and Italian Colleagues, in
administration of NSAIDs could prevent the subsequent long-last-          particular Giovanni Di Minno, in Naples.81-86
ing platelet inhibition by aspirin.65,66 On the other hand, salicylate
                                                                          Platelet turnover and aspirin in type 2 diabetes



                                                                               e
administration prevented platelet inhibition by indomethacin.67
The current clinical implications of these pioneering observations,
are at present clinically well established.68                            us    In the early Eighties Catalano and Smith, at the Cardeza
                                                                          Foundation in Philadelphia, had reported a discrepancy between
                                                                          the entry of new platelets into the circulation (as determined by
                                                                    al
                                                                          monitoring the return of TxB2 in serum, after the ingestion of
The pre-systemic first-pass de-acetylation                                100 mg aspirin) and the disappearance of radiolabelled platelets
                                                     ci

of oral aspirin                                                           from the circulation (turnover).81 Because inhibition of platelet
                                                   er


                                                                          aggregation by aspirin is irreversible, the return after an interval
    The necessity to consider the pharmacokinetics of aspirin was         of time of the ability to form thromboxane by platelets in circu-
                                                  m



strengthened by the observation that, in subjects taking even rel-        lating blood should reflect the entry into the circulation of
                                       om
                                 -c
                         on
                    N




Figure 3. The pre-systemic first-pass de-acetylation of oral aspirin.


                                          Bleeding, Thrombosis and Vascular Biology 2024; 3:121
6                                                         G. de Gaetano and C. Cerletti




platelets whose cyclo-oxygenase activity had not been affected               3. Born GVR, Cross MJ. The aggregation of blood platelets. J
by aspirin.82                                                                   Physiol 1963;168:178-95.
     It had also been suggested that aspirin might also inhibit              4. O’Brien JR. Platelet aggregation. Part II. Some results from
megakaryocyte cyclo-oxygenase. To test this possibility, aspirin                a new method of study. J Clin Pathol 1962;15:446-9.
or saline were administered to rats made thrombocytopenic                    5. Beaumont JL, Caen J, Bernard J. Influence de l’acide acetyl
(platelet count less than 5% of basal value) by a specific an-                  salicylique dans les maladies hémorrhagiques. Sang
tiplatelet antiserum. By 24 h after thrombocytopenia was induced,               1956;27:243-8.
platelet count was about 15% of basal values in both control and             6. Blatrix C. Allongement du temps de saignement sous
aspirin-treated rats. However, while in controls TxB2 production                l’influence de certain médicaments. Nouv Rev Franç Hémat
was restored to about 20% of basal values, in aspirin-treated rats              1963;3:346-51.
less than 5% TxB2 was detected. A marked difference between                  7. Quick AJ. Salicylates and bleeding: the aspirin tolerance test.
the two groups was still found 96 h after induction of thrombocy-               Am J Med Sci 1966;252:265-9.
topenia, when platelet count restoration was similar. Since aspirin          8. Weiss HJ, Aledort LM. Impaired platelet-connective tissue
disappeared very rapidly from the circulation, the delay in recov-              reaction in man after aspirin ingestion. Lancet 1967;2:495-7.
ery of cyclo-oxygenase activity supported the hypothesis of a                9. Zucker MB, Peterson J. Inhibition of adenosine diphosphate-
megakaryocyte effect of this drug.83                                            induced secondary aggregation and other platelet functions
     In the mid Eighties the possibility of a trial with aspirin in di-         by acetylsalicylic acid ingestion. Proc Soc Exp Biol Med
abetic angiopathy was planned by Di Minno’s team in Naples, in                  1968;127:547-51.




                                                                                                       ly
collaboration with Melvin Silver, from Philadelphia, who was in a           10. O'Brien JR. Effect of salicylates on human platelets. Lancet




                                                                                               on
sabbatical year at the Federico II University. The return of malon-             1968;1:1431.
dialdehyde, a reliable index of arachidonic acid metabolism by              11. Evans G, Packham MA, Nishizawa EE, et al. The effect of
cyclo-oxygenase, after cessation of the regimen of a single daily               acetylsalicylic acid on platelet function. J Exp Med




                                                                                          e
dose of 100 mg aspirin for 1 month, indicated that the time at which            1968;128:877-94.
circulating platelets had recovered 50% of their ability to form such
metabolite was 4.5 days in controls but only 2 days in diabetic pa-
tients.84 Thus, the data were interpreted to indicate that a daily
                                                                                 us
                                                                            12. de Gaetano G, Donati MB, Vermylen J. A simple method to
                                                                                study the “stabilising” effect of chemicals and drugs on the
                                                                                platelet membrane. Thromb Res 1972;1:631-6.
                                                                          al
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