COLC, an anti-inflammatory alkaloid, is now a pivotal therapy in the management of cardiovascular inflammation [20]. COLC recently gained traction as an emerging therapy as an anti-inflammatory agent following the 2019 Colchicine Cardiovascular Outcomes Trial (COLCOT), which demonstrated a 1.6% absolute reduction in major adverse cardiovascular events (MACE) in patients with recent acute coronary syndrome (ACS) [21]. In patients with chronic coronary syndrome (CCS), as per the Low-dose Colchicine 2 (LoDoCo 2) Trial, COLC similarly reduced MACE over a median follow-up of 28.6 months [22]. These two landmark trials were crucial in its regulatory approval by the FDA, being labeled as the first targeted anti-inflammatory drug for CAD [6, 23]. Several systematic reviews have summarized the available evidence on COLC for both ACS and CCS, with a recent meta-analysis pooling data from all of the major trials alluding to a 32% reduction in MACE among patients treated with COLC [24,25,26]. Despite this evidence, COLC has not been shown to confer any survival benefit in these trials, and its net risk–benefit profile requires further investigation [27, 28].

COLC primarily inhibits microtubule assembly but also demonstrates myriad other mechanistic effects, such as inhibiting a complex and intricate milieu comprising (1) endothelial cell dysfunction and inflammation, (2) smooth muscle cell proliferation and migration, (3) macrophage chemotaxis, migration, and adhesion, and (4) platelet activation. At lower doses, it inhibits microtubule dynamics and cell migration, while at higher doses, it impedes cell division [28]. COLC also impedes the translocation of tissue factor via cytoskeletal tracks associated with the microtubule arrays, along with other intracellular traffic of secreted and transmembrane proteins in vesicles [28].

On a molecular basis, it also attenuates proinflammatory cytokine release. It inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation, the latter of which is crucial in cell repair [29]. COLC also mitigates intracellular transport of the adaptor molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD), attenuating the release of interleukin-1β, which is a pivotal cytokine involved in the network of immune inflammation linked to CAD [28]. COLC has stable interaction with the adenosine triphosphate (ATP) binding pocket of the neuronal apoptosis inhibitor protein (NAIP), which can decrease pore formation and ATP-mediated NLRP3 inflammasome activation [28]. Physiological concentrations of COLC inhibit collagen- and calcium ionophore-induced platelet aggregation and internal signaling [9]. Many of these in vitro studies demonstrated COLC effects on platelet function only at supraphysiological concentrations.

This study assessed whether there were any pleiotropic effects on platelet reactivity in patients with stable CAD on DAPT using 0.5 mg of COLC once daily for 14 days. The VerifyNow™ (VN) (Werfen, Bedford, MA, USA) is a rapid, commonly used point-of-care analyzer that determines platelet reactivity by assessing light transmittance induced by platelet aggregation in response to specific agonists [30, 31]. Some agonists that are crucial in platelet aggregation pathways include thrombin, collagen, serotonin, adenosine diphosphate (ADP), and thromboxane A2 (TXA2). Aspirin and clopidogrel are both cornerstone antiplatelet therapies in the management of CAD and block the formation of TXA2- and P2Y12-mediated ADP stimulation, respectively [32]. The P2Y1 receptor affects platelet morphology with transient aggregation, whereas the P2Y12 receptor is integral in the cascade amplification of platelet aggregation and thrombus stabilization [33].

In a mechanistic study by Cirillo et el., platelets from 35 patients with stable CAD on DAPT were pre-incubated with COLC 10 µM before being stimulated with ADP 20 µM or thrombin receptor activating peptide (TRAP) 25 µM at several time points (0, 30, 60 and 90 min) to assess maximal aggregation by light transmission aggregometry (LTA). It was observed that COLC significantly attenuated TRAP-induced platelet aggregation in both clopidogrel responders and those with HPR, with the latter subgroup also displaying a similar direction with respect to ADP-induced platelet aggregation. Overall, it was demonstrated that COLC inhibited platelet aggregation in patients with HPR despite DAPT [34]. In our study, the median ARU baseline score was 463, and post-COLC it was 436, which was not statistically significant (p = 0.485). The mean difference in scores was −18.31 (95% CI −74.34 to 37.71, p = 0.504). At baseline, 27.3% of the patients had aspirin resistance, compared to 13.6% post-COLC (p = 0.423). The median baseline PRU score was 210, and post-COLC it was 199, which was also not statistically significant (p = 0.581). The mean difference in scores was −7.31 (95% CI −31.1 to 16.5, p = 0.530). At baseline, 50% of the patients had clopidogrel resistance, compared to 45.5% post-COLC (p = 0.999). Our study’s results did not replicate the findings seen in Cirillo’s study, albeit with several caveats, as ours was an in vivo, pragmatic, real-world clinical study with a nominal COLC dose used in recent seminal trials, namely COLCOT and LoDoCo 2.

A study by Shah et al. assessed the effects of varying concentrations of COLC on platelet activity in vitro, and a clinically relevant 1.8-mg dose was administered to 10 healthy participants. It was determined that COLC addition in vitro reduced LTA aggregation only at supratherapeutic concentrations but decreased monocyte- (MPA) and neutrophil-platelet aggregation (NPA) at therapeutic concentrations. The administration of 1.8 mg COLC to healthy patients had no observed effect on LTA aggregation; however, it reduced the degree of MPA, NPA, platelet surface expression of PAC-1 and P-selectin, and platelet adhesion to collagen 2 h post-COLC. Overall, in clinically pertinent concentrations, COLC decreased surface expression markers and inhibited some facets of platelet aggregation. This study revealed that a nominal loading dose of COLC attenuates platelet activity with respect to platelet activation and the platelet–leukocyte interface, but may not impact platelet–platelet interactions [10]. Our study did not utilize a loading dose of 1.8 mg or variable doses, and platelet function was only assessed with respect to ARUs and PRUs, as it did not include platelet activity surface markers.

In a study performed by Raju et al., no difference was noted in platelet aggregation in response to ADP, arachidonic acid, or collagen in a subgroup of 49 patients. There was also no difference in platelet function assessed using platelet aggregation with ADP (5 μmol), arachidonic acid (0.5 mmol), collagen (1 µg/mL), and collagen (5 µg/mL), and urine dehydrothromboxane B2 [35]. This study enrolled patients with ACS and acute stroke with COLC 1 mg as the trial intervention. In contrast, our patient cohort included a stable panel of chronic coronary syndromes and adopted the FDA-approved dosage used in the milestone trials.

As mentioned earlier, COLC may, directly and indirectly, impact platelet aggregation via several pathways, including reducing platelet aggregation in response to collagen, ADP, and TRAP, inhibiting platelet degranulation and the formation of platelet-derived extracellular vesicles, reducing reactive oxygen species generation in response to glycoprotein VI stimulation, and modulating cytoskeleton rearrangement by inhibiting cofilin and LIM domain kinase 1 [9]. Based on the study results, we cannot ascertain whether COLC has any effect on these pathways, given that they were not statistically significant, and the study utilized the standard CVD dose without assessing other biochemical and physiologic parameters.

Currently, given the revitalization of COLC in the cardiovascular armamentarium, there is a paucity of in vivo clinical studies evaluating mechanistic pathways and pharmacodynamic and pharmacokinetic characteristics. The COLC–platelet interaction has the potential to attenuate MACE, but further high-fidelity studies are required [9, 36]. While this study, in addition to the studies mentioned above, did not display a robust antiplatelet effect, given their intrinsic limitations of study size, platelet function testing, COLC dosing, and clinical endpoints, it may prompt other investigators to seek alternative mechanisms or pathways of the COLC-derived mortality benefit observed in the COLCOT and LoDoCo 2 studies.

This study was not primarily designed for clinical endpoints but was sufficiently powered based on ARU and PRU data from preexisting studies from our group in this Caribbean setting. Thus, the generalizability and applicability of these results may not translate to clinical effectiveness, efficacy, or safety.

As with previous studies, the vast majority of patients enrolled were of Caribbean South Asian descent, implying an inherent selection bias. This has been a recurrent feature of these clinical studies performed in this region, all hovering with near-identical ethnic proportions [4, 5, 15, 37, 38]. The same can be said of the relatively high prevalence of T2DM, approaching 80% in this study group, which is inextricably linked with accentuated platelet reactivity [39, 40].

Patients were on maintenance DAPT with aspirin and clopidogrel, and platelet function profiles on more novel and potent antithrombotic agents, such as ticagrelor and apixaban, were not evaluated. Prespecified subgroup analyses evaluating any interaction effect of other therapies with COLC were not performed, such as sodium-glucose cotransporter-2 inhibitor (SGLT2i), which can also impact platelet reactivity [15, 38]. This was due to the pilot sample size, with even smaller subgroups—for example, six patients on SGLT2i—and the concern for data dredging or mining. Employing VN as the solitary platelet function test is also a drawback, as it relies on a fixed concentration of a limited number of agonists, of which arachidonic acid is non-physiological. Ideally, assessing responses to varying concentrations of an array of physiological agonists may have proven more informative. Comprehensive platelet function testing, including flow cytometry, thromboelastography, and novel markers such as FcγRIIa, may prove informative; however, it remains unavailable in this setting due to several logistical issues [41,42,43]. Additionally, platelet function testing did not entail assessing other doses of COLC or several time points, as performed in the aforementioned mechanistic studies. Ideally, a large-scale, double-blind, randomized controlled trial would have been optimal; however, many challenges currently exist in implementing such a venture in a limited-resource setting.