Abstract
Background
Coronavirus disease 2019 (COVID-19) is associated with an inflammatory cytokine burst and a prothrombotic coagulopathy. Platelets may contribute to microthrombosis, and constitute a therapeutic target in COVID-19 therapy.
Aim
To assess if platelet activation influences mortality in COVID-19.
Methods
We explored two cohorts of patients with COVID-19. Cohort A included 208 ambulatory and hospitalized patients with varying clinical severities and non-COVID patients as controls, in whom plasma concentrations of the soluble platelet activation biomarkers CD40 ligand (sCD40L) and P-selectin (sP-sel) were quantified within the first 48 hours following hospitalization. Cohort B was a multicentre cohort of 2878 patients initially admitted to a medical ward. In both cohorts, the primary outcome was in-hospital mortality.
Results
In cohort A, median circulating concentrations of sCD40L and sP-sel were only increased in the 89 critical patients compared with non-COVID controls: sP-sel 40,059 (interquartile range 26,876–54,678) pg/mL; sCD40L 1914 (interquartile range 1410–2367) pg/mL (P < 0.001 for both). A strong association existed between sP-sel concentration and in-hospital mortality (Kaplan-Meier log-rank P = 0.004). However, in a Cox model considering biomarkers of immunothrombosis, sP-sel was no longer associated with mortality, in contrast to coagulopathy evaluated with D-dimer concentration (hazard ratio 4.86, 95% confidence interval 1.64–12.50). Moreover, in cohort B, a Cox model adjusted for co-morbidities suggested that prehospitalization antiplatelet agents had no significant impact on in-hospital mortality (hazard ratio 1.05, 95% CI 0.80–1.37; P = 0.73).
Conclusions
Although we observed an association between excessive biomarkers of platelet activation and in-hospital mortality, our findings rather suggest that coagulopathy is more central in driving disease progression, which may explain why prehospitalization antiplatelet drugs were not a protective factor against mortality in our multicentre cohort.
Keywords: COVID-19, Platelets, Coagulopathy, Biomarkers, Antiplatelet drugs
Background
Coronavirus disease 2019 (COVID-19) is a primary respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. There are multiple cardiovascular consequences of COVID-19, with a substantial thrombotic burden translated into a high frequency of vascular micro- and macrothromboembolic events compared with other acute medical respiratory infections [2], [3]. Moreover, autopsy case series have reported extensive diffuse microthrombi within peripheral capillaries and arterioles in the lungs, heart and other organs, resulting in an acute respiratory distress syndrome and multiorgan failure, leading to critical forms of COVID-19 [4].
The conjunction of acute inflammation and direct viral infection of endothelial cells induces endothelial activation and inflammation (i.e., vasculitis) [5], [6], thus promoting coagulopathy and subsequent pulmonary vessel obstruction [3]. Consistently, the intensity of coagulopathy evaluated by D-dimer concentrations has been associated with COVID-19 severity at admission, with thrombotic events and with mortality, independent of thrombotic events [7], [8], [9]. In addition to coagulation cascade activation, platelets are prominent circulating actors of thrombus formation, and a set of indicators favours platelet hyperactivity in patients with COVID-19. Indeed, activated platelets and circulating platelet-leukocyte aggregates, which may promote platelet deposition and microthrombosis in damaged blood vessels, have been reported in patients infected by SARS-CoV-2, and could be associated with disease severity and worsened outcomes [10], [11]. Therefore, the influence of antiplatelet agents (APAs) has been actively pursued in clinical trials to improve outcomes [12], [13], [14], [15]. However, no consensus has been reached on the clear-cut improvement of clinical outcomes by APAs in COVID-19.
The first aim of our study was to thoroughly assess the platelet activation profile displayed by patients infected by SARS-CoV-2, with a particular focus on determining the factors most associated with platelet activation, and the relationship between the degree of platelet activation and mortality. To do so, in a cross-section at admission, we measured circulating soluble P-selectin (sP-sel) and soluble CD40 ligand (sCD40L), to characterize platelet activation in association with biomarkers reflecting coagulopathy and inflammation degree, in a retrospective cohort of patients with COVID-19. Indeed, both sP-sel and sCD40L are predominantly shed from activated platelets, and are therefore robust markers of platelet activation in vivo, even though a subsidiary origin exists from activated endothelial cells for sP-sel [16] and from stimulated lymphocytes for sCD40L [17]. Second, using data from a second multicentre cohort, we aimed to determine if APA treatment before hospitalization had an impact on COVID-19 mortality.
Methods
Study populations
We relied on two cohorts of patients with COVID-19. Cohort A was composed of 208 adult (aged ≥ 18 years) ambulatory and hospitalized patients with SARS-CoV-2 infection confirmed by reverse transcription polymerase chain reaction (RT-PCR), enrolled in two French hospitals (Hôpital Européen Georges-Pompidou and Hôpital Cochin-Hotel Dieu). In addition, 29 patients with an original clinical suspicion of COVID-19 ruled out by a negative RT-PCR result served as controls. Patients were included between 13 March and 26 June 2020 (ClinicalTrials.gov identifier: NCT04624997). According to World Health Organization guidance, patients were classified as non-critical (requiring oxygen therapy; score 5–7) or critical (requiring mechanical ventilation; score 8–9) in the first 48 hours following admission [18]. Outpatients were patients with COVID-19 without hospitalization criteria.
Cohort B included 2878 consecutive adult patients with COVID-19 hospitalized originally in medical wards in 24 French healthcare centres, from 26 February to 20 April 2020 (ClinicalTrials.gov identifier: NCT04344327), as previously described [19], [20].
Local investigators collected baseline and major event data in an electronic case report form via REDCap software (Research Electronic Data Capture; Vanderbilt University, Nashville, TN, USA). Inclusions were conducted in accordance with the Declaration of Helsinki, and all patients (or trusted relatives) signed a written consent form at the time of enrolment. In both cohorts, the primary outcome was in-hospital mortality.
Platelet-activation biomarker assessment
Platelet-activation biomarkers were assessed in all patients with COVID-19 and non-COVID-19 controls in cohort A. Venous blood was collected at hospital admission (i.e., in the first 48 hours following admission) in 0.129 M trisodium citrate tubes (9NC BD; Vacutainer, Plymouth, UK). Platelet-poor plasma was obtained after a double centrifugation at 2500 × g for 15 minutes and stored at –80 °C until analysis. Plasma concentrations of sP-sel and sCD40L were quantified in plasma using a Human Magnetic Luminex Assay (R&D Systems, Minneapolis, MN, USA). Data were analysed with the Bio-Plex 200 using Bio-Plex Manager 5.0 software (Bio-Rad, Marnes-la-Coquette, France).
Statistical analysis
Continuous data are expressed as medians (interquartile ranges [IQRs]) and categorical data as proportions. For comparison between two groups, we used the Mann-Whitney test and Fisher's exact test for continuous and categorical variables, respectively. For comparison between two groups or more, we used the Kruskal-Wallis and Cochran-Armitage tests for trends for continuous and categorical variables, respectively. For survival analysis among patients hospitalized for COVID-19, the start of the study was triggered by the diagnosis of SARS-CoV-2 infection. The end of the study was defined by the patient's death during hospitalization or discharge alive from the hospital. We used the Kaplan-Meier representation to estimate the survival function from diagnosis to in-hospital death according to the median sP-sel concentrations of hospitalized patients in cohort A. We used the Cox proportional hazards model to investigate the relationships between the increase in sP-sel levels (cohort A) or APA treatment status (cohort B) and in-hospital mortality. All analyses were two-sided, and a P value of < 0.05 was deemed statistically significant. Statistical analysis was performed using R studio software, including R, version 3.6.3 (RStudio, PBC, Boston, MA, USA).
Results
sP-sel and sCD40L plasma concentrations are increased in critical patients with COVID-19
We explored platelet activation in the 237 individuals from cohort A, 62.0% of whom were male, with a median age of 62 (IQR 50.0–72.0) years, including 29 non-COVID-19 controls and 23 outpatients. Among the 185 hospitalized patients, 89 (48.1%) had critical forms of COVID-19, were under mechanical ventilation and were treated in intensive care units (ICUs), whereas 96 (51.9%) were hospitalized in medical wards at admission. Among the hospitalized patients, 34 (18.4%) had venous thromboembolism throughout hospitalization, and 41 (22.2%) died within 60 days after hospitalization. Demographics, co-morbidities and biological characteristics and outcomes of cohort A patients are summarized in Table 1 . Median platelet count at admission did not differ between non-COVID-19 individuals (243 g/L, IQR 212–299 g/L) and patients with COVID-19, regardless of their clinical severity, namely outpatients (215 g/L, IQR 189–234 g/L; P = 0.98), non-critical patients (238 g/L, IQR 175–310 g/L; P > 0.99) and critical patients (249 g/L, IQR 179–329 g/L; P > 0.99; Fig. 1A ). In contrast, median sCD40L and sP-sel concentrations were only increased in critical patients with COVID-19 (sP-sel 40,059 pg/mL, IQR 26,876–54,678 pg/mL; sCD40L 1914 pg/mL, IQR 1410–2367 pg/mL; P < 0.001 for both) compared with non-critical patients (sP-sel 20,606 pg/mL, IQR 14,634–30,041 pg/mL; sCD40L 1298 pg/mL, IQR 947–1672 pg/mL; P < 0.001 for both), outpatients (sP-sel 19,277 pg/mL, IQR 15,915–22,879 pg/mL; sCD40L 1205 pg/mL, IQR 882–1361 pg/mL; P < 0.001 for both) and individuals without COVID-19 (sP-sel 20,450 pg/mL, IQR 18,457–25,585 pg/mL; sCD40L 1298 pg/mL, IQR 948–1410; P < 0.001 for both; Fig. 1B and Fig. 1C). Both sP-sel and sCD40L were significantly associated with biomarkers reflective of immunothrombosis: D-dimer concentrations (Fig. 1E and Fig. 1F) and C-reactive protein (CRP) (Fig. 1H and Fig. 1I) or organ failure assessed with creatinine (Fig. 1K and Fig. 1L) and high-sensitivity cardiac troponin I (Hs-cTnI) (Fig. 1N and Fig. 1O). In contrast, there was no association between platelet count and D-dimer (Fig. 1D), CRP (Fig. 1G), creatinine (Fig. 1J) and Hs-cTnI (Fig. 1M).
Table 1.
Demographic, clinical and biological characteristics of patients with and without coronavirus disease 2019 at admission included in cohort A.
| Non-COVID-19 (n = 29) | COVID-19 |
P | |||
|---|---|---|---|---|---|
| Outpatients (n = 23) | Non-critical (n = 96) | Critical (n = 89) | |||
| Male sex | 12 (41.4) | 9 (39.1) | 55 (57.3) | 65 (73.0) | 0.002 |
| Age (years) | 39.0 (32.0–46.0) | 40.0 (34.0–46.5) | 65.0 (53.0–78.0) | 62.0 (53.0–71.0) | 0.14 |
| BMI (kg/m2) | 24.5 (22.1–28.1) | 23.3 (21.6–24.6) | 24.4 (23.2–28.5) | 28.1 (26.0–33.7) | < 0.001 |
| Prehospitalization APAs | |||||
| Aspirin only | 0 (0.0) | 0 (0.0) | 13 (13.5) | 13 (14.6) | 0.16 |
| Clopidogrel only | 0 (0.0) | 0 (0.0) | 1 (1.0) | 4 (4.5) | 0.25 |
| Aspirin & clopidogrel | 0 (0.0) | 0 (0.0) | 2 (2.1) | 4 (4.5) | 0.42 |
| Admission biological variables | |||||
| Platelet count (g/L) | 243 (212–299) | 215 (189–234) | 238 (175–310) | 249 (179–329) | 0.56 |
| Platelet volume (fL) | 9.9 (9.5–10.4) | 10.6 (10.4–11.3) | 10.3 (10.1–11.1) | 12.2 (10.4–12.3) | < 0.001 |
| sP-sel (pg/mL) | 20,450 (18,457–25,585) | 19,277 (15,915–22,879) | 20,606 (14,634–30,041) | 40,059 (26,876–54,678) | < 0.001 |
| sCD40L (pg/mL) | 1298 (948–1410) | 1205 (882–1361) | 1298 (947–1672) | 1914 (1410–2367) | < 0.001 |
| D-dimer (ng/mL) | 214 (172–291) | 295 (140–438) | 1089 (798–1889) | 4186 (2498–7292) | < 0.001 |
| CRP (mg/L) | NA | 28.0 (14.8–33.4) | 63.6 (32.0–117.9) | 189.1 (121.5–259.9) | < 0.001 |
| Creatinine (μmol/L) | NA | 64.00 (64.0–64.0) | 68.0 (56.5–83.5) | 102.0 (72.6–214.3) | < 0.001 |
| Hs-cTnI (ng/L) | 6.8 (5.6–9.1) | 7.2 (5.6–8.8) | 8.1 (5.0–15.1) | 28.80 (15.3–51.7) | < 0.001 |
| Clinical outcomes | |||||
| Length of hospitalization (days) | NA | NA | 8.5 (5.0–16.0) | 26.0 (15.0–39.0) | < 0.001 |
| Mechanical ventilation | 0 (0.0) | 0 (0.0) | 4 (4.2) | 89 (100.0) | < 0.001 |
| VTEa | 0 (0.0) | 0 (0.0) | 11 (11.5) | 23 (25.8) | < 0.001 |
| In-hospital mortality | 0 (0.0) | 0 (0.0) | 2 (2.1) | 39 (43.8) | < 0.001 |
Data are expressed as number (%) or median (interquartile range). APAs: antiplatelet agents; BMI: body mass index; COVID-19: coronavirus disease 2019; CRP: C-reactive protein; Hs-cTnI: high-sensitivity cardiac troponin I; NA: not available; sCD40L: soluble CD40 ligand; sP-sel: soluble P-selectin; VTE: venous thrombosis event.
VTE was defined as deep venous thrombosis and/or pulmonary embolism during hospitalization.
Fig. 1.
Association of platelet count and plasma platelet-activation biomarker concentrations at admission with coronavirus disease 2019 (COVID-19) severity in cohort A. A–C. Boxplots of platelet count, soluble P-selectin (sP-sel) concentration and soluble CD40 ligand (sCD40L) concentration at admission for individuals not infected with COVID-19 (non-COVID) and hospitalized patients with COVID-19 with varying clinical severities (outpatient, non-critical and critical). Data points represent individual measurements, and horizontal bars represent medians with interquartile ranges. The P value refers to the Kruskal-Wallis test between the four clinical severity groups (control, outpatients, non-critical and critical). D–O. Scatter plots showing the correlation between platelet count, sP-sel concentration, sCD40L concentration and the concentrations of biomarkers of multiple organ failure (C-reactive protein [CRP], D-dimer, creatinine and high-sensitivity cardiac troponin I [Hs-cTnI]). All analyses were two-sided and a P value < 0.05 was considered statistically significant. r: Spearman's rank correlation coefficient.
Pre-hospitalization APAs did not interfere with platelet-activation biomarkers in COVID-19
We then aimed to determine if APAs could mitigate the excessive platelet activation associated with critical COVID-19. In cohort A, among hospitalized patients, 37 (20.0%) were treated with APAs at admission, including 26 (70.3%) with aspirin, five (13.5%) with clopidogrel and six (16.2%) with a combination of clopidogrel plus aspirin. In APA-treated patients, the median platelet count (236 g/L, IQR 170–306 g/L), sP-sel concentration (23,272 pg/mL, IQR 16,897–41,396 pg/mL) and sCD40L concentration (1672 pg/mL, IQR 1302–2132) did not differ from those in non-APA-treated patients: platelet count 248 g/L (IQR 184–311 g/L) (P = 0.72); sP-sel 25,036 pg/mL (IQR 17,804–36,709) (P = 0.99); sCD40L 1384 pg/mL (IQR 1302–2132) (P = 0.18) (Fig. 2A , Fig. 2B and Fig. 2C). As we showed that platelet activation was linked with clinical severity, we compared platelet count, sP-sel and sCD40L concentrations separately in critical and non-critical patients according to APA treatment, and found no difference within subgroups (Fig. 2A, Fig. 2B and Fig. 2C).
Fig. 2.
Relationship between antiplatelet agent (APA) treatment, platelet-activation biomarker concentrations and in-hospital mortality in cohort A. A–C. Boxplots of platelet count and plasma concentrations of soluble CD40 ligand (sCD40L) and soluble P-selectin (sP-sel) at admission, according to prehospitalization treatment with APAs. Data points represent individual measurements, and horizontal bars represent medians with interquartile ranges. Comparisons between patients with or without APA treatment were performed using the Mann-Whitney test. D–E. Survival curves according to plasma soluble sP-sel and sCD40L concentrations at admission using a Kaplan-Meier estimator in hospitalized patients in cohort A. Cut-offs for sC40L (1593 pg/mL) and sP-sel (28216 pg/mL) were defined using the median concentrations for hospitalized patients in cohort A. The start of the study was triggered by the diagnosis of severe acute respiratory syndrome coronavirus 2 infection. The number of patients at risk in the two groups at days 0, 20, 40 and 60 are displayed. The end of the study was defined either by patient's death during hospitalization or by discharge alive from the hospital. Survival curves were compared using the log-rank test. F: forest plot of the Cox proportional hazards model in cohort A hospitalized patients, showing the Cox proportional hazards model for sP-sel concentrations adjusted for body mass index (BMI), C-reactive protein (CRP) and D-dimer concentrations. The red dot represents the adjusted hazard ratio. The horizontal lines show the 95% confidence intervals (CIs). Cut-offs were defined as the median value of the population. All analyses were two-sided, and a P value < 0.05 was considered to be statistically significant.
Platelet activation is no longer associated with mortality after adjustment for coagulopathy
Even if unaffected by APAs, platelet activation could be valuable in the detection of patients most at risk of death. In cohort A, higher than median sCD40L concentration at admission was not significantly predictive of in-hospital mortality in a Kaplan-Meier estimator (log-rank P = 0.14; Fig. 2D), contrary to admission sP-sel concentrations (log-rank P = 0.004; Fig. 2E). Moreover, we evaluated sP-sel association in a Cox proportional hazards model adjusted on D-dimer and CRP, in which sP-sel was no longer predictive of in-hospital mortality (adjusted hazard ratio [aHR] 1.91, 95% confidence interval [CI] 0.68–5.40; Fig. 2F), in contrast to D-dimer concentration (aHR 4.86, 95% CI 1.64–12.50; P = 0.029). In this model, D-dimer concentration was the unique variable that was significantly predictive of in-hospital mortality (aHR 3.86, 95% CI 1.14–13.01; P = 0.029), suggesting that coagulopathy evaluated with D-dimer level is the best predictor of in-hospital mortality when adjusted for inflammation and platelet activation.
APAs did not decrease in-hospital mortality in a large multicentre cohort of hospitalized patients with COVID-19
Cohort B was composed of 2878 consecutive patients initially hospitalized in medical wards for SARS-CoV-2 infection. Briefly, 1666 (57.9%) were men, with a mean age of 67 years. A total of 627 of the 2878 (21.9%) patients had been treated with APAs before hospitalization. As expected, compared with patients free of APAs at admission, APA-treated patients were significantly older (P < 0.001) and had a higher proportion of men (P < 0.001), a higher body mass index (P = 0.008) and were more likely to have Marne's cardiovascular co-morbidities, notably hypertension (P < 0.001), hyperlipidaemia (P < 0.001), diabetes (P < 0.001), history of coronary artery disease or myocardial infarction (P < 0.001) or stroke (P < 0.001). The detailed characteristics of patients included in cohort B are summarized in Table 2 . Regarding clinical outcomes, 370 (12.9%) patients were transferred to ICUs, and were placed under mechanical ventilation during their hospital stay, and 362 (12.6%) died from COVID-19. The raw mortality rate was higher in patients with prehospitalization treatment with APAs (21.1%) than in patients without APAs (10.2%, P < 0.001). However, after adjustment for confounding factors, including demographics (age, sex), co-morbidities (hypertension, diabetes, hyperlipidaemia, stroke, chronic kidney disease, overweight) and biological variables (platelet count), prehospitalization APA treatment had no significant impact on COVID-19-associated mortality in a Cox model (hazard ratio 1.05, 95% CI 0.80–1.37; P = 0.73; Fig. 3 ).
Table 2.
Demographic, clinical and biological characteristics of patients with and without coronavirus disease 2019 at admission included in cohort B, according to prehospitalization antiplatelet agent treatment.
| No prehospitalization APAs (n = 2251) | Prehospitalization APAs (n = 627) | P | |
|---|---|---|---|
| Male sex | 1252 (55.6) | 414 (66.0) | < 0.001 |
| Age (years) | 64.12 ± 17.29 | 75.65 ± 11.99 | < 0.001 |
| BMI (kg/m2) | 28.00 ± 6.12 | 27.23 ± 5.67 | 0.008 |
| Platelet count (g/L) | 221.31 ± 100.10 | 217.35 ± 96.00 | 0.38 |
| Co-morbidities | |||
| Hypertension | 973 (43.5) | 480 (76.9) | < 0.001 |
| Hyperlipidaemia | 445 (19.9) | 355 (56.9) | < 0.001 |
| Diabetes | 410 (18.3) | 267 (42.9) | < 0.001 |
| Coronary artery disease or MI | 293 (13.0) | 334 (53.3) | < 0.001 |
| Stroke | 130 (5.9) | 123 (9.8) | < 0.001 |
| Asthma | 167 (7.4) | 22 (3.5) | < 0.001 |
| COPD | 98 (4.4) | 66 (10.5) | < 0.001 |
| Chronic kidney disease | 249 (11.2) | 156 (25.3) | < 0.001 |
| Active or history of malignancy | 290 (12.9) | 125 (19.9) | < 0.001 |
| History of VTEa | 164 (7.3) | 48 (7.7) | 0.82 |
| Clinical outcomes | |||
| Median length of hospitalization (days) | 8.65 ± 5.61 | 9.58 ± 6.31 | 0.005 |
| Mechanical ventilation | 293 (13.0) | 77 (12.3) | 0.68 |
| In-hospital mortality | 230 (10.2) | 132 (21.1) | < 0.001 |
Data are expressed as number (%) or mean ± standard deviation. APAs: antiplatelet agents; BMI: body mass index; COPD: chronic obstructive pulmonary disease; MI: myocardial infarction; VTE: venous thrombosis event.
VTE was defined as deep venous thrombosis and/or pulmonary embolism during hospitalization.
Fig. 3.
Relationship between antiplatelet agent (APA) treatment and in-hospital mortality in cohort B. Forest plot of Cox proportional hazards model in the entire population of cohort B (n = 2748) for in-hospital mortality. Red squares represent adjusted hazard ratios (aHR). Horizontal lines show 95% confidence intervals (CIs). All analyses were two-sided, and a P value < 0.05 was considered to be statistically significant. BMI: body mass index.
Discussion
In this study, we confirmed an association between platelet activation evaluated with soluble P-selectin and in-hospital mortality. However, we demonstrated, for the first time, interference of COVID-19-associated coagulopathy with platelet activation, which sheds a new light on the kinetics of microthrombosis in COVID-19, and may explain why APAs failed to improve the course of the disease. Indeed, using these two large French cohorts of patients with COVID-19, we also demonstrated that APA treatment before hospitalization does not reduce in-hospital mortality and does not modify the level of platelet activation at admission.
Our study first observed that the circulating platelet activation biomarkers sP-sel and sCD40L were both increased in critical patients with COVID-19 treated in an ICU. Soluble P-selectin (or CD62P) is an adhesion molecule expressed mostly on the surface of activated platelets and, to a lesser extent, on the surface of activated endothelial cells. Therefore, sP-sel is thought to arise predominantly from platelets and to reflect platelet activation [21]. CD40 ligand transmembrane protein, a member of the tumour necrosis factor family, is rapidly expressed on the platelet surface following activation. sCD40L is mainly generated by the cleavage of CD40L from the surface of activated platelets, and is therefore considered as a platelet activation marker [21]. The significant platelet-activation profile that we observed in severe forms of COVID-19 is consistent with the results of previous studies. Indeed, both Goshua et al. and Yatim et al. reported increased sP-sel and sCD40L concentrations in patients with COVID-19 cared for in ICUs compared with patients not admitted to an ICU [22], [23]. Hyperactivated platelets in patients with COVID-19 have been found using several approaches, including in vitro platelet aggregation assessment [4], platelet transcriptional signature [5] or the direct quantification of activated platelet aggregates in the bloodstream [24]. More than just severity markers, longitudinal follow-up of sCD40L and sP-sel could be a more accurate means of evaluating each patient's degree of platelet activation during the whole course of the disease [25]. Likewise, Campo et al. showed that the trends in sCD40L and sP-sel concentrations measured in hospitalized patients with COVID-19 during the first 2 weeks were associated with in-hospital mortality [26]. Furthermore, beyond their central role in haemostasis, platelets also have a major inflammatory and immune function, through the release of proinflammatory mediators, such as interleukin-1β, RANTES or sCD40L, and the direct activation of immune cells via the CD40L/CD40 complex [27]. Consistently, it has been demonstrated that platelets derived from critical COVID-19 are more prone to release interleukin-1β upon exposure to thrombin, an agonist that can trigger platelet activation [28]. In line with this, an in vitro study showed that the SARS-CoV-2 spike protein was able to trigger platelet activation directly and to promote platelet-monocyte communication, leading to proinflammatory cytokine production by monocytes [29]. Interestingly, Barrett et al. found in vitro an hyperactivated platelet phenotype in COVID-19 at the origin of endotheliopathy [30]. Therefore, platelets probably act as a link between an excessive immunoinflammatory response against SARS-CoV-2 and the widespread microthrombosis, a hallmark of severe forms of the disease. Platelets may thus be the central drivers in immunothrombosis – a prominent clinical feature precipitating organ injury and failure.
However, prehospitalization APAs (mainly aspirin in our cohort), the main drug class designed to block platelet activation, did not affect sP-sel or sCD40L concentrations. Aspirin is a well-known inhibitor of the platelet activation and amplification pathway [31]. Studies have shown that just a single week of low-dose aspirin significantly reduces plasma concentrations of sP-sel and sCD40L, even in acute pathological conditions, such as in patients with end-stage heart failure on mechanical circulatory support [32]. In contrast with our results, several studies have demonstrated that aspirin can reduce the hyperactive phenotype of platelets from patients with COVID-19 [11], [33]. However, these results were obtained after incubating platelets with aspirin in vitro, and therefore did not consider the complex interactions between platelets, endothelium, immune cells and shear stress. Therefore, the observed inability of APAs to hinder the hyperactivated platelet profile in critical patients with COVID-19 could align with an overwhelmed activation, distinct from classic platelet activation pathways, which makes the decreased activation impossible to circumvent.
sP-sel concentrations were strongly associated with CRP and D-dimer concentrations, and after adjustments for these two biomarkers, sP-sel concentrations did not discriminate survivors from non-survivors, in contrast with D-dimer concentrations. This finding suggests a more key role for coagulation activation in thrombotic microangiopathy, consistent with our current understanding of COVID-19 pathophysiology, in which the triggering event of microthrombosis is endothelial damage and vasculitis, which activates the coagulation cascade [34], [35]. Furthermore, Denorme et al. observed that platelets isolated from patients with COVID-19 had a reduced ability to become procoagulant compared with those from matched healthy donors, which may be in line with a subsidiary role of platelets in COVID-19 microthrombosis [36]. In light of our results, platelets could more be collateral damage than a microthrombosis-initiating event.
Our study also explored the real-life impact of chronic treatment with APAs on a large multicentre cohort B of hospitalized patients with COVID-19 and found no effect of APAs on mortality. Several studies have reported conflicting findings regarding the impact of APAs on COVID-19 prognosis. In a propensity score-matched cohort of 17,347 patients, Chow et al. reported that in-hospital mortality was significantly lower in patients receiving prehospital APAs [13]. In contrast, the randomized controlled open-label RECOVERY trial concluded that the addition of 150 mg aspirin once daily in addition to the COVID-19 standard of care (i.e., therapeutic heparin) was not associated with a reduction in the risk of progressing to invasive mechanical ventilation or mortality, and increased the rate of bleeding events [14]. Furthermore, the recent ACTIV-4a clinical trial showed that the combination of a therapeutic dose of heparin plus a P2Y12 inhibitor in non-critical patients with COVID-19 had no effect in reducing mortality or the rate of thrombotic events [12]. An important limitation of these prospective studies is that the potential benefit of APAs in COVID-19 may depend on the timing of treatment initiation, particularly if microthrombosis is already constituted at the time of admission. Therefore, the lack of benefit of APAs observed in these trials may be biased because APAs were introduced after the point at which most benefit could be gained. However, in cohort B, all patients treated with APAs were on long-term treatment long before being infected with SARS-CoV-2, weakening this hypothesis.
Study limitations
One of the main limitations of our study is the period of patient inclusion. Indeed, between the first months of the pandemic and the current situation, much changed regarding the standard of care of patients hospitalized with COVID-19 (dexamethasone and different anticoagulation regimens), the emergence of SARS-CoV-2 variants and vaccination. Given all those potential confounding factors, platelet activation relevance, in particular concerning the immunothrombotic phenomenon, should be analysed in current critical forms of patients with COVID-19, taking into account the variants and the presence of neutralizing antibodies as a result of previous infection and/or vaccination.
Conclusions
All in all, although we have confirmed an association between excessive platelet activation and in-hospital mortality, we propose here a chain of events, starting with coagulopathy and driving platelet activation. This hypothesis may explain why APAs failed to improve the course of the disease. A better understanding of the pathophysiology of microthrombosis is an essential prerequisite to identify new therapeutic approaches and, in particular, new combinations and/or doses of antithrombotic strategies. Our findings argue for early targeting of coagulation activation and endotheliopathy to keep the extent of microthrombosis to a minimum (Central Illustration).
Central Illustration. Platelets may contribute to microthrombosis, and constitute a therapeutic target in COVID-19 therapy, notably through the use of antiplatelet agents (APAs). First, using a monocentric cohort of patients with COVID-19 with varying clinical severities in whom plasma biomarkers of platelet activation (soluble P-selectin, and soluble CD40 ligand) were quantified, we demonstrated platelet biomarkers levels were not associated with mortality, in contrast to coagulopathy evaluated with D-dimer concentration. Then, in a second large multicentre cohort of COVID-19 patients, we found out that prehospitalization antiplatelet agents had no significant impact on in-hospital mortality after adjustment for co-morbidities. APAs: antiplatelet agents; BMI: body mass index; CI: confidence interval; COVID-19: coronavirus disease 2019; CRP: C-reactive protein; sCD40L: soluble CD40 ligand; sPsel, soluble P-selectin.
Disclosure of interest
The authors declare that they have no competing interest.
Funding
This work was funded with grants from French national agency for research ANR SARCODO (Fondation de France), Mécénat Covid AP–HP and Crédit Agricole d’Île-de-France Mécénat.
Acknowledgements
We thank AP–HP for promotion of the SARCODO project. We thank the clinical research unit URC HEGP CIC-EC1418 (Natacha Nohile, Pauline Jouany and Dr Juliette Djadi-Prat) and Helene Cart-Grandjean from AP–HP for their involvement in the SARCODO project. We would like to thank Crédit Agricole Île de France for their patronage and support. We would like to acknowledge all nurses and technicians involved in the vascular medicine, internal medicine, respiratory medicine, intensive care and haematology departments of the Hôpital Européen Georges-Pompidou for their help in taking care of patients and including them in the study. We would like to acknowledge all physicians involved in the recruitment or follow-up of patients.
Footnotes
Tweet: platelet activation in #COVID19. In 2 large cohorts of COVID-19 patients, Philippe et al. show soluble P-selectin plasma levels are not associated with mortality. Pre-hospitalization antiplatelet agents were not associated with lower mortality. @DavidMSmadja @RichardChocron @guilbon @NaderYatim @HadjadjJerome @TristanMirault @MarcSamama @TerrierBen @PlanquetteSante @SanchezOlivie11 @gendronnicolas @HopitalPompidou @UP_Medecine.
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