Platelet activation in critically ill COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic [1]. Severe disease is characterized by an acute respiratory distress syndrome (ARDS), respiratory failure and death in about 1% of cases [2, 3]. Most important risk factors for severe disease include age, overweight, diabetes, hypertension and history of cardiovascular disease [4, 5]. Severe and critical patients were shown to develop arterial and venous thrombotic complications, such as pulmonary embolism, stroke and myocardial infarction [6, 7]. Markers of coagulation activation, in particular increased D-dimer, fibrinogen and von Willebrand factor (vWF) levels were found to be associated with critical illness, whereas only minor changes were noted in prothrombin time and platelet counts [8‐11]. In addition, autopsy series described multiple thrombosis in deceased COVID-19 patients [12, 13]. These findings suggest vascular micro-thrombotic disease as a primary factor for mortality in critically ill COVID-19 [6, 14]. Therefore, some authors supported the systematic use of curative anticoagulation upon admission to intensive care unit (ICU) [15], a strategy that has been reported to decrease mortality in severe COVID-19 [8, 16‐18].

Mechanisms underlying increased thrombotic events are still unclear but accumulating evidence point to a key role for endothelial and platelet activation [19, 20]. As viral inclusions were described in endothelial cells, it has been hypothesized that endothelial cell injury and activation could drive platelet activation and subsequent coagulopathy [13, 21]. Therefore, dissecting the contribution of platelets to COVID-19 critical illness is key to understand SARS-CoV-2 infection pathogenesis and identify novel therapeutic strategies.

Platelet P-selectin is a key thromboinflammatory molecule involved in platelet activation and function. It has been demonstrated to play a crucial role in primary hemostasis by regulating platelet–leukocyte interactions, fibrin and tissue factor recruitment into platelet aggregates and thrombus formation [22]. Its soluble form, sP-selectin, is released upon platelet and/or endothelial cell activation and measurement of sP-selectin has been proposed as a reliable marker of in vivo platelet activation [23]. Moreover, sP-selectin levels have been shown to correlate with acute lung injury severity score and related death [24].

The aim of our study was to assess, in hospitalized COVID-19 patients, the ability of sP-selectin to predict requirement for mechanical ventilation and in-hospital mortality. Next, using whole-blood transcriptional data, we uncovered a platelet activation transcriptional signature associated with critical illness.

Dysregulated hemostasis is emerging as a key factor in COVID-19 pathogenesis and severity. The present study provides new insights into the contribution of platelets to disease severity with the identification of a unique hemostasis signature in critically ill patients. We identified sP-selectin as a soluble marker associated with in-hospital mortality, and elevated SELPLG and PPBP RNA levels as strong negative predictors of mechanical ventilation in hospitalized patients.

Elevated sP-selectin levels promote leukocyte–endothelial and leucocyte–platelet adhesion [31], release of citrullinated histones and neutrophil extracellular traps (NETs) formation [32], promoting formation of platelet–neutrophil or –monocyte aggregates via binding of P-selectin to its ligand PSGL-1 (encoded by SELPLG) expressed on the surface of leukocytes [33]. Accordingly, elevated sP-selectin and increase in platelet–monocyte aggregates and have been recently described in COVID-19 patients [33‐35], the latter being effectively blocked by platelet P-selectin neutralization [33]. Our study supports these findings and highlights the key role of platelet activation in critical patients.

Importantly, while signatures related to innate immune activation and impaired interferon activity distinguished mild-to-moderate disease from severe and critical disease [27], we show that only a primary hemostasis-related gene signature could further distinguish severe from critical patients (Fig. 2a), especially the specific platelet chemokine-encoding gene PPBP/CXCL7 and SELPLG, encoding for the P-selectin glycoprotein ligand-1 (PSGL-1), which also predicted clinical outcome. Pro-platelet basic protein (PPBP), also known as CXCL7, is the most abundant platelet chemokine [29, 36], expressed within platelets as an inactive precursor and activated after cleavage during thrombus formation by enzymes released by neutrophils. PSGL-1 is expressed by leukocytes, allowing the formation of platelet–leukocyte conjugates and adhesion of leukocytes to activated endothelium through its interaction with P- or E-selectin [29, 37]. PBPP/CXCL7 has been shown to be essential for neutrophil migration into the thrombus [38], and the formation of platelet–neutrophil aggregates and murine models of acute lung injury showed that deletion of PPBP/CXLC7 protected the mice from lung disease [36], as well as blocking platelet activation [39]. Overall, our data support a model, whereby platelet activation induces PPBP/CXCL7 release and P-selectin upregulation followed by neutrophil attraction into sites of injury and the formation of platelet–leucocyte aggregates, precipitating organ injury and failure.

In addition to their role in primary hemostasis, platelets are an integral part of the immune response to pathogens. The functional interdependence and the coordinated activation of both processes, designated as thrombo-inflammation, may drive adverse effects, such as thrombosis, multiple organ failure and death [40]. We support the hypothesis that deregulated primary hemostasis, in association to inflammation, could drive COVID-19 progression into critical disease. Viral infection and sepsis models have been associated with platelet gene expression and functional alterations [41, 42]. Supporting this generalizable thrombo-inflammation model, we found similar elevated sP-selectin in non-COVID septic ICU patients. However, some unique features may characterize SARS-CoV-2-mediated platelet dysregulation. Authors have shown that sP-selectin seemed to be more strongly associated COVID-19-related in-hospital mortality [43, 44]. Frazer et al. [45] found significantly elevated sP-selectin in ICU patients in comparison to non-COVID-19 ICU patients at day 3 post-admission (and not at days 1 and 2), that persisted until day 7. This finding suggests that persistent platelet activation may characterize COVID-19-associated coagulopathy. In addition, Manne et al. showed that platelet gene expression profile was unique in COVID-19 patients in comparison to H1N1 pandemic patients. Altogether, these data demonstrate that COVID-19 critical patients share important similarities with other critical sepsis, but some features may uniquely contribute to platelet activation during SARS-CoV-2 infection.

Prediction of illness trajectories after the onset of symptoms is difficult but remains critical. Clinical and biological factors have been shown to predict poor outcome, including arterial blood gazes values (e.g., hypoxemia, hypercapnia, P/F ratio, lactate), inflammatory markers (e.g., CRP, IL-6), coagulation markers (e.g., VWF, D-dimer) and cellular blood composition (e.g., neutrophil-to-lymphocyte ratio). These factors have led to the development of prognosis scores to predict critical illness [46]. Our findings suggest that markers of dysregulated hemostasis could be evaluated for their predictive prognostic value.

Finally, these findings suggest that a subset of patient may benefit from drugs preventing platelet activation, such as antiplatelet agents or P-selectin inhibitors [47], which have also been proposed in other septic settings [48, 49]. These anti-platelets agents in combination with anti-inflammatory drugs (glucocorticoids, anti-IL-6) could have substantial impact on thrombo-inflammation.

The benefit of anti-platelet agents is, however, challenged by recent findings from Althaus and coll. [50] which proposed that the induction of a procoagulant platelet subpopulation may be a key driver of critical COVID-19. Procoagulant platelets displayed increased P-selectin, integrin αIIb and GPIbα expression on their surface [51], in line with biomarkers (sP-selectin, PPBP, SELPLG) and signature (ITG2AB, GP1BB, PPBP and SELPLG) we identified. However, in this scenario, the benefits of aspirin and P2Y₁₂ inhibitors would be limited; blockade of P2Y₁ and P2Y₁₂ does not inhibit procoagulant platelets activation [52] and pretreatment with aspirin has minimal effect on their generation [53]. In contrast, Manne et al. [35] did not find evidence for an increased procoagulant platelet subpopulation, and an additional study concluded that procoagulant platelets may, on the contrary, have a protective effect during SARS-CoV-2-related pneumonia [54]. Therefore, the mechanisms underlying platelets activation, and subsequently, the potential benefit of anti-platelet therapy remain controversial. The REMA-CAP trial is currently investigating the use of aspirin or a P2Y₁₂ inhibitor (clopidogrel, prasugrel, or ticagrelor) [55], and results from the RECOVERY trial on aspirin will soon be unraveled [56]. So far, one retrospective trial suggested a potential benefit of aspirin use on critical care and in-hospital mortality [57].

Our study has some limitations. We used two independent cohorts with a small sample size from two centers. The two cohorts were not homogenous in regard to timing of measurements and severity of patients. In addition, the study was not designed as a longitudinal study, so no sequential measurement was available. Indeed, sP-selectin association with clinical outcome was moderate and serial measurement over time may optimize its outcome prediction performance. Moreover, longitudinal analysis of sP-selectin levels may provide insights on the causal role of platelets activation in driving critical disease. Our transcriptomic analysis was performed on whole-blood RNA, so we cannot evaluate the contribution of each cell population to hemostasis dysregulation. Separate transcriptional profiling of platelets vs other circulating populations and analysis of cell expression phenotypes (P-selectin, phosphatidyl-serine exposure) and evaluation of platelets functions may provide further insights into the contribution of each population to thrombo-inflammation and dissect the mechanisms of platelet–neutrophil or platelet–monocyte aggregates.

Overall, this exploratory study sheds light onto the role of thrombo-inflammation in critical patients. We identified platelet activation markers sP-selectin, SELPLG and PPBP/CXCL7 as potential biomarkers of critical worsening. Additional studies dedicated to evaluate the predictive performance of these biomarkers are required to both validate our findings and optimize their ability to predict progression to critical disease.