Two independent cohorts were analyzed for this study. Cohort 1 consisted of 60 COVID-19 patients that were admitted to European Georges Pompidou Hospital and included upon admission. Clinical and biological characteristics are previously reported in (20) and described in Table 1. Briefly, median age was 58.5 years (IQR, 49 to 72) and 76.7% were male. Patients were analyzed after a median duration of 6 days (IQR, 4 to 7) after onset of first symptoms. Fever was present in 97% of the patients, and other most common symptoms were cough (83.3%), dyspnea (71.7%), fatigue (70.0%), myalgia (40.0%) and diarrhea (26.7%). Degree of COVID-19 severity was categorized as mild-to-moderate in 35 patients (clinical symptoms associated with dyspnea and requiring a maximum of 3L/min), severe in 10 patients (respiratory distress requiring more than 3 L/min of oxygen and no other organ failure) and critical in 15 patients (respiratory failure requiring mechanical ventilation).
Eleven out of 35 patients with mild-to-moderate disease and 3/10 patients with severe disease experienced clinical worsening and required mechanical ventilation. Thrombotic events were noted in 8 patients (3 mild-to-moderate, 2 severe and 3 critical).
Cohort 2 consisted of 50 patients with various degree of COVID-19 admitted to Cochin hospital and 18 healthy controls, and was described in (21). For the purpose of the present study, only patients with whole-blood transcriptional analysis were included, i.e. 32 COVID-19 patients and 13 healthy controls. Characteristics of the patients are described in Table 2. Median age was 56 years (IQR, 51 to 65) and 78% were male, while median age of healthy controls was 59 years (IQR, 41 to 60) and 77% were male. Patients were analyzed after a median duration of 10 days (IQR, 9 to 11) after onset of first symptoms. Fever was present in 100%, and other most common symptoms were dyspnea (100%), fatigue (32%), cough (97%), myalgia (97%) and diarrhea (34%). Degree of COVID-19 severity was categorized as mild-to-moderate in 11 patients (median oxygen requirement 1.5 L/min), severe in 10 patients (median oxygen requirement 5 L/min) and critical in 11 patients. No patients with mild-to-moderate disease required admission to an ICU, while 5 out of 10 patients with severe disease were eventually admitted to the ICU. Thrombotic events were noted in 3 patients (1 severe and 2 critical).
Increased plasma sP-selectin on admission to the hospital is associated with disease severity and is a predictor of mechanical ventilation and death
Because a high proportion of critically ill patients present clinical and biological evidence of activated coagulation, we measured plasma sP-selectin in Cohort 1. Increased plasma sP-selectin levels on admission to the hospital was significantly associated with critical disease (Figure 1a). Moreover, sP-selectin levels were positively correlated with inflammatory parameters, i.e. CRP levels (r = 0.30, p = 0.017, Figure 1b), but not with platelet count (r = 0.1, p = 0.43, Figure 1c),
We next evaluated the ability of sP-selectin, normalized to platelet counts, to predict intubation (Figure 1d) or death (Figure 1e) using a receiver operating characteristic (ROC) curve. We found that outcomes were significantly associated with higher sP-selectin values and significant area under the curve (AUC = 0.67, p = 0.028 for intubation and AUC = 0.74, p = 0.0047 for death). An optimal cutoff value of 150 NC (normalized concentration: concentration in pg/mL normalized to 106 platelets/mL) was predictive of intubation with 66% sensitivity, 61% specificity, 66% negative predictive value (NPV) and 61% positive predictive value (PPV), and of death with 82% sensitivity, 60% specificity, 90% NPV and 55% PPV. Together, these data suggested that sP-selectin, a reliable marker of platelet activation, was strongly associated with death and may identify patients at higher risk of admission to the ICU.
Critically ill COVID-19 patients display a dysregulated primary hemostasis transcriptional signature
We previously performed a whole-blood immunological transcriptional characterization on 32 patients with laboratory-confirmed COVID-19 displaying various disease severity (cohort 2) (21). We therefore next analyzed genes involved in hemostasis using an unbiased gene set enrichment analysis comparing severe to critical COVID-19 patients. We found that critically ill patients displayed an elevated expression of genes related to primary hemostasis (Figure 2a). Hierarchical clustering identified genes that were upregulated in a grade-dependent manner, thereby driving the hemostasis signature in critically ill patients (Figure 2b). These genes included ITG2AB, GP1BB, PPBP and SELPLG (Figure 3a), which recapitulated multiple steps of platelet activation (22,23). Importantly, and similarly to sP-selectin, RNA levels more strongly correlated with CRP level (Figure 3b) than with platelet counts (Figure 3c).
We next evaluated the ability of these genes to predict clinical outcome after normalization with platelets. Among patients that did not initially require critical care upon inclusion (n = 21), 5 (24%) presented respiratory failure afterwards requiring mechanical ventilation (Table 2). PPBP and SELPLG were found to be the best predictor of clinical worsening, with an AUC of 0.8 for both outcomes (p = 0.048) (Figure 4a, b and c). An optimal cutoff value for PBPP of 5 NRC (normalized RNA counts : counts normalized to 106 platelets/mL) was predictive of intubation with 100% sensitivity, 63% specificity, 100% NPV and 45% PPV, while a cutoff value of 2.65 NRC for SELPLG was predictive of intubation with 100% sensitivity, 69% specificity, 100% NPV and 50% PPV. Overall, these markers provide excellent negative predictive values, with no patient below the threshold value requiring future mechanical ventilation.