A large amount of pathological evidence from autopsies is revealing that thrombosis is an important consequence of COVID-19 disease(21). The development of thrombosis in patients with COVID-19 is due to the fact that after the infection with the virus, the body reacts with an extreme immune response and a "cytokine storm", leading to the release of "messenger substances" that induce pneumonia. These substances are the ones causing thrombosis and blood vessel blockage(21). This work focused on the relationship between COVID-19 related pneumonia and thrombosis, by the evaluation of several parameters related to the risk of thrombosis in COVID-19 patients, and the dynamic changes of these indicators in patients with different outcomes.
In addition, our study revealed that several indicators were related to the severity of the disease, including platelet associated parameters (PLT, PCT, PDW, MPV, P-LCR), cytokine (IL-6), coagulopathy parameters (PT, PTA, INR, D-dimer, FDP), and thrombosis-related indicators (IP-10, MCP-1).
Many clinical studies showed that COVID-19 is associated with coagulopathy, but it is different from the disseminated intravascular coagulation with normal platelets, PT and fibrinogen. A report demonstrated that the platelet count is lower in non-survivors than survivors(22), and our study confirmed this result, although we additionally demonstrated that more platelet associated parameters differed between the two groups. Some studies(23) showed that non-survivors have significantly higher levels of D-dimer and FDP, longer PT and live APTT than survivors at admission. In addition, 71.4% most of the non-survivors showed disseminated intravascular coagulation during hospitalization compared to survivors, with abnormal coagulation results in the late stage of the disease(23). Our results are consistent with these results previously published, confirming the abnormal coagulation function in COVID-19 patients. Therefore, we further evaluated the coagulation and thrombosis-related indicators in COVID-19 patients using ELISA.
Huang et al.(22) reported that patients infected with 2019-nCoV show a significant increase in serum proinflammatory cytokine levels, especially IL1β, IFNγ, IP-10 and MCP-1, which may cause the activation of the T-helper-1 (Th1) cell response. In addition, patients who require ICU admission have higher GCSF, IP-10, MCP-1, MIP1a, and TNFα concentrations than patients who do not require ICU admission, suggesting that the cytokine storm is associated with disease severity(22). Moreover, Qin et.al(24) reported that several inflammatory cytokines such as IL-2R, IL-6, IL-8, IL-10 and TNF-α were increased in severe patients compared with their level in the non-severe patients.
Our study did not find a difference in serum IL-1β level between severe and critically ill patients, but our results revealed a difference in IL-6 level between these two groups. IL-6 is a potent inducer of the acute phase response. Indeed, it is an endogenous pyrogen mainly produced in the acute and chronic inflammatory sites, causing fever in people with autoimmune diseases or infections. IL-6 is then secreted into the serum to induce transcriptional inflammation through the interleukin 6 receptor alpha. Furthermore, increased IL-6 can cause a cytokine storm(25, 26).
IP-10 and MIP1a, as well as IL-1β, IL-6, IL-8, IL-10, and TNFα, are also inflammatory cytokines, and therefore showed a strong positive correlation with each other. IP-10, MCP-1, and MIP1a are parameters related to thrombosis, thus having a significant correlation with the coagulation parameters.
The level of both IP-10 and MCP-1 was higher in critically ill patients than that in serious patients. Therefore, in this study, the 74 enrolled patients were divided according to the level of IP-10 and MCP-1. Our results showed an increased IL-6 level in the IP-10 + MCP-1 increased group compared to the IP-10 + MCP-1 decreased group. PT, INR increased, and PTA decreased in the IP-10 + MCP-1 increased group compared to the IP-10 + MCP-1 decreased group, also confirming the previous statement. Moreover, the proportion of critically ill patients in the IP-10 + MCP-1 increased group was higher than that in the IP-10 + MCP-1 decreased group, further indicating that IP-10 and MCP-1 are biomarkers for predicting the severity of COVID-19 disease. When the IP-10 and MCP1 level was compared between the survival group and the death group, no significant difference was found, which might be due to the fact that the selected patients were severe or critically ill, resulting in a too high mortality rate, with no difference between survival and death. In addition, several previous studies in Wuhan showed that the D-dimer level in non-survivors are higher than that in survivors(23, 27), suggesting that the increased D-dimer level is an independent risk factors of death in COVID-19 patients (28). Therefore, patients were grouped according to the D-dimer level and the results showed that regardless of the clinical feature, the increased D-dimer group had higher IP-10 and MCP-1 level than the decreased group, while MIP1a was not statistically significant between the two groups. Our further speculation was that IP-10 and MCP-1 could be related to the risk of progress to death in COVID-19 patients.
A report demonstrated that CXCL10 (IP-10) inhibits endothelial recovery independently of any other inflammatory factor, and anti-CXCL10 antibody is under validation in a clinical trial to prevent cardiovascular events (13) because the more severe the COVID-19 patient is, the higher the serum IP-10 level is. Therefore, anti-IP-10 antibody treatment may represent a new approach in COVID-19 patients, especially the ones with thrombotic events.
Patients whose multi-point indicators were greater than three time points were selected for dynamic analysis. The analysis of dynamic changes revealed that the overall index of the death group was higher than that in the survival group. In addition, the indicators remarkably increase in patients with a poor outcome, while some indicators decreased in a later time, suggesting a disease change to a pathophysiological model, although further studies are needed to explain this phenomenon.
This is the first study comparing the coagulation and thrombosis-related ELISA indicators, platelet-related parameters, routinely tested cytokines and coagulation indicators according to the guidelines when serious and critically ill patients are grouped. Furthermore, this study compared the dynamic changes of multiple indicators in the serum of patients with multi-point detection.
This study is a single-center retrospective study, thus these results might not be representative, in addition to the fact that all the included patients were severe and critically ill. Thus, these results could not be compared with the results in mild patients.
However, there are some limitations in the present study. First, the sample size is small due to the limited time and number of patients allocated to PUMCH. Second, only patients with more than three measurements were included in the dynamic changes analysis. Although the more time-points available, the better characterization of dynamics over time is allowed, this approach leaves only 14 patients for the analysis, and it may introduce some bias, as the patients who had blood samples obtained most frequently may also be the most critically ill and may thus not be representative for the entire cohort. More multi-center studies are needed in the future to verify these results and for a comprehensive interpretation of the clinical results.
In conclusion, the level of both IP-10 and MCP-1 in the serum of critically ill patients was higher than that in severe patients, proving that IP-10 and MCP-1 are biomarkers predicting the severity of COVID-19 disease. Moreover, IP-10 and MCP-1 level increased in the D-dimer increased group compared with the decreased group, suggesting that IP-10 and MCP-1 could be related to the risk of death in COVID-19 patients. Thus, anti-IP-10 antibody treatment may represent a new approach in COVID-19 patients, especially the ones with thrombotic events. However, since the selected patients were severe or critically ill, the results did not show any difference between survival and death, suggesting the need of further research.