Background: Management of anticoagulant therapy in COVID-19 patients is critical. Low-molecular-weight heparin (LMWH) thromboprophylaxis is already recommended, and anti-Factor Xa (anti-FXa) monitoring has been used to titrate LMWH doses. Methods: Through a cross-sectional study, we evaluated anti-FXa activity in patients admitted to the ICU, receiving intermediate dose (30, 40, 50 mg, subcutaneously [SC], twice daily) or therapeutic dose (1 mg/kg, SC, Q12h) of enoxaparin to find whether the patients in these two groups achieved anti-FXa levels in the accepted thromboprophylaxis range. Results: The occurrence of deep vein thrombosis was 26% in the therapeutic-dose group and 17% in the intermediate-dose group. D-dimer values were nearly 3.5-fold higher in those who received a therapeutic dose of anticoagulants than in those who received intermediate-dose thromboprophylaxis. Patients in the therapeutic-dose group had significantly higher IL-6 levels (p ≤ 0.001). More than one-third of the patients in the therapeutic-dose group (n = 8; 42.18%) and approximately half of the patients in the intermediate-dose group (n = 12; 52.2%) achieved the target range level of anti-FXa. Patients who received therapeutic doses were more likely to have anti-FXa levels above the expected range (47.4 vs 13% in the intermediate-dose group; p < 0.05). Conclusion: Therapeutic dose of enoxaparin in critically ill COVID-19-infected patients did not reduce the incidence of thromboembolic events and, on the other hand, may predispose these patients to increased risk of bleeding by increasing anti-FXa activity above the desired level. Administration of intermediate-dose thromboprophylaxis is suggested to achieve anti-FXa levels in the accepted thromboprophylaxis range.
Virus-induced endothelial dysfunction followed by cytokine storm and hyperinflammation contributes to the pro-coagulative state and thrombotic complications and results in an increased risk of thromboembolic events in COVID-19 patients . Therefore, optimal management of anticoagulant therapy in COVID-19 patients has a fundamental role. Guidelines already recommend the use of low-molecular-weight heparin (LMWH) for hospitalized COVID-19 patients . Its short half-life, relatively predictable pharmacokinetic profile, fewer known drug-drug interactions, and ease of monitoring have made LMWH the preferred anticoagulants in hospitalized critically ill patients . Dosing of LMWH for thromboprophylaxis in patients with severe COVID-19 is still being debated . A prophylactic dose of LMWH is often used, while some studies suggest higher thromboprophylaxis dosing such as enoxaparin 40 mg twice daily versus 40 mg once daily for intensive therapy unit (ITU) patients . Monitoring for prophylaxis is not routinely used; however, authors advised that anticoagulant treatment should be guided by anti-factor Xa (anti-FXa) activity . The appropriate target anti-FXa level for the prophylactic dose of enoxaparin has been suggested as between 0.2 and 0.4/0.5 IU/mL . Middeldorp and colleagues reviewed anti-FXa activity in COVID-19-infected patients admitted to the hospital, all receiving thromboprophylaxis enoxaparin (40 mg once daily), and compared 4-h anti-FXa activity levels. They found that 95% of ITU patients failed to achieve a target anti-FXa activity (0.2–0.4 IU/mL) . When standard and intermediate dose of enoxaparin are compared in critically ill patients with COVID-19, intermediate-dose thromboprophylaxis is suggested to achieve target thromboprophylaxis anti-FXa levels . To determine the appropriate dose of thromboprophylaxis and the validity of target anti-FXa activity of 0.2–0.5 U/mL, we investigated anti-FXa levels in two groups of critically ill COVID-19-infected patients who received intermediate or therapeutic doses of enoxaparin to find whether the patients of these two groups achieve anti-FXa levels in the accepted thromboprophylaxis range and compared the incidence of deep vein thrombosis (DVT) and central line thrombosis in them.
In this cross-sectional study, we enrolled all 18-year-old to 80-year-old patients with a confirmed positive COVID-19 determined by real-time polymerase chain reaction, admitted to the adult intensive care units of Namazi Hospital, the largest academic medical center in the south of Iran, for 2 months (July and August 2021). Pregnant women, current users of oral contraceptive pills, patients with BMI > 30, GFR <30 mL/min, positive history of heparin-induced thrombocytopenia or allergic reaction to LMWH, dual antiplatelet therapy (aspirin + plavix), platelet count <50 × 103/μL, and history of venous thromboembolism (VTE) or pulmonary thromboembolism were excluded.
The study was endorsed by the Ethics Committee of the Shiraz University of Medical Sciences, Shiraz, Iran. Electronic medical records (EMR) were accessed to retrieve demographic information, medical history, and comorbidities. The severity of illness was categorized into four levels: mild, moderate, severe, and critical types, according to the WHO COVID-19 Disease Severity Classification. The APACHE II (Acute Physiology and Chronic Health Evaluation) SCORE was measured within the first 24 h of ICU admission using the software.
DVT risk factors (according to American Society of Hematology [ASH] Risk assessment models), platelet count, and markers of inflammation including C-reactive protein, erythrocyte sedimentation rate, lactate dehydrogenase, interleukin-6 (IL-6), and procalcitonin were also obtained from patients’ records.
The incidence of VTE in the lower extremities (femoral and popliteal) and around the central venous catheter was assessed using Doppler sonography on ICU admission days 1 and 7. Patients suspected of pulmonary embolism were also subjected to CT angiography of pulmonary vessels with intravenous contrast. The use of vasopressors or antiplatelet agents, renal replacement therapy, the incidence of thrombosis and sepsis, and the final status of patients including death, discharge, and ICU length of stay >7 days were also recorded.
The patients received the following two enoxaparin regimens in the ICU according to illness severity and physician order: (1) “intermediate-dose”: enoxaparin 30, 40, or 50 mg, subcutaneously (SC), 2 times a day according to BMI and creatinine clearance according to the ASH (American Society of Hematology); (2) “therapeutic-dose”: enoxaparin 1 mg/kg (100 U/kg), SC, Q12h according to BMI and creatinine clearance .
Plasma anti-FXa activity was measured 24–36 h after ICU admission and 4 h after enoxaparin injection. Based on previous studies investigating the efficacy of anti-FXa in non-COVID-19 patients, the target range of enoxaparin for VTE prophylaxis was considered 0.2–0.5 unit/mL .
The differences between groups were analyzed by the Mann-Whitney U test. For categorical variables, they were presented as numbers and percentages, and χ2 and Fisher exact tests were used to estimate the significant values. The data were analyzed using the statistical package for social science (version 23; SPSS Inc., Chicago, IL, USA), and a p value < 0.05 was considered statistically significant.
Baseline clinical characteristics and comorbidities of study groups are presented in Table 1. Patients’ median age was 54 (interquartile range [IQR] = 51–65), and 62% of individuals were male. There were no differences in anticoagulant or antiplatelet agents before ICU admission between groups. The median time from onset of symptoms to ICU admission (COVID day) was 11 days in both groups. There were no differences between the two groups regarding comorbidities (Table 1). Over the study period, 112 critically ill patients with real-time polymerase chain reaction-confirmed COVID-19 were admitted to the intensive care unit within 60 days. Fifty-two patients met the inclusion criteria. Of them, 10 cases were excluded due to delayed delivery of the blood sample, dose change, or discontinuation of enoxaparin. Finally, 19 patients (45.2%) received a therapeutic dose, and 23 patients (54.7%) received an intermediate dose of enoxaparin. The overall occurrence of DVT was 21.4% (26% in the therapeutic-dose group and 17% in the intermediate-dose group), and the incidence of central line thrombosis was 4.8% (1 patient in each group; 5.3% in the therapeutic-dose group and 4.3% in the intermediate-dose group). No cases of pulmonary embolism were observed among the suspected patients.
Comparison of Patients among the Two Anticoagulant Regimens
D-dimer values were nearly 3.5-fold higher in those who received therapeutic doses of anticoagulants (median: 2.12 mg/L; IQR: 0.77–5.27 mg/L) than in those who received intermediate-dose thromboprophylaxis (median: 0.61 mg/L; IQR: 0.35–1.29 mg/L; p < 0.001). Patients in the therapeutic-dose group had significantly higher IL-6 levels (p ≤ 0.001) and non-significantly higher levels of C-reactive protein, erythrocyte sedimentation rate, lactate dehydrogenase, and procalcitonin. Five patients (26%) in the therapeutic-dose group were on vasopressors, and none of them required renal replacement therapy. There were no significant differences in death, discharge, and ICU length of stay of more than 7 days between groups (Table 2).
Anti-FXa Level Targets
Anti-FXa levels were checked in all studied patients. As it is depicted in Table 3, some patients had anti-FXa levels out of the appropriate range in each group. More than one-third of the patients in the therapeutic-dose group (n = 8; 42.18%) and approximately half of the patients in the intermediate-dose group (n = 12; 52.2%) achieved the target range level of anti-FXa. Patients who received therapeutic dose were more likely to have anti-FXa levels above the expected range (47.4 vs. 13% in the intermediate-dose group. p < 0.05). Anti-FXa levels were below the expected range in 10.5% (2 patients) of the therapeutic-dose group and 34.8% (8 patients) in the intermediate-dose group, which were not statistically different.
Our findings showed no significant differences in demographic characteristics or premedical history. The illness severity and DVT risk factors were the same in both groups of our study. In addition, D-dimer values were nearly 3.5-fold higher in those who received therapeutic dose than in those who received intermediate dose. Patients in the therapeutic-dose group had significantly higher IL-6 levels. Consistent with our findings, elevated plasma levels of D-dimer and IL-6 have been repeatedly demonstrated in patients with COVID-19  and are associated with increased thrombotic risk and poor outcomes in patients hospitalized with COVID-19 . We compared anti-FXa activity levels 4-h after Enoxaparin administration and found that almost half of the patients in both groups achieved anti-FXa levels within the appropriate range.
Virus-induced vessel wall injury and consequent cytokine storm along with immobility and hypoxia result in an increased risk of thromboembolic events and a higher risk of mortality in COVID-19-infected patients . Therefore, management of the anticoagulant therapy in these patients has a critical role. Guidelines already recommend the use of LMWH for thromboprophylaxis [10, 11], and anti-Factor Xa (anti-FXa) monitoring has been used to titrate LMWH doses for prophylactic or therapeutic anticoagulation in critically ill patients. However, the appropriate dose of thromboprophylaxis and the validity of the target anti-FXa activity of 0.2–0.5 U/mL have not been rigorously approved.
According to the INSPIRATION study, a randomized clinical trial among 562 COVID-19 patients admitted to ICU in 10 academic centers in Iran who received intermediate-dose or standard-dose prophylactic anticoagulation, routine use of empirical intermediate-dose prophylactic anticoagulation is not recommended. They showed that there is no difference in venous or arterial thrombosis between these two groups .
To our knowledge, there are limited studies that have evaluated anti-FXa levels in two groups of critically ill COVID-19-infected patients who received intermediate or therapeutic dose of enoxaparin to find out whether the patients in these two groups achieve anti-FXa levels in the accepted thromboprophylaxis range. We studied anti-FXa activity in patients admitted to ICU, receiving intermediate or therapeutic doses of enoxaparin.
Rappaport et al.  reported that intermediate-dose thromboprophylaxis led to the target range of anti-FXa activity in most of the studied patients. Starting intermediate-dose thromboprophylaxis for critically ill patients with COVID-19 is recommended to achieve anti-FXa levels in the accepted thromboprophylaxis range .
We also observed that 35% of patients receiving intermediate-dose and 10.5% of patients in the therapeutic-dose group had anti-FXa levels <0.2IU/mL. In accordance, Middeldorp and colleagues reviewed anti-FXa activity in COVID-19-infected patients admitted to the hospital, all receiving thromboprophylaxis with enoxaparin (40 mg once daily), and compared 4-h anti-FXa activity levels. They found that 95% of ITU patients failed to achieve a target anti-FXa activity (0.2–0.4 IU/mL). Low anti-FXa levels in some of our patients can be explained as follows: biological factors, inflammatory markers, hyperbilirubinemia, extremes of age and weight, and the use of vasopressors may reduce the absorption of LMWH and decrease the effectiveness of LMWH .
Although low anti-FXa activity may be associated with increased pro-coagulative state and thrombotic complications and an increased risk of mortality, there were no significant differences in the occurrence of DVT, the incidence of central line thrombosis, mortality rate, and ICU length of stay of more than 7 days between the two groups of our study. In the HEP-COVID randomized clinical trial, therapeutic-dose LMWH did not reduce major thromboembolism events and death compared with standard heparin thromboprophylaxis among ICU patients with COVID-19 which is consistent with the results of our study .
As an additional finding, we found that there was no difference in the need for a vasopressor between the therapeutic and intermediate-dose thromboprophylaxis groups. This finding was in accordance with a multi-platform randomized clinical trial (REMAP-CAP, ACTIV-4a, and ATTACC) from 9 counties that showed that “In critically ill patients with COVID-19, an initial strategy of therapeutic-dose anticoagulation with heparin did not result in a greater probability of survival to hospital discharge or a greater number of days free of cardiovascular or respiratory organ support than did usual-care pharmacologic thromboprophylaxis.” .
A noticeable finding in our study was that in the therapeutic-dose group, most of the patients had anti-FXa levels above the target range. It means that higher prophylactic doses of LMWH not only did not reduce the incidence of DVT in COVID-19-infected patients but also may have increase the chance of bleeding.
A previous meta-analysis for the non-COVID population reported that the risk of bleeding with therapeutic doses of enoxaparin was four times higher than with prophylactic doses, especially in patients with renal impairment . Some recent studies in critically ill COVID-19 patients also reported significant bleeding complications, particularly in those receiving therapeutic doses of the anticoagulants [17, 18].
Therapeutic-dose of enoxaparin versus intermediate dose in critically ill COVID-19 patients did not reduce the incidence of the thrombotic events and may predispose them to increased risk of bleeding by increased anti-FXa activity above the desired level. Moreover, the administration of intermediate dose is suggested to achieve anti-FXa levels in the accepted thromboprophylaxis range.
This study has some limitations. Conventional ranges of anti-factor Xa activity may not be accurate and appropriate as a predictor of bleeding in critically ill COVID-19 patients. The results of this single-center study may not be appropriate to confirm that the target anti-FXa activity is a reliable predictive factor for bleeding in COVID-19 patients, particularly those receiving the therapeutic-dose anticoagulant. Larger prospective studies are required to assess the risk of bleeding and other complications in these populations. Furthermore, this study was underpowered to test for all relevant covariate interactions reliably.
This study was supported by Shiraz University of Medical Sciences. The authors would like to thank the patients who participated in this trial.
Statement of Ethics
The study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.sums.med.rec.1400.461). Written informed consent was taken from all the patients.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
The present article was extracted from the thesis written by Dr. Sina Azadikhah. There was no financial support for this study. The author declared that they have no financial interest.
Mansour Masjedi and Farid Zand: conception and design of the study and critical revision of the manuscript. Sina adikhah, Golnar Sabetian, and Mina Ostovan: clinical data collection and analysis. Vida Naderi-boldaji: analysis and interpretation of data and drafting and revising of the manuscript. Naeimehossadat Asmarian: statistical analysis.
Data Availability Statement
All data generated during this study are included in this article, and further inquiries can be directed to the corresponding author.