key: cord-0900634-0t9s1hgj authors: Campello, Elena; Simion, Chiara; Bulato, Cristiana; Radu, Claudia M.; Gavasso, Sabrina; Sartorello, Francesca; Saggiorato, Graziella; Zerbinati, Patrizia; Fadin, Mariangela; Spiezia, Luca; Simioni, Paolo title: Absence of hypercoagulability after nCoV-19 vaccination: An observational pilot study date: 2021-06-25 journal: Thromb Res DOI: 10.1016/j.thromres.2021.06.016 sha: 6b04cc3ad932fbe6d6bfbd8dbf24abb5eb13da6b doc_id: 900634 cord_uid: 0t9s1hgj BACKGROUND: It is still unknown whether COVID-19 vaccines induce a prothrombotic state or increase the hypercoagulable condition in subjects with a predisposition to thrombosis. OBJECTIVES: We evaluated the coagulation profile in a series of healthy subjects who received the first dose of the BNT162b2 or the ChAdOx1 vaccines and assessed whether hypercoagulability developed. PATIENTS/METHODS: Volunteers among the staff of the University of Padua or health care professionals in the Padua University Hospital who had received either the ChAdOx1 or BNT162b2 vaccine in the previous 10 ± 2 days were eligible. A cohort of unvaccinated volunteers among family members of the University staff acted as control group. Global coagulation monitoring was assessed by whole blood rotational thromboelastometry, whole blood impedance aggregometry and thrombin generation. Platelet count was also obtained. RESULTS: One hundred and ninety subjects were enrolled: 101 (53.2%) received the ChAdOx1 vaccine and 89 (46.8%) the BNT162b2 vaccine. Twenty-eight non-vaccinated subjects acted as controls. Thromboelastometry parameters were all comparable among groups. Thrombin receptor activating peptide (TRAP)-, ADP- and ASPI-induced platelet aggregation were similar among groups, as well as platelet count. Endogenous thrombin potential (ETP) was comparable among groups. The results were confirmed after controlling for age, gender and hormonal. Considering women taking combined oral contraceptives or thrombophilia carriers, no differences were detected in thromboelastometry or thrombin generation parameters between subjects who received ChAdOx1 vs. BNT162b2 vaccines. CONCLUSIONS: No significant activation of fibrinogen-driven coagulation, plasma thrombin generation or clinically meaningful platelet aggregation after ChAdOx1 or BNT162b2 vaccination was observed. The European Medicines Agency (EMA) has approved five vaccines against coronavirus Recent observations have documented several cases of unusual thrombotic events in combination with thrombocytopenia after receiving the ChAdOx1 vaccine [3, 4] . The underlying mechanism is partly understood as it appears, in many cases, to mimic that of an atypical Heparin-induced thrombocytopenia [5] . It is still unknown whether there may be a predisposition to develop this rare complication. Although rare, these observations induced profound concern in people candidate to receive nCoV-19 vaccination. A large number of requests for medical examinations come every day from healthy subjects to the outpatients clinics in order to exclude possible risk factors for vaccination. Additionally, patients with thrombophilia conditions have further concern. To date it remains to be demonstrated whether COVID-19 vaccines may induce a transient hypercoagulable state or increase the hypercoagulable condition in subjects with a predisposition to thrombosis. We performed an observational study to evaluate the coagulation profile in a series of healthy subjects who had received the first dose of the BNT162b2 or the ChAdOx1 vaccines and assessed whether hypercoagulability developed. J o u r n a l P r e -p r o o f Journal Pre-proof Volunteers among the staff of the University of Padua or health care professionals in the Padua University Hospital who had received either the ChAdOx1 or BNT162b2 vaccine in the previous 10±2 days were eligible and offered coagulation monitoring. A cohort of unvaccinated volunteers among family members of the staff of the University of Padua acted as control group for coagulation monitoring. Exclusion criteria were: i) SARS-Cov2 infection within the previous three months; ii) infection, hospitalization, surgery within the previous month; iii) pregnancy/postpartum; iv) ongoing anticoagulant therapy; v) active cancer (recent diagnosis or radio-chemotherapy in the previous three months; vi) past venous thrombotic events. Cases and controls underwent fasting venous sampling of 9 mL of blood into citratecontaining vacutainer tubes. Platelet-poor plasma (PPP) for thrombin generation was prepared within 1 h by double centrifugation (2 x 10 min at 1500 g) at room temperature. Aliquots (1 mL) were immediately frozen and then stored at -80 • . Rotational thromboelastometry-ROTEM is based on the viscoelastic method. Upon activation by calcium, phospholipids and ellagic acid or tissue factor, clot formation is achieved and thereby decrease the rotational potential of a pin (i.e., viscoelastometry) [6] . The increase in viscoelastic force is proportional to the capability of clot formation in intrinsic (INTEM), extrinsic coagulation (EXTEM) pathways and fibrinogen contribution to blood clot (FIBTEM). Evaluation of platelet aggregation by Multiplate® is based on the impedance method. Upon activation by different agonists, platelets adhere to the sensor wires and thereby increase the electrical resistance (i.e., impedance). The increase is proportional to the capability of platelets to aggregate on each wire. Results are expressed as Area Under the Curve (AUC, AU*min) [7] . The higher the AUC value, the greater the capability of platelets to aggregate. Particularly, platelets were stimulated with 3 different agonists: (1) thrombin receptor activating peptide-6 (TRAP-6) 32 μmol/L, which is the most potent platelet activator and stimulates platelet aggregation via the thrombin receptor PAR-1 (TRAP test-Roche Diagnostics GmbH, Mannheim, Germany); (2) ADP 6.5 μmol/L (ADP test-Roche Diagnostics GmbH); (3) arachidonic acid 500 μmol/L, which allows the evaluation of cyclooxygenase-dependent aggregation (ASPI test-Roche Diagnostics GmbH). Thromboelastometry and platelet aggregometry were performed in whole blood within 2 h after sample draw by trained members of the research team, as previously reported [7, 8] . Thrombin generation was assessed in PPP with the CAT method (Thrombinoscope BV) [8, 9] . Briefly, 80 μL of plasma were dispensed into the wells of a 96-well microtiter plate TM is the main cofactor in the thrombin-induced activation of the natural anticoagulant protein C. In normal plasma, the addition of TM leads to reduced thrombin generation (reduction of ETP). In this study, the concentration of TM (5 nmol/L) was to reduce the ETP by 51 ± 13% in normal pool plasma (resulting in an ETP ratio of 0.49 ± 0.13). Plasma from 45 healthy subjects was also tested to evaluate the effects of TM on ETP and acted as controls for TGA. This group consisted of 22 males and 23 females without history of cardiovascular, autoimmune and acute diseases and not taking antithrombotic, antibiotic, or hormonal therapy. All tests were performed in duplicate. The ETP ratio was calculated as follows: ETP with TM/ETP without TM, and it reflects the -resistance‖ to the anticoagulant effect of protein C. The lower the ETP ratio, the better preserved the level and the function of protein C. Conversely, a higher ETP ratio means more severe protein C resistance and a potentially greater predisposition to thrombosis. All participants gave written informed consent for coagulation monitoring and the use of data for research purposes. Qualitative data were described as frequencies and percentages. Quantitative data were described as mean ± standard deviation (SD). Comparisons between the independent groups were performed by the Mann Whitney U test and Kruskal-Wallis test with post-hoc J o u r n a l P r e -p r o o f Journal Pre-proof analysis (Dunn) for quantitative variables, and the Chi-square test or Fisher's exact test for categorical variables. Multiple linear regression analysis was run in order to control for potential confounding factors (i.e. age, gender, and hormonal therapy). Statistical significance was set at p ≤ 0.05. All analyses were completed using SPSS software version 26.0. One hundred and ninety subjects who met the inclusion criteria were enrolled: 101 Thrombin generation was performed in 132 vaccinated subjects (70% of the cohort) and in all the non-vaccinated subjects. ETP was similar among groups (Figure 1 , panel A) even after controlling for age, gender and hormonal therapy. ETP with TM was slightly increased in subjects vaccinated with BNT162b2 as compared to the other two groups (p=0.037), leading to a slightly increased in the ETP ratio (p=0.007; Figure 1, Among women taking COC therapy (9.5% of the vaccinated population), no differences were detected in thromboelastometry (MCF in FIBTEM) or in thrombin generation parameters (ETP and ETP ratio) between subjects who received ChAdOx1 vs. BNT162b2 vaccines (Table 3) . Among thrombophilia carriers (11.6% of the vaccinated population), no differences were detected in thromboelastometry (MCF in FIBTEM) or in thrombin generation parameters (ETP and ETP ratio) between subjects who received ChAdOx1 vs. BNT162b2 vaccines (Table 3) . Finally, among subjects with autoimmune diseasesmostly Hashimoto's thyroiditis -(13.7% of the vaccinated population) no differences were detected in coagulation parameters between subjects who received ChAdOx1 vs. BNT162b2 vaccines (Table 3) . The COVID-19 vaccine ChAdOx1 (AstraZeneca) has been associated with rare thrombotic complications such as cerebral venous sinus thrombosis (CVST) [3, 4, 10] . It has been reported that these rare complications may occur approximately 5 to 20 days after vaccination and are immune-related leading to severe thrombocytopenia mediated by platelet-activating antibodies and thrombotic manifestations [3, 4, 11] . This mechanism is rather peculiar and dissimilar to those commonly involved in the development of venous or arterial thrombosis due to more common prothrombotic conditions, such as congenital or acquired thrombophilia [12] . The possible occurrence of severe thrombosis as a complication of the nCoV-19 vaccination arose great concern among people waiting to be called for the vaccination. In order to reassure people about the safety of the vaccination, we performed an observational pilot study to evaluate the potential presence of hypercoagulability after nCoV-19 vaccination using global coagulation assays. The coagulation monitoring of this pilot cohort of vaccinated subjects excluded the presence of hypercoagulability at a mean time of nine days after ChAdOx1 or BNT162b2 vaccination. Using thromboelastometry, impedance aggregometry and thrombin generation, we can exclude the presence of fibrinogen-driven hypercoagulability, platelet hyperactivity and increased plasma thrombin generation induced by the vaccination. In addition, no reduction in platelet count was detected. Although the sample size was very low, we can beckon that a few females showed increased thrombin generation linked to combined oral contraceptives [13] , regardless of the type of vaccine received. Additionally, the comparison of a small cohort of carriers of J o u r n a l P r e -p r o o f inherited thrombophilia showed a similar coagulation profile between the two types of vaccination. The same is true for subjects suffering from autoimmune conditions, mainly thyroidites. We would be remiss if we did not mention some of the limitations of our study. Firstly, though the number of subjects included is small, it is representative of the population eligible for COVID-19 vaccines in Italy at this time. Additionally, we agree that a longitudinal design of the study with coagulation evaluation before and after the vaccine would have been stronger; however, the presence of a non-vaccinated control group can compensate for the lack of pre-vaccine coagulation determinations. Besides the enrollment of a non-vaccinated group as control, we showed that thromboelastometry, aggregometry, as well as thrombin generation parameters in vaccinated subjects were all within the reference ranges. Secondly, our conclusions should be limited to ChAdOx1 or BNT162b2, the only two vaccines considered in our study. Thirdly, we did not perform coagulation tests in order to exclude other possible mechanisms of vaccine-induced hypercoagulability (e.g. D-dimer or coagulation inhibitors levels) but we chose to only perform global coagulation tests in order to explore overall whole blood and plasma coagulation profile, as well as platelet function. Lastly, subjects were recruited on a voluntary basis for coagulation monitoring and this may account for the relatively high prevalence of inherited thrombophilia in the otherwise asymptomatic cohort (11.6%) vs. the general population (2-4%) [14] . Following news of a possible association between vaccination and thrombosis, subjects with known thrombophilia might have adhered more promptly to our coagulation monitoring on a voluntary basis. Needless to say, the absence of hypercoagulability also shown in this small subgroup of thrombophilic patients after vaccination appears reassuring. The same holds true of women taking oral contraceptives in whom vaccination had no additional effect in increasing the expected mild hypercoagulability related to the hormonal treatment [13] . J o u r n a l P r e -p r o o f Journal Pre-proof In conclusion, we observed no significant activation of fibrinogen-driven coagulation, plasma thrombin generation or clinically meaningful platelet aggregation after ChAdOx1 or BNT162b2 vaccination. We observed no onset of hypercoagulability in healthy subjects nor increased hypercoagulability in subjects with inherited or acquired thrombophilia after vaccination, which suggests that other pathogenetic mechanisms may contribute to the development of CVST. Although this is a pilot study, these preliminary results may be useful for Clinicians to reassure people referring to our Centres every day for an opinion on the prothrombotic risk associated with the nCoV-19 vaccination. We acknowledge that longitudinal studies are needed to better understand to what extent second doses of ChAdOx1 or BNT162b2, as well as other available COVID-19 vaccines may cause hypercoagulability in healthy subjects or increase hypercoagulability in those with inherited or acquired thrombophilia, and to clarify the underlying mechanisms. None. J o u r n a l P r e -p r o o f Thrombosis and Thrombocytopenia after ChAdOx1 nCoV-19 Vaccination Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination Autoimmune heparin-induced thrombocytopenia Hypercoagulability detected by whole blood thromboelastometry (ROTEM®) and impedance aggregometry (MULTIPLATE®) in obese patients Influence of Hepatocellular Carcinoma on Platelet Aggregation in Cirrhosis Calibrated automated thrombin generation measurement in clotting plasma Thrombotic Thrombocytopenia after Ad26.COV2.S Vaccination Thrombophilia testing and venous thrombosis Effect of oral contraceptives on thrombin generation measured via calibrated automated thrombography risk factors and prevention