Blood Coagulation and Beyond: Position Paper from the Fourth Maastricht Consensus Conference on Thrombosis.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

Vascular Function, Systemic Inflammation, and Coagulation Activation 18 Months after COVID-19 Infection: An Observational Cohort Study.

INTRODUCTION: Among its effect on virtually all other organs, COVID-19 affects the cardiovascular system, potentially jeopardizing the cardiovascular health of millions. Previous research has shown no indication of macrovascular dysfunction as reflected by carotid artery reactivity, but has shown sustained microvascular dysfunction, systemic inflammation, and coagulation activation at 3 months after acute COVID-19. The long-term effects of COVID-19 on vascular function remain unknown. MATERIALS AND METHODS: This cohort study involved 167 patients who participated in the COVAS trial. At 3 months and 18 months after acute COVID-19, macrovascular dysfunction was evaluated by measuring the carotid artery diameter in response to cold pressor testing. Additionally, plasma endothelin-1, von Willebrand factor, Interleukin(IL)-1ra, IL-6, IL-18, and coagulation factor complexes were measured using ELISA techniques. RESULTS: The prevalence of macrovascular dysfunction did not differ between 3 months (14.5%) and 18 months (11.7%) after COVID-19 infection (p = 0.585). However, there was a significant decrease in absolute carotid artery diameter change, 3.5% ± 4.7 vs. 2.7% ± 2.5, p-0.001, respectively. Additionally, levels of vWF:Ag were persistently high in 80% of COVID-19 survivors, reflecting endothelial cell damage and possibly attenuated endothelial function. Furthermore, while levels of the inflammatory cytokines interleukin(IL)-1RA and IL-18 were normalized and evidence of contact pathway activation was no longer present, the concentrations of IL-6 and thrombin:antithrombin complexes were further increased at 18 months versus 3 months (2.5 pg/mL ± 2.6 vs. 4.0 pg/mL ± 4.6, p = 0.006 and 4.9 µg/L ± 4.4 vs. 18.2 µg/L ± 11.4, p < 0.001, respectively). DISCUSSION: This study shows that 18 months after COVID-19 infection, the incidence of macrovascular dysfunction as defined by a constrictive response during carotid artery reactivity testing is not increased. Nonetheless, plasma biomarkers indicate sustained endothelial cell activation (vWF), systemic inflammation (IL-6), and extrinsic/common pathway coagulation activation (FVII:AT, TAT) 18 months after COVID-19 infection.

Serial thrombin generation and exploration of alternative anticoagulants in critically ill COVID-19 patients: Observations from Maastricht Intensive Care COVID Cohort.

Background: COVID-19 associated coagulopathy (CAC) is associated with an increase in thromboembolic events. Current guidelines recommend prophylactic heparins in the management of CAC. However, the efficacy of this strategy in the intensive care population remains uncertain. Objective: We aimed to measure thrombin generation (TG) to assess CAC in intensive care unit (ICU) patients receiving thromboprophylaxis with low molecular weight heparin (LMWH) or unfractionated heparin (UFH). In addition, we performed statistical modeling to link TG parameters to patient characteristics and clinical parameters. Lastly, we studied the potency of different anticoagulants as an alternative to LMWH treatment in ex vivo COVID-19 plasma. Patients/Methods: We included 33 patients with confirmed COVID-19 admitted at the ICU. TG was measured at least twice over the course of 6 weeks after admission. Thrombin generation parameters peak height and endogenous thrombin potential (ETP) were compared to healthy controls. Results were subsequently correlated with a patient characteristics and laboratory measurements. In vitro spiking in TG with rivaroxaban, dabigatran, argatroban and orgaran was performed and compared to LMWH. Results: Anti-Xa levels of all patients remained within the therapeutic range throughout follow-up. At baseline, the mean (SE) endogenous thrombin potential (ETP) was 1,727 (170) nM min and 1,620 (460) nM min for ellagic acid (EA) and tissue factor (TF), respectively. In line with this we found a mean (SE) peak height of 353 (45) nM and 264 (96) nM for EA and TF. Although fluctuating across the weeks of follow-up, TG parameters remained elevated despite thromboprophylaxis. In vitro comparison of LMWHs and direct thrombin inhibitors (e.g., agratroban, dabigatran) revealed a higher efficacy in reducing coagulation potential for direct thrombin inhibition in both ellagic acid (EA) and tissue factor (TF) triggered TG. Conclusion: In a sub-group of mechanically ventilated, critically ill COVID-19 patients, despite apparent adequate anti-coagulation doses evaluated by anti-Xa levels, thrombin generation potential remained high during ICU admission independent of age, sex, body mass index, APACHE II score, cardiovascular disease, and smoking status. These observations could, only partially, be explained by (anti)coagulation and thrombosis, inflammation, and multi-organ failure. Our in vitro data suggested that direct thrombin inhibition compared with LMWH might offer an alternate, more effective anticoagulant strategy in COVID-19.

The Composition and Physical Properties of Clots in COVID-19 Pathology.

Hemostasis is a finely tuned process of which dysregulation can lead either to bleeding or thrombotic complications. The latter is often caused by the hypercoagulable state as it is also seen in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, i.e., in COVID-19 patients. COVID-19 patients requiring hospitalization often suffer from thrombotic events that could not be predicted using routine coagulation assays. Recently, several studies have reported ROtational ThromboElastoMetry (ROTEM) as a promising tool to predict outcomes in COVID-19 patients. In this review we give an overview of ROTEM with a particular focus on the interpretation of the symmetrical clot formation curve in relation to coagulopathy in COVID-19 patients. Furthermore, we have introduced new parameters that might help to better distinguish between COVID-19 patients and outcomes.

ChAdOx1 vaccination, blood coagulation, and inflammation: No effect on coagulation but increased interleukin-6.

BACKGROUND: Vaccination is the leading approach in combatting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. ChAdOx1 nCoV-19 vaccination (ChAdOx1) has been linked to a higher frequency of rare thrombosis and thromboembolism. This study aimed to explore markers related to the blood coagulation system activation and inflammation, before and after ChAdOx1 vaccination. PATIENTS AND METHODS: An observational cohort study including 40 health care workers. Whole blood samples were collected before, and either 1 or 2 days after vaccination. Activated coagulation factors in complex with their natural inhibitors were determined by custom ELISAs, including thrombin:antithrombin (T:AT), kallikrein:C1-esterase-inhibitor (PKa:C1Inh), factor(F)IXa:AT, FXa:AT, FXIaAT, FXIa:alpha-1-antitrypsin (α1AT), FXIa:C1inh, and FVIIa:AT. Plasma concentrations of interleukin (IL)-6 and IL-18 were quantified via ELISA. Analyses were performed using Wilcoxon signed-rank test. RESULTS: Levels of FVIIa:AT decreased with a median (IQR) of 707 (549-1028) pg/ml versus 598 (471-996) pg/ml, p = 0.01; and levels of IL-6 increased, 4.0 (1.9-6.8) pg/ml versus 6.9 (3.6-12.2) pg/ml, p = 0.02, after vaccination. No changes were observed in T:AT, PKa:C1Inh, FIXa:AT, FXa:AT, FXIaAT, FXIa:α1AT, FXIa:C1inh, and IL-18. CONCLUSION: ChAdOx1 leads to an inflammatory response with increased levels of IL-6. We did not observe activation of the blood coagulation system 1-2 days following vaccination.

Serial markers of coagulation and inflammation and the occurrence of clinical pulmonary thromboembolism in mechanically ventilated patients with SARS-CoV-2 infection; the prospective Maastricht intensive care COVID cohort.

BACKGROUND: The incidence of pulmonary thromboembolism is high in SARS-CoV-2 patients admitted to the Intensive Care. Elevated biomarkers of coagulation (fibrinogen and D-dimer) and inflammation (c-reactive protein (CRP) and ferritin) are associated with poor outcome in SARS-CoV-2. Whether the time-course of fibrinogen, D-dimer, CRP and ferritin is associated with the occurrence of pulmonary thromboembolism in SARS-CoV-2 patients is unknown. We hypothesise that patients on mechanical ventilation with SARS-CoV-2 infection and clinical pulmonary thromboembolism have lower concentrations of fibrinogen and higher D-dimer, CRP, and ferritin concentrations over time compared to patients without a clinical pulmonary thromboembolism. METHODS: In a prospective study, fibrinogen, D-dimer, CRP and ferritin were measured daily. Clinical suspected pulmonary thromboembolism was either confirmed or excluded based on computed tomography pulmonary angiography (CTPA) or by transthoracic ultrasound (TTU) (i.e., right-sided cardiac thrombus). In addition, patients who received therapy with recombinant tissue plasminogen activator were included when clinical instability in suspected pulmonary thromboembolism did not allow CTPA. Serial data were analysed using a mixed-effects linear regression model, and models were adjusted for known risk factors (age, sex, APACHE-II score, body mass index), biomarkers of coagulation and inflammation, and anticoagulants. RESULTS: Thirty-one patients were considered to suffer from pulmonary thromboembolism ((positive CTPA (n = 27), TTU positive (n = 1), therapy with recombinant tissue plasminogen activator (n = 3)), and eight patients with negative CTPA were included. After adjustment for known risk factors and anticoagulants, patients with, compared to those without, clinical pulmonary thromboembolism had lower average fibrinogen concentration of - 0.9 g/L (95% CI: - 1.6 - - 0.1) and lower average ferritin concentration of - 1045 µg/L (95% CI: - 1983 - - 106) over time. D-dimer and CRP average concentration did not significantly differ, 561 µg/L (- 6212-7334) and 27 mg/L (- 32-86) respectively. Ferritin lost statistical significance, both in sensitivity analysis and after adjustment for fibrinogen and D-dimer. CONCLUSION: Lower average concentrations of fibrinogen over time were associated with the presence of clinical pulmonary thromboembolism in patients at the Intensive Care, whereas D-dimer, CRP and ferritin were not. Lower concentrations over time may indicate the consumption of fibrinogen related to thrombus formation in the pulmonary vessels.

Serial EXTEM, FIBTEM, and tPA Rotational Thromboelastometry Observations in the Maastricht Intensive Care COVID Cohort-Persistence of Hypercoagulability and Hypofibrinolysis Despite Anticoagulation.

Background: Coronavirus Disease 2019 (COVID-19) patients often present with thromboembolic events. In COVID-19 patients, routine hemostatic assays cannot correctly identify patients at risk for thromboembolic events. Viscoelastic testing with rotational thromboelastometry (ROTEM) might improve the characterization of COVID-19-associated coagulopathy. Objective: To unravel underlying coagulopathy and fibrinolysis over time as measured by serial assessment heparin-independent (FIBTEM and EXTEM) and fibrinolysis illustrating (tissue plasminogen activator; tPA) ROTEM assays. Patients/Methods: Between April 23 and June 12, consecutive adult patients enrolled within the Maastricht Intensive Care COVID (MaastrICCht) cohort were included, and a comprehensive set of clinical, physiological, pharmaceutical, and laboratory variables were collected daily. Twice per week, EXTEM, FIBTEM, and tPA ROTEM were performed. Clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), lysis onset time (LOT), and lysis time (LT) were determined to assess clot development and breakdown and were compared to routine hemostatic assays. Results: In 36 patients, 96 EXTEM/FIBTEM and 87 tPA ROTEM tests were performed during a 6-week follow-up. CT prolongation was present in 54% of EXTEM measurements, which were not matched by prothrombin time (PT) in 37%. Respectively, 81 and 99% of all EXTEM and FIBTEM MCF values were above the reference range, and median MCF remained elevated during follow-up. The ROTEM fibrinolysis parameters remained prolonged with median LOT consequently >49 min and unmeasurable LT in 56% of measurements, suggesting a severe hypofibrinolytic phenotype. Conclusion: ROTEM tests in COVID-19 ICU patients show hypercoagulability and severe hypofibrinolysis persisting over at least 6 weeks.

Suggestions for global coagulation assays for the assessment of COVID-19 associated hypercoagulability.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection is associated with a clear prothrombotic phenotype. Although the exact pathophysiological mechanisms are not yet fully understood, thrombosis is clearly a highly important in the prognosis and outcome of COVID-19. As such, there is a need for diagnostic analysis and quantification of the coagulation potential in these patients, both at diagnosis and follow-up. Global coagulation assays like thrombin generation (TG) and rotational thromboelastometry (ROTEM) might be suitable in estimating COVID-19 associated coagulopathy and thrombosis risk. Therefore, we aimed at validating both assays for samples with high levels of fibrinogen and in the presence of anticoagulant heparins, such as commonly observed for COVID-19 ICU patients. MATERIALS AND METHODS: Calibrated Automated Thrombography (CAT) was optimized to assess plasma thrombin generation in the presence of heparins. The final conditions with either 10 µg/mL Ellagic acid (EA) or PPP Reagent HIGH (high tissue factor; HPPH) were validated according to the EP5 protocol for within-run and between-run variability. Overall variability was well below 10%. To estimate the influences of heparins and high fibrinogen levels, CAT was performed on spiked plasma aliquots from 13 healthy volunteers. Comparable to the CAT method, tPA-ROTEM was used to validate the effect of high fibrinogen and heparins on clotting time, clot firmness and clot lysis parameters. RESULTS: Our adjusted COVID-19 assay showed a heparin dose dependent decrease in peak height and endogenous thrombin potential (ETP) for both EA and HPPH triggered variants. High fibrinogen did not alter the inhibitory effect of either LMWH or UFH, nor did it influence the peak height or ETP in any of the conditions. The tPA-ROTEM showed a significant prolongation in clotting time with the additions of heparin, which normalized with the addition of high fibrinogen. MCF was markedly increased in all hyperfibrinogenemic conditions. A trend towards increased lysis time and, thus, decreased fibrinolysis was observed. CONCLUSION: Thrombin generation and tPA-ROTEM protocols for measurements in the COVID-19 populations were adjusted and validated. The adjusted thrombin generation assay shows good sensitivity for measurements in heparin spiked plasma. High levels of fibrinogen did not alter the assay or the effectiveness of heparins as measured in this assay. t-PA ROTEM was effective in measurement of both high fibrinogen and heparins spiked samples and was sensitive to the expected relevant coagulant changes by these conditions. No clear fibrinolytic effect was observed in different conditions.