Only Subclinical Alterations in the Haemostatic System of People with Diabetes after COVID-19 Vaccination.

People with diabetes have an increased risk of experiencing adverse COVID-19 outcomes. COVID-19 vaccination is, therefore, highly recommended. However, people with diabetes have an inherently elevated risk of thrombotic events and the impact of the vaccination on the coagulation system in this patient population remains to be elucidated. The aim of this study was to investigate the impact of COVID-19 vaccination on the haemostatic system in people with type 1 or type 2 diabetes. We evaluated the effects of COVID-19 vaccination (BioNTech Pfizer, Moderna, AstraZeneca) on standard coagulation parameters, whole blood coagulation (Thrombelastometry), platelet function (impedance aggregation), and thrombin generation (calibrated automated thrombography) in people with type 1 diabetes mellitus (n = 41) and type 2 diabetes mellitus (n = 37). Blood sampling points were prior to vaccination and two weeks after the respective vaccination. Thrombelastometry measurements indicated moderately increased clot formation post-vaccination in people with type 1, as well as with type 2, diabetes: "Clot formation times" were significantly shorter, and both "maximum clot firmness" and "alpha angles" were significantly higher, as compared to the respective pre-vaccination values. Therefore, TEM parameters were not altered after vaccination in patients receiving ASA. Moreover, platelet aggregation was enhanced in people with type 1 diabetes, and plasma levels of D-Dimer were increased in people with type 2 diabetes, following COVID-19 vaccination. All other standard coagulation parameters, as well as thrombin generation, were not affected by the vaccination. The coagulation responses of people with diabetes to COVID-19 vaccination were only subclinical and comparable to those observed in healthy individuals. Our findings suggest that people with diabetes do not face an increased activation of the coagulation post-vaccination.

Decreased protein C activity, lower ADAMTS13 antigen and free protein S levels accompanied by unchanged thrombin generation potential in hospitalized COVID-19 patients.

INTRODUCTION: COVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown. PATIENTS AND METHODS: In 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications. RESULTS: COVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE. CONCLUSIONS: In COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.

Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome.

BACKGROUND: Post-COVID syndrome (PCS) affects millions of people worldwide, causing a multitude of symptoms and impairing quality of life months or even years after acute COVID-19. A prothrombotic state has been suggested; however, underlying mechanisms remain to be elucidated. OBJECTIVES: To investigate thrombogenicity in PCS using a microfluidic assay, linking microthrombi, thrombin generation, and the von Willebrand factor (VWF):a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13 (ADAMTS13) axis. METHODS: Citrated blood was perfused through microfluidic channels coated with collagen or an antibody against the VWF A3 domain, and thrombogenicity was monitored in real time. Thrombin generation assays were performed and α(2)-antiplasmin, VWF, and ADAMTS13 activity levels were also measured. RESULTS: We investigated thrombogenicity in a cohort of 21 patients with PCS with a median time following symptoms onset of 23 months using a dynamic microfluidic assay. Our data show a significant increase in platelet binding on both collagen and anti-VWF A3 in patients with PCS compared with that in controls, which positively correlated with VWF antigen (Ag) levels, the VWF(Ag):ADAMTS13 ratio (on anti-VWF A3), and inversely correlated with ADAMTS13 activity (on collagen). Thrombi forming on collagen presented different geometries in patients with PCS vs controls, with significantly increased thrombi area mainly attributable to thrombi length in the patient group. Thrombi length positively correlated with VWF(Ag):ADAMTS13 ratio and thrombin generation assay results, which were increased in 55.5% of patients. α(2)-Antiplasmin levels were normal in 89.5% of patients. CONCLUSION: Together, these data present a dynamic assay to investigate the prothrombotic state in PCS, which may help unravel the mechanisms involved and/or establish new therapeutic strategies for this condition.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Point-of-Care Platform for Diagnosis of Venous Thrombosis by Simultaneous Detection of Thrombin Generation and D-Dimer in Human Plasma.

Venous thromboembolism (VTE) refers to a blood clot that starts in a vein. The risk of developing VTE is highest after major surgery or a major injury, or when someone has heart failure, cancer, or infectious disease (e.g., COVID-19). Without prompt treatment to break up clots and prevent more from forming, VTE can restrict or block blood flow and oxygen, which can damage the body tissue or organs. VTE can occur without any obvious signs, and imaging technologies are used. Alternatively rapid measurement of thrombin generation (TG) and D-dimer could be used to make a fast, portable, and easy-to-use diagnostic platform for VTE. Here, we have demonstrated a diagnostic sensing platform with the ability of simultaneous detection of TG and D-dimer in human plasma. Modifications were made to both the assay protocols to eliminate the need for sample dilution and incubation steps. Using a substantially reduced sample volume, the measurement results show comparable performance to the gold standard method. Our platform is able to deliver accurate and cost-effective results for both TG and D-dimer assays when using undiluted plasma in under 15 min. The assays presented are therefore a good candidate technology for use in a point-of-care platform to diagnose VTE.

Prognostic value of an automated thrombin generation assay in COVID-19 patients entering hospital: A multicentric, prospective observational study.

INTRODUCTION: The prognostic significance of the thrombin generation assay (TGA) with a thrombomodulin (TM) challenge in patients entering hospital with severe COVID-19 is uncertain. METHODS: We prospectively evaluated an automated TGA (aTGA) using the ST-ThromboScreen® assay and ST-Genesia® analyser in 179 patients with severe COVID-19 during their admission to 2 university hospitals. The primary outcome was early survival at Day 28 (D28). Secondary outcomes were late survival at Day 90 (D90), later transfer to an intensive care unit (ICU), and occurrence of any thrombotic complications during hospitalisation. RESULTS: Among the 174 patients, 50 were initially admitted to ICUs. Forty-two were transferred to ICUs before D28. Fourteen patients, all in ICUs, died before D28, and 20 before D90, all but 1 in ICUs. None of the aTGA-derived results were associated with vital status either at D28 or D90. Nine patients had a thrombotic event with no association with the aTGA results. Later transfer to the ICU was associated with higher velocity index, thrombin peak height and endogenous thrombin potential (ETP) values of the aTGA performed with TM, and mainly with a lower TM-induced decrease in ETP (odds ratio 15.5 (2.15-132), p = 0.009). CONCLUSIONS: aTGA, a global assay supposed to evidence coagulopathy, could predict neither early or late survival, nor thrombotic events, in hospitalised COVID-19 patients. Its clinical justification in that setting is thus unlikely. A relative resistance of the ETP to TM was associated with later transfer to the ICU and deserves further investigation.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis.

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell-cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5'-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

A highly selective and sensitive competitive aptasensor capable of quantifying thrombin elevated with side effects in severe COVID-19.

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10-320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.

SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide.

SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.

Sustained VWF-ADAMTS-13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction.

BACKGROUND: Prolonged recovery is common after acute SARS-CoV-2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS-13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. PATIENTS AND METHODS: Fifty patients were reviewed at a minimum of 6 weeks following acute COVID-19. ADAMTS-13, Weibel Palade Body (WPB) proteins, and angiogenesis-related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID-19 patients (n = 36). RESULTS: ADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. CONCLUSION: Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.

Hypercoagulability, endotheliopathy, and inflammation approximating 1 year after recovery: Assessing the long-term outcomes in COVID-19 patients.

Sustained hypercoagulability and endotheliopathy are present in convalescent COVID-19 patients for up to 4 months from recovery. The hemostatic, endothelial, and inflammatory profiles of 39 recovered COVID-19 patients were evaluated up to 16 months after recovery from COVID-19. These values were compared with a control group of healthy volunteers (n = 124). 39 patients (71.8% males, median age 43 years) were reviewed at a mean of 12.7 ± 3.6 months following recovery. One patient without cardiovascular risk factors had post COVID-19 acute ischaemic limb. Elevated D-dimer and Factor VIII levels above normal ranges were noted in 17.9% (7/39) and 48.7% (19/39) of patients respectively, with a higher median D-dimer 0.34 FEU µg/mL (IQR 0.28, 0.46) (p < .001) and Factor VIII 150% (IQR 171, 203) (p = .004), versus controls. Thrombin generation (Thromboscreen) showed a higher median endogenous thrombin potential (ETP) of 1352 nM*min (IQR 1152, 1490) (p = .002) and a higher median peak height of 221.4 nM (IQR 170.2, 280.4) (p = 0.01) and delayed lag time 2.4 min (1.42-2.97) (p = 0.0002) versus controls. Raised vWF:Ag and ICAM-1 levels were observed in 17.9% (7/39) and 7.7% (3/39) of patients respectively, with a higher median VWF:Ag 117% (IQR 86, 154) (p = 0.02) and ICAM-1 54.1 ng/mL (IQR 43.8, 64.1) (p = .004) than controls. IL-6 was noted to be raised in 35.9% (14/39) of patients, with a higher median IL-6 of 1.5 pg/mL (IQR 0.6, 3.0) (p = 0.004) versus controls. Subgroup analysis stratifying patients by COVID-19 severity and COVID-19 vaccination preceding SARS-CoV-2 infection did not show statistically significant differences. Hypercoagulability, endothelial dysfunction, and inflammation are still detectable in some patients approximately 1 year after recovery from COVID-19.

Outcomes With Direct and Indirect Thrombin Inhibition During Extracorporeal Membrane Oxygenation for COVID-19.

Anticoagulation during extracorporeal membrane oxygenation (ECMO) for Coronovirus Disease 2019 (COVID-19) can be performed by direct or indirect thrombin inhibitors but differences in outcomes with these agents are uncertain. A retrospective, multicenter study was conducted. All consecutive adult patients with COVID-19 placed on ECMO between March 1, 2020 and April 30, 2021 in participating centers, were included. Patients were divided in groups receiving either a direct thrombin inhibitor (DTI) or an indirect thrombin inhibitor such as unfractionated heparin (UFH). Overall, 455 patients with COVID-19 from 17 centers were placed on ECMO during the study period. Forty-four patients did not receive anticoagulation. Of the remaining 411 patients, DTI was used in 160 (39%) whereas 251 (61%) received UFH. At 90-days, in-hospital mortality was 50% (DTI) and 61% (UFH), adjusted hazard ratio: 0.81, 95% confidence interval (CI): 0.49-1.32. Deep vein thrombosis [adjusted odds ratio (aOR): 2.60, 95% CI: 0.90-6.65], ischemic (aOR: 1.58, 95% CI: 0.18-14.0), and hemorrhagic (aOR:1.22, 95% CI: 0.39-3.87) stroke were similar with DTI in comparison to UFH. Bleeding requiring transfusion was lower in patients receiving DTI (aOR: 0.40, 95% CI: 0.18-0.87). Anticoagulants that directly inhibit thrombin are associated with similar in-hospital mortality, stroke, and venous thrombosis and do not confer a higher risk of clinical bleeding in comparison to conventional heparin during ECMO for COVID-19.

Nanomaterial-Based Label-Free Electrochemical Aptasensors for the Detection of Thrombin.

Thrombin plays a central role in hemostasis and its imbalances in coagulation can lead to various pathologies. It is of clinical significance to develop a fast and accurate method for the quantitative detection of thrombin. Electrochemical aptasensors have the capability of combining the specific selectivity from aptamers with the extraordinary sensitivity from electrochemical techniques and thus have attracted considerable attention for the trace-level detection of thrombin. Nanomaterials and nanostructures can further enhance the performance of thrombin aptasensors to achieve high sensitivity, selectivity, and antifouling functions. In highlighting these material merits and their impacts on sensor performance, this paper reviews the most recent advances in label-free electrochemical aptasensors for thrombin detection, with an emphasis on nanomaterials and nanostructures utilized in sensor design and fabrication. The performance, advantages, and limitations of those aptasensors are summarized and compared according to their material structures and compositions.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Combining Heparin and a FX/Xa Aptamer to Reduce Thrombin Generation in Cardiopulmonary Bypass and COVID-19.

Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. We take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets factor X/Xa, with UFH (or low molecular weight heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. This aptamer-UFH combination (1) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, (2) allows for a reduced level of UFH to be employed, (3) more effectively limits thrombin generation compared to UFH alone, and (4) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation.

Assessing Clinically Meaningful Hypercoagulability after COVID-19 Vaccination: A Longitudinal Study.

A large number of daily requests to exclude possible prothrombotic risk factors for coronavirus disease 2019 (COVID-19) vaccines were received. Our aim was to longitudinally evaluate coagulation profiles in a series of healthy subjects who received COVID-19 vaccination and assess hypercoagulability thereafter. Volunteers awaiting a first or second dose of either the ChAdOx1 or BNT162b2 vaccine were enrolled. Venous samples were obtained at baseline (before the vaccine) and longitudinally 3 ± 2 days (T1) and 10 ± 2 days after the vaccine (T2). Global coagulation monitoring was assessed via platelet count, whole blood thromboelastometry and impedance aggregometry, plasma thrombin generation, and anti-platelet factor 4 (PF4)/heparin immunoglobulin G antibodies. One hundred and twenty-two subjects were enrolled (61 [50%] ChAdOx1 and 61 BNT162b2). The ChAdOx1 cohort showed a slight but transient increase in thrombin generation (mainly endogenous thrombin potential [ETP] with thrombomodulin and ETP ratio) at T1, which promptly decreased at T2. In addition, the second dose of either vaccine was associated with increased thrombin peak, ETP with thrombomodulin, and ETP ratio. At baseline, 3.2% of the ChAdOx1 cohort and 1.6% BNT162b2 cohort were positive for PF4/heparin antibodies with a stable titer through T1 and T2. No relevant differences were detected in platelet count and aggregation, or thromboelastometry parameters. No thrombotic or hemorrhagic events occurred. We can confirm that no clinically meaningful hypercoagulability occurred after either vaccine, albeit keeping in mind that thrombin generation may increase in the first days after the second dose of either vaccine and after the first dose of the ChAdOx1 vaccine.

Inflammation and Platelet Activation After COVID-19 Vaccines - Possible Mechanisms Behind Vaccine-Induced Immune Thrombocytopenia and Thrombosis.

Introduction of vaccines against COVID-19 has provided the most promising chance to control the world-wide COVID-19 pandemic. However, the adenovirus-vector based Oxford/AstraZeneca [ChAdOx1] (AZ) and Johnson & Johnson [Ad26.CoV2.S] COVID-19 vaccines have been linked with serious thromboembolic events combined with thrombocytopenia, denominated Vaccine-induced Immune Thrombocytopenia and Thrombosis (VITT). The pathogenesis of COVID-19 VITT remain incompletely understood; especially the initial events that trigger platelet activation, platelet factor (PF)4 release, complex formation and PF4 antibody production are puzzling. This is a prospective study investigating the impact of different COVID-19 vaccines on inflammation (CRP, TNF-α, IL-1ß, IL-6, IL-8, IL-10), vascular endothelial activation (syndecan-1, thrombomodulin, E-selectin, ICAM-1, ICAM-3, VCAM-1), platelet activation (P-selectin, TGF-ß, sCD40L) and aggregation (Multiplate® impedance aggregometry), whole blood coagulation (ROTEM®), thrombin generation and PF4 antibodies to reveal potential differences between AZ and mRNA vaccines in individuals without VITT. The study included 80 (55 AZ and 55 mRNA) vaccinated individuals and 55 non-vaccinated age- and gender matched healthy controls. The main findings where that both vaccines enhanced inflammation and platelet activation, though AZ vaccination induced a more pronounced increase in several inflammatory and platelet activation markers compared to mRNA vaccination and that post-vaccination thrombin generation was higher following AZ vaccination compared to mRNA vaccination. No difference in neither the PF4 antibody level nor the proportion of individuals with positive PF4 antibodies were observed between the vaccine groups. This is the first study to report enhanced inflammation, platelet activation and thrombin generation following AZ vaccination compared to mRNA vaccination in a head-to-head comparison. We speculate that specific components of the AZ adenovirus vector may serve as initial trigger(s) of (hyper)inflammation, platelet activation and thrombin generation, potentially lowering the threshold for a cascade of events that both trigger complications related to excessive inflammation, platelet and coagulation activation as observed in epidemiological studies and promote development of VITT when combined with high-titer functionally active PF4 antibodies.

Increased histone-DNA complexes and endothelial-dependent thrombin generation in severe COVID-19.

Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Circulating histone-DNA complexes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). Using calibrated automated thrombography, thrombin generation was altered in COVID-19 patient plasma samples. Despite having increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. While healthy control plasma samples did not generate measurable thrombin after 60 min, plasma samples from COVID-19+ patients formed thrombin (mean lag time ~20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation may contribute to coagulopathy in COVID-19.

Clinical use of thrombin generation assays.

Determining patient's coagulation profile, i.e. detecting a bleeding tendency or the opposite, a thrombotic risk, is crucial for clinicians in many situations. Routine coagulation assays and even more specialized tests may not allow a relevant characterization of the hemostatic balance. In contrast, thrombin generation assay (TGA) is a global assay allowing the dynamic continuous and simultaneous recording of the combined effects of both thrombin generation and thrombin inactivation. TGA thus reflects the result of procoagulant and anticoagulant activities in blood and plasma. Because of this unique feature, TGA has been widely used in a wide array of settings from both research, clinical and pharmaceutical perspectives. This includes diagnosis, prognosis, prophylaxis, and treatment of inherited and acquired bleeding and thrombotic disorders. In addition, TGA has been shown to provide relevant information for the diagnosis of coagulopathies induced by infectious diseases, comprising also disturbance of the coagulation system in COVID-19, or for the assessment of early recurrence in breast cancer. This review article aims to document most clinical applications of TGA.