Current and potentially novel antithrombotic treatment in acute ischemic stroke.

Acute ischemic stroke (AIS) is the most common type of stroke and requires immediate reperfusion. Current acute reperfusion therapies comprise the administration of intravenous thrombolysis and/or endovascular thrombectomy. Although these acute reperfusion therapies are increasingly successful, optimized secondary antithrombotic treatment remains warranted, specifically to reduce the risk of major bleeding complications. In the development of AIS, coagulation and platelet activation play crucial roles by driving occlusive clot formation. Recent studies implicated that the intrinsic route of coagulation plays a more prominent role in this development, however, this is not fully understood yet. Next to the acute treatments, antithrombotic therapy, consisting of anticoagulants and/or antiplatelet therapy, is successfully used for primary and secondary prevention of AIS but at the cost of increased bleeding complications. Therefore, better understanding the interplay between the different pathways involved in the pathophysiology of AIS might provide new insights that could lead to novel treatment strategies. This narrative review focuses on the processes of platelet activation and coagulation in AIS, and the most common antithrombotic agents in primary and secondary prevention of AIS. Furthermore, we provide an overview of promising novel antithrombotic agents that could be used to improve in both acute treatment and stroke prevention.

The intrinsic coagulation pathway plays a dominant role in driving hypercoagulability in ANCA-associated vasculitis.

ABSTRACT: The risk of a venous thrombotic event (VTE) is increased in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); however, a detailed understanding of the underlying mechanisms of hypercoagulability is limited. We assessed prospectively different coagulation parameters in 71 patients with active AAV at baseline and after 6 months of follow-up. D-dimers and fibrinogen were increased in most patients at presentation and remained elevated in half of the patients. Particularly, thrombin-antithrombin (T:AT) complex and activated coagulation factors in complex with their natural inhibitors of the intrinsic coagulation pathway (ie, activated FXII:C1 esterase inhibitor [FXIIa:C1Inh], FXIa:AT, and FXIa:alpha1-antitrypsin [FXIa:α1AT]) were profoundly elevated in patients at baseline. Thrombin formation was dominantly correlated with coagulation factors of the intrinsic pathway (ie, FXIIa:AT, FXIa:AT, FXIa:α1AT, and FXIa:C1Inh) compared to the extrinsic pathway (ie, FVIIa:AT). Hypercoagulability correlated with higher disease activity, ANCA levels, C-reactive protein, serum creatinine, and proteinuria. VTEs were observed in 5 out of 71 (7%) patients within 1 month (interquartile range, 1-5) after inclusion. Baseline T:AT levels were significantly higher in patients with VTE than in those without VTE (P = .044), but other clinical or laboratory markers were comparable between both groups. Hypercoagulability is dominantly characterized by activation of the intrinsic coagulation pathway and elevated D-dimers in active AAV. The driving factors of hypercoagulability are yet to be studied but are most likely related to an interplay of increased disease activity, vascular inflammation, and endothelial damage. Future targets for intervention could include inhibitors of the intrinsic coagulation pathway and compounds specifically reducing the hyperinflammatory state.

Platelet biology and function: plaque erosion vs. rupture.

The leading cause of heart disease in developed countries is coronary atherosclerosis, which is not simply a result of ageing but a chronic inflammatory process that can lead to acute clinical events upon atherosclerotic plaque rupture or erosion and arterial thrombus formation. The composition and location of atherosclerotic plaques determine the phenotype of the lesion and whether it is more likely to rupture or to erode. Although plaque rupture and erosion both initiate platelet activation on the exposed vascular surface, the contribution of platelets to thrombus formation differs between the two phenotypes. In this review, plaque phenotype is discussed in relation to thrombus composition, and an overview of important mediators (haemodynamics, matrix components, and soluble factors) in plaque-induced platelet activation is given. As thrombus formation on disrupted plaques does not necessarily result in complete vessel occlusion, plaque healing can occur. Therefore, the latest findings on plaque healing and the potential role of platelets in this process are summarized. Finally, the clinical need for more effective antithrombotic agents is highlighted.

Higher levels of circulating desphospho-uncarboxylated matrix Gla protein over time are associated with worse survival: the prospective Maastricht Intensive Care COVID cohort.

BACKGROUND: Extra-hepatic vitamin K-status, measured by dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP), maintains vascular health, with high levels reflecting poor vitamin K status. The occurrence of extra-hepatic vitamin K deficiency throughout the disease of COVID-19 and possible associations with pulmonary embolism (PE), and mortality in intensive care unit (ICU) patients has not been studied. The aim of this study was to investigated the association between dp-ucMGP, at endotracheal intubation (ETI) and both ICU and six months mortality. Furthermore, we studied the associations between serially measured dp-ucMGP and both PE and mortality. METHODS: We included 112 ICU patients with confirmed COVID-19. Over the course of 4 weeks after ETI, dp-ucMGP was measured serially. All patients underwent computed tomography pulmonary angiography (CTPA) to rule out PE. Results were adjusted for patient characteristics, disease severity scores, inflammation, renal function, history of coumarin use, and coronary artery calcification (CAC) scores. RESULTS: Per 100 pmol/L dp-ucMGP, at ETI, the odds ratio (OR) was 1.056 (95% CI: 0.977 to 1.141, p = 0.172) for ICU mortality and 1.059 (95% CI: 0.976 to 1.059, p = 0.170) for six months mortality. After adjustments for age, gender, and APACHE II score, the mean difference in plasma dp-ucMGP over time of ICU admission was 167 pmol/L (95% CI: 4 to 332, p = 0.047). After additional adjustments for c-reactive protein, creatinine, and history of coumarin use, the difference was 199 pmol/L (95% CI: 50 to 346, p = 0.010). After additional adjustment for CAC score the difference was 213 pmol/L (95% CI: 3 to 422, p = 0.051) higher in ICU non-survivors compared to the ICU survivors. The regression slope, indicating changes over time, did not differ. Moreover, dp-ucMGP was not associated with PE. CONCLUSION: ICU mortality in COVID-19 patients was associated with higher dp-ucMGP levels over 4 weeks, independent of age, gender, and APACHE II score, and not explained by inflammation, renal function, history of coumarin use, and CAC score. No association with PE was observed. At ETI, higher levels of dp-ucMGP were associated with higher OR for both ICU and six month mortality in crude and adjusted modes, although not statistically significantly.

Biomarkers of sustained systemic inflammation and microvascular dysfunction associated with post-COVID-19 condition symptoms at 24 months after SARS-CoV-2-infection.

Introduction: Comprehensive studies investigating sustained hypercoagulability, endothelial function, and/or inflammation in relation to post-COVID-19 (PCC) symptoms with a prolonged follow-up are currently lacking. Therefore, the aim of this single-centre cohort study was to investigate serum biomarkers of coagulation activation, microvascular dysfunction, and inflammation in relation to persisting symptoms two years after acute COVID-19. Methods: Patients diagnosed with acute SARS-CoV-2 infection between February and June 2020 were recruited. Outcome measures included the CORona Follow-Up (CORFU) questionnaire, which is based on an internationally developed and partially validated basic questionnaire on persistent PCC symptoms. Additionally, plasma biomarkers reflecting coagulation activation, endothelial dysfunction and systemic inflammation were measured. Results: 167 individuals were approached of which 148 (89%) completed the CORFU questionnaire. At 24 months after acute infection, fatigue was the most prevalent PCC symptom (84.5%). Over 50% of the patients experienced symptoms related to breathing, cognition, sleep or mobility; 30.3% still experienced at least one severe or extreme (4 or 5 on a 5-point scale) PCC symptom. Multiple correlations were found between several PCC symptoms and markers of endothelial dysfunction (endothelin-1 and von Willebrand factor) and systemic inflammation (Interleukin-1 Receptor antagonist). No positive correlations were found between PCC symptoms and coagulation complexes. Discussion: In conclusion, this study shows that at 24 months after acute COVID-19 infection patients experience a high prevalence of PCC symptoms which correlate with inflammatory cytokine IL-1Ra and markers of endothelial dysfunction, especially endothelin-1. Our data may provide a rationale for the selection of treatment strategies for further clinical studies. Trial registration: This study was performed in collaboration with the CORona Follow-Up (CORFU) study (NCT05240742, https://clinicaltrials.gov/ct2/show/ NCT05240742).

Tissue factor expression in monocyte subsets during human immunothrombosis, endotoxemia and sepsis.

INTRODUCTION: Tissue factor expression on monocytes is implicated in the pathophysiology of sepsis-induced coagulopathy. How tissue factor is expressed by monocyte subsets (classical, intermediate and non-classical) is unknown. METHODS: Monocytic tissue factor surface expression was investigated during three conditions. Primary human monocytes and microvascular endothelial cell co-cultures were used for in vitro studies. Volunteers received a bolus of lipopolysaccharide (2 ng/kg) to induce endotoxemia. Patients with sepsis, or controls with critical illness unrelated to sepsis, were recruited from four intensive care units. RESULTS: Contact with endothelium and stimulation with lipopolysaccharide reduced the proportion of intermediate monocytes. Lipopolysaccharide increased tissue factor surface expression on classical and non-classical monocytes. Endotoxemia induced profound, transient monocytopenia, along with activation of coagulation pathways. In the remaining circulating monocytes, tissue factor was up-regulated in intermediate monocytes, though approximately 60 % of individuals (responders) up-regulated tissue factor across all monocyte subsets. In critically ill patients, tissue factor expression on intermediate and non-classical monocytes was significantly higher in patients with established sepsis than among non-septic patients. Upon recovery of sepsis, expression of tissue factor increased significantly in classical monocytes. CONCLUSION: Tissue factor expression in monocyte subsets varies significantly during health, endotoxemia and sepsis.

Plasma Kallikrein as a Forgotten Clotting Factor.

For decades, it was considered that plasma kallikrein's (PKa) sole function within the coagulation cascade is the activation of factor (F)XII. Until recently, the two key known activators of FIX within the coagulation cascade were activated FXI(a) and the tissue factor-FVII(a) complex. Simultaneously, and using independent experimental approaches, three groups identified a new branch of the coagulation cascade, whereby PKa can directly activate FIX. These key studies identified that (1) FIX or FIXa can bind with high affinity to either prekallikrein (PK) or PKa; (2) in human plasma, PKa can dose dependently trigger thrombin generation and clot formation independent of FXI; (3) in FXI knockout murine models treated with intrinsic pathway agonists, PKa activity results in increased formation of FIXa:AT complexes, indicating direct activation of FIX by PKa in vivo. These findings suggest that there is both a canonical (FXIa-dependent) and non-canonical (PKa-dependent) pathway of FIX activation. These three recent studies are described within this review, alongside historical data that hinted at the existence of this novel role of PKa as a coagulation clotting factor. The implications of direct PKa cleavage of FIX remain to be determined physiologically, pathophysiologically, and in the context of next-generation anticoagulants in development.

Predictive value for increased activated factor XI activity in acute venous thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) is associated with excessive coagulation activity, which in part can be attributed to activation of contact system. However, the knowledge regarding the impact of contact activation in acute VTE is limited. OBJECTIVE: To unravel the involvement of contact activation in acute VTE. METHODS: Contact activation was investigated in patients with acute VTE (n = 321) and population controls without a history of VTE (n = 300). For comparison, Factor XI(a) levels, activity, and plasma kallikrein (PKa) activity were determined in plasma samples with an activated partial thromboplastin time- or thrombin generation-based assay (free FXI concentration [FXI:c] and calibrated automated thrombogram:FXIa, respectively) and with enzyme-linked immunosorbent assays for enzyme-inhibitor complexes (FXIa:alpha-1-antitrypsin [α1AT], FXIa:antithrombin [AT], FXIa:C1-inhibitor [C1Inh], and PKa:C1-inh). RESULTS: In patients with VTE, higher FXI:c levels (124 ± 37% vs 114 ± 28%), but lower calibrated automated thrombogram:FXIa levels were apparent. This was accompanied by increased FXIa:α1AT, FXIa:AT, and PKa:C1-inh levels in patients compared with controls (312pM [238-424] vs 203pM [144-288]; 29pM [23-38] vs 23pM [20-30]; 1.9nM [1.2-4.7] vs 1.4nM [0.7-3.5], respectively), whereas FXIa:C1-inh levels did not differ. Logistic regression models showed good discriminatory values for FXI:c and FXIa:α1AT (area under the curve = 0.64 [0.6/0.69] and 0.73 [0.69/0.77], respectively). After a 2-year follow-up, 81 recurrent VTE events or deaths occurred in the patient cohort, for which the baseline levels of FXIa:α1AT and FXIa:C1Inh had a significant prognostic value (Hazard ratios per SD [95% CI], 1.26 [1.10-1.45]; p =.0012 and 1.19 [1.05-1.36]; p =.0082, respectively). CONCLUSION: Our study revealed elevated FXIa levels and activity in acute VTE, which was also associated with recurrent VTE, suggesting an important risk contribution of FXI activation to VTE. The evidence provided by this study supports the utility of FXIa inhibition in the setting of acute VTE.

Clinical utility of the 4S-AF scheme in predicting progression of atrial fibrillation: data from the RACE V study.

AIMS: The recent 4S-AF (scheme proposed by the 2020 ESC AF guidelines to address stroke risk, symptom severity, severity of AF burden and substrate of AF to provide a structured phenotyping of AF patients in clinical practice to guide therapy and assess prognosis) scheme has been proposed as a structured scheme to characterize patients with atrial fibrillation (AF). We aimed to assess whether the 4S-AF scheme predicts AF progression in patients with self-terminating AF. METHODS AND RESULTS: We analysed 341 patients with self-terminating AF included in the well-phenotyped Reappraisal of Atrial Fibrillation: Interaction between HyperCoagulability, Electrical remodelling, and Vascular Destabilization in the Progression of AF (RACE V) study. Patients had continuous monitoring with implantable loop recorders or pacemakers. AF progression was defined as progression to persistent or permanent AF or progression of self-terminating AF with >3% burden increase. Progression of AF was observed in 42 patients (12.3%, 5.9% per year). Patients were given a score based on the components of the 4S-AF scheme. Mean age was 65 [interquartile range (IQR) 58-71] years, 149 (44%) were women, 103 (49%) had heart failure, 276 (81%) had hypertension, and 38 (11%) had coronary artery disease. Median CHA2DS2-VASc (the CHA2DS2-VASc score assesses thromboembolic risk. C, congestive heart failure/left ventricular dysfunction; H, hypertension; A2, age ≥ 75 years; D, diabetes mellitus; S2, stroke/transient ischaemic attack/systemic embolism; V, vascular disease; A, age 65-74 years; Sc, sex category (female sex)) score was 2 (IQR 2-3), and median follow-up was 2.1 (1.5-2.6) years. The average score of the 4S-AF scheme was 4.6 ± 1.4. The score points from the 4S-AF scheme did not predict the risk of AF progression [odds ratio (OR) 1.1 95% CI 0.88-1.41, C-statistic 0.53]. However, excluding the symptoms domain, resulting in the 3S-AF (4S-AF scheme without the domain symptom severity, only including stroke risk, severity of AF burden and substrate of AF) scheme, predicted the risk of progression (OR 1.59 95% CI 1.15-2.27, C-statistic 0.62) even after adjusting for sex and age. CONCLUSIONS: In self-terminating AF patients, the 4S-AF scheme does not predict AF progression. The 3S-AF scheme, excluding the symptom domain, may be a more appropriate score to predict AF progression. TRIAL REGISTRATION NUMBERS: Clinicaltrials.gov NCT02726698 for RACE V.

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.

Integrating Mechanisms in Thrombotic Peripheral Arterial Disease.

Peripheral arterial disease (PAD), a manifestation of systemic atherosclerosis, is underdiagnosed in the general population. Despite the extensive research performed to unravel its pathophysiology, inadequate knowledge exists, thus preventing the development of new treatments. This review aims to highlight the essential elements of atherosclerosis contributing to the pathophysiology of PAD. Furthermore, emphasis will be placed on the role of thrombo-inflammation, with particular focus on platelet and coagulation activation as well as cell-cell interactions. Additional insight will be then discussed to reveal the contribution of hypercoagulability to the development of vascular diseases such as PAD. Lastly, the current antithrombotic treatments will be discussed, and light will be shed on promising new targets aiming to aid the development of new treatments.

Lower levels of vWF are associated with lower risk of cardiovascular disease.

Objective: The current study was undertaken to prospectively explore whether having low levels of von Willebrand factor (vWF) antigen and vWF activity reduce the risk for cardiovascular disease and death. Methods: VWF antigen and vWF activity were measured by enzyme-linked immunosorbent assay and an immunological-based assay, respectively, in a subsample of 4857 individuals aged between 35 and 74 years old, enrolled between April 2007 and October 2008 in the population-based Gutenberg Health Study. VWF antigen and activity below the 20th percentile was set as a measure of "low vWF." Adjusted robust Poisson regression models were used to analyze the relation between low vWF and the incidence of cardiovascular disease (CVD). Consequent adjusted cox regression models as well as cumulative incidence plots were calculated to explore the relation between all-cause and cardiovascular mortality and low vWF. Results: VWF activity levels <20th percentile (i.e., <76.2%) were associated with a decreased relative risk for CVD (RR: 0.59, 95% CI: 0.37-0.95), despite adjusting for age and sex. After adjusting for levels of F-VIII, the association persisted (RR: 0.60, 95% CI: 0.36-0.99). The cumulative incidence plots demonstrated that vWF antigen <20th percentile significantly correlated with decreased cardiovascular mortality. VWF antigen<20th percentile (i.e., <83%) was significantly associated with lower risk of all-cause mortality, despite adjusting for clinical factors (RR: 61, 95% CI: 0.41-0.91). Conclusion: The study demonstrated that having low vWF activity levels were associated with a lower risk for CVD. Additionally, it revealed a decreased risk of cardiovascular and all-cause mortality in individuals with low levels of vWF antigen, shining new light on vWF as a potential target for novel therapies.

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.

Subtype-specific plasma signatures of platelet-related protein releasate in acute pulmonary embolism.

INTRODUCTION: There is evidence that plasma protein profiles differ in the two subtypes of pulmonary embolism (PE), isolated PE (iPE) and deep vein thrombosis (DVT)-associated PE (DVT-PE), in the acute phase. The aim of this study was to determine specific plasma signatures for proteins related to platelets in acute iPE and DVT-PE compared to isolated DVT (iDVT). METHODS: Within the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism (GMP-VTE) Project, a multicenter prospective cohort study of 693 confirmed VTE cases, a highly sensitive targeted proteomics approach based on dual-antibody proximity extension assay was applied. LASSO-regularized logistic regression analysis selected 33 and 30 of 135 platelet-related candidate proteins in iPE and DVT-PE vs. iDVT, respectively. RESULTS: All regulated proteins were well associated with six prominently released platelet proteins and the majority showed specificity for iPE and DVT-PE compared to iDVT. While iPE-specific proteins were assigned to be predominantly released via shedding mechanisms and extracellular vesicles, granule secretion was identified as a major release mechanism assigned to DVT-associated PE-specific proteins. Network analysis demonstrated three interconnected clusters of specifically regulated proteins in iPE linked to immunoreceptor signaling, pathogen clearance and chemotaxis, whereas for DVT-associated PE one cluster linked to tissue remodeling and leukocyte trafficking. Machine learning-based analysis reveals specific plasma signatures and differential release mechanisms of proteins related to platelets in acute iPE and DVT-associated PE. CONCLUSION: These data suggest that the platelet protein releasate contributes to the differential regulation of plasma proteins in acute PE compared to iDVT, which may be associated with different platelet activation patterns.

Dual pathway inhibition as compared to acetylsalicylic acid monotherapy in relation to endothelial function in peripheral artery disease, a phase IV clinical trial.

Objective: Dual pathway inhibition (DPI) by combining acetylsalicylic acid (ASA) with low-dose rivaroxaban has been shown to reduce cardiovascular events in patients with peripheral arterial disease (PAD) when compared to ASA monotherapy. A potential explanation is that inhibition of factor Xa improves endothelial function through crosstalk between coagulation and inflammatory pathways, subsequently attenuating the occurrence of cardiovascular events. We hypothesize that the addition of rivaroxaban to ASA in PAD patients leads to improved endothelial function. Design: An investigator-initiated, multicentre trial investigating the effect of DPI on endothelial function. Methods: Patients, diagnosed with PAD, were enrolled in two cohorts: cohort A (Rutherford I-III) and cohort B (Rutherford IV-VI). Participants received ASA monotherapy for a 4-weeks run-in period, followed by 12 weeks of DPI. Macro- and microvascular endothelial dysfunction were studied by measuring carotid artery reactivity upon sympathetic stimulus and by measuring plasma endothelin-1 concentrations, respectively. All measurements were performed during the use of ASA (baseline) and after 12 weeks of DPI. Results: 159 PAD patients (111 cohort A, 48 cohort B) were enrolled. Twenty patients discontinued study drugs early. Carotid artery constriction upon sympathetic stimulation at baseline (ASA) and after 12 weeks of DPI was similar in the total group, 22.0 vs. 22.7% (p = 1.000), and in the subgroups (Cohort A 22.6 vs. 23.7%, p = 1.000; cohort B 20.5 vs. 20.5%, p = 1.000), respectively. The mean concentration of plasma endothelin-1 at baseline and after 12 weeks of DPI did not differ, 1.70 ± 0.5 vs. 1.66 ± 0.64 pmol/L (p = 0.440) in the total group, 1.69 ± 0.59 vs. 1.62 ± 0.55 pmol/L in cohort A (p = 0.202), and 1.73 ± 0.53 vs. 1.77 ± 0.82 pmol/L in cohort B (p = 0.682), respectively. Conclusion: Macro- and microvascular endothelial dysfunction, as reflected by carotid artery reactivity and plasma endothelin-1 concentrations, are not influenced in PAD patients by addition of low-dose rivaroxaban to ASA monotherapy for 12 weeks. Trial registration: https://clinicaltrials.gov/ct2/show/NCT04218656.

Relationships between coagulation factors and thrombin generation in a general population with arterial and venous disease background.

BACKGROUND: The current study aims to identify the relationships between coagulation factors and plasma thrombin generation in a large population-based study by comparing individuals with a history of arterial or venous thrombosis to cardiovascular healthy individuals. METHODS: This study comprised 502 individuals with a history of arterial disease, 195 with history of venous thrombosis and 1402 cardiovascular healthy individuals (reference group) from the population-based Gutenberg Health Study (GHS). Calibrated Automated Thrombography was assessed and coagulation factors were measured by means of BCS XP Systems. To assess the biochemical determinants of TG variables, a multiple linear regression analysis, adjusted for age, sex and antithrombotic therapy, was conducted. RESULTS: The lag time, the time to form the first thrombin, was mainly positively associated with the natural coagulant and anti-coagulant factors in the reference group, i.e. higher factors result in a longer lag time. The same determinants were negative for individuals with a history of arterial or venous thrombosis, with a 10 times higher effect size. Endogenous thrombin potential, or area under the curve, was predominantly positively determined by factor II, VIII, X and IX in all groups. However, the effect sizes of the reported associations were 4 times higher for the arterial and venous disease groups in comparison to the reference group. CONCLUSION: This large-scale analysis demonstrated a stronger effect of the coagulant and natural anti-coagulant factors on the thrombin potential in individuals with a history of arterial or venous thrombosis as compared to healthy individuals, which implicates sustained alterations in the plasma coagulome in subjects with a history of thrombotic vascular disease, despite intake of antithrombotic therapy.

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia.

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.

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.