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Background: A significant proportion of patients with peripheral artery disease (PAD) displays a poor response to aspirin and/or the platelet P2Y 12 receptor antagonist clopidogrel. This phenomenon is reflected by high on-treatment platelet reactivity (HTPR) in platelet function assays in vitro and is associated with an increased risk of adverse cardiovascular events., Objective: This study aimed to elucidate specific plasma protein signatures associated with HTPR to aspirin and clopidogrel in PAD patients., Methods and Results: Based on targeted plasma proteomics, 184 proteins from two cardiovascular Olink panels were measured in 105 PAD patients. VerifyNow ASPI- and P2Y 12 -test values were transformed to a continuous variable representing HTPR as a spectrum instead of cut-off level-defined HTPR. Using the Boruta random forest algorithm, the importance of 3 plasma proteins for HTPR in the aspirin, six in clopidogrel and 10 in the pooled group (clopidogrel or aspirin) was confirmed. Network analysis demonstrated clusters with CD84, SLAMF7, IL1RN and THBD for clopidogrel and with F2R, SELPLG, HAVCR1, THBD, PECAM1, TNFRSF10B, MERTK and ADM for the pooled group. F2R, TNFRSF10B and ADM were higher expressed in Fontaine III patients compared to Fontaine II, suggesting their relation with PAD severity., Conclusions: A plasma protein signature, including eight targets involved in proatherogenic dysfunction of blood cell-vasculature interaction, coagulation and cell death, is associated with HTPR (aspirin and/or clopidogrel) in PAD. This may serve as important systems-based determinants of poor platelet responsiveness to aspirin and/or clopidogrel in PAD and other cardiovascular diseases and may contribute to identify novel treatment strategies., Competing Interests: Declaration of competing interest SH is an employee of Bayer AG. The study was sponsored inter alia by Bayer AG. The sponsors had no role in the design or conduct of the research. HtC received research funding outside the present study from Bayer and received outside the present study honoraria for consultation and/or advisory board participation, from Bayer, Alveron, Galapagos, Portola and Alexion. All reimbursements were transferred to the CARIM institute. HtC and HMHS are shareholders with Coagulation Profile, a university spinoff small diagnostic company not involved in the present study. PSW has received research funding outside the present study from Boehringer Ingelheim, Sanofi-Aventis, Bayer Healthcare, Daiichi Sankyo Europe and Novartis and received outside the present study honoraria for lectures or consulting from Boehringer Ingelheim, Bayer HealthCare, Evonik, AstraZeneca and Sanofi-Aventis. PSW is principal investigator of the DIASyM research core (BMBF 161L0217A). All other authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Residual venous obstruction (RVO) after deep vein thrombosis (DVT) is considered a risk factor of recurrent venous thromboembolism (VTE), arterial events and post-thrombotic syndrome (PTS). We hypothesized thrombo-inflammatory markers might be associated with RVO and clinical outcomes., Materials and Methods: In a DVT cohort with routine RVO-assessment and 5-year follow-up, patients were invited for blood withdrawal after stopping anticoagulants. Thrombin generation potential, coagulation enzyme:inhibitor complexes, soluble platelet markers and clinical markers were measured in platelet-poor plasma. Associations were represented as odds ratio (OR) or hazard ratio (HR) per standard deviation., Results: Patients with RVO (102/306, 33 %) had higher rates of PTS (24 vs. 12 %, p = 0.008), but similar rates of recurrence (16 vs. 15 %, p = 0.91) and arterial events (7 vs. 4 %, p = 0.26). RVO was associated with thrombin peak height (OR 1.40 [1.04-1.88]), endogenous thrombin potential (ETP, OR 1.35 [1.02-1.79]), and CRP (OR 1.74 [1.10-2.75]). Recurrent VTE was associated with ETP (HR 1.36 [1.03-1.81]), FXIa:C 1 -inhibitor (HR 1.34 [1.04-1.72]), thrombin:antithrombin (HR 1.36 [1.16-1.59]), soluble P-selectin (HR 2.30 [1.69-3.11]), soluble glycoprotein VI (sGPVI, HR 1.30 [1.01-1.69]), D-dimer (HR 1.56 [1.31-1.86]), and factor VIII (HR 1.44 [1.15-1.82]). Arterial events were associated with sGPVI (HR 1.80 [1.25-2.59]). PTS was not associated with any marker., Conclusions: Our findings indicate RVO was associated with thrombo-inflammation, but this did not predict clinical outcomes in this setting. Importantly, we found recurrent VTE was associated with ongoing coagulation and platelet activation in patients well beyond the acute phase of DVT. Furthermore, sGPVI indicated an increased risk of arterial events, highlighting the role of platelets in arterial thrombosis following DVT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Endothelial damage and thrombosis caused by COVID-19 may imperil cardiovascular health. More than a year since the WHO declared COVID-19 pandemic, information on its effects beyond the acute phase is lacking. We investigate endothelial dysfunction, coagulation and inflammation, 3 months post-COVID-19., Materials and Methods: A cohort study was conducted including 203 patients with prior COVID-19. Macrovascular dysfunction was assessed by measuring the carotid artery diameter in response to hand immersion in ice-water. A historic cohort of 312 subjects served as controls. Propensity score matching corrected for baseline differences. Plasma concentrations of endothelin-1 were measured in patients post-COVID-19, during the acute phase, and in matched controls. Coagulation enzyme:inhibitor complexes and inflammatory cytokines were studied., Results and Conclusions: The prevalence of macrovascular dysfunction did not differ between the COVID-19 (18.6%) and the historic cohort (22.5%, RD -4%, 95%CI: -15-7, p = 0.49). Endothelin-1 levels were significantly higher in acute COVID-19 (1.67 ± 0.64 pg/mL) as compared to controls (1.24 ± 0.37, p < 0.001), and further elevated 3 months post-COVID-19 (2.74 ± 1.81, p < 0.001). Thrombin:antithrombin(AT) was high in 48.3%. Markers of contact activation were increased in 16-30%. FVIIa:AT (35%) and Von Willebrand Factor:antigen (80.8%) were elevated. Inflammatory cytokine levels were high in a majority: interleukin(IL)-18 (73.9%), IL-6 (47.7%), and IL-1ra (48.9%). At 3 months after acute COVID-19 there was no indication of macrovascular dysfunction; there was evidence, however, of sustained endothelial cell involvement, coagulation activity and inflammation. Our data highlight the importance of further studies on SARS-CoV-2 related vascular inflammation and thrombosis, as well as longer follow-up in recovered patients., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Cardiac arrest and cardiopulmonary resuscitation (CPR) are associated with activated coagulation and microvascular fibrin deposition with subsequent multiorgan failure and adverse outcome., Objectives: Activated Factor XI-antithrombin (FXIa-AT) complex, activated Factor IX-antithrombin (FIXa-AT) complex and thrombin-antithrombin (TAT) complex were measured as markers of coagulation activation, and evaluated as independent prognostic indicators in out-of-hospital cardiac arrest (OHCA) patients., Methods: From February 2007 until December 2010 blood samples were collected in close approximation to CPR from patients with OHCA of assumed cardiac origin. Follow-up samples in survivors were drawn 8-12 h and 24-48 h after hospital admission. All measurements were determined by ELISA., Results: Thirty-seven patients presented with asystole and 77 with ventricular fibrillation as first recorded heart rhythm. At 30-days follow-up, 70 patients (61.4%) had died. All patients had elevated levels of FXIa-AT complex, FIXa-AT complex and TAT. Initial levels were significantly higher in non-survivors compared to 30-days survivors. A significant increase in risk of 30-days all-cause mortality was observed through increasing quartiles of all three biomarkers in univariate Cox regression analysis. Compared to the lowest quartile (Q1), only FXIa-AT complex levels in Q3 (HR 3.17, p = 0.011) and Q2 (HR 3.02, p = 0.016) were independently associated with all-cause mortality in the multivariable analysis. FIXa-AT complex and TAT-complex did not behave as independent predictors., Conclusions: Complexes of FXIa-AT were independently associated with 30-days survival in OHCA-patients., Clinical Trial Registration: ClinicalTrials. gov, NCT02886273., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification., Methods: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems., Results: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma., Conclusion: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Blood coagulation comprises a complex cellular and molecular mechanism that maintains vascular integrity, protects against bleeding (hemostasis) and responds to injury. However, several elements of the coagulation system, including several coagulation factors and platelets, are also involved in other physiological and pathological processes. Tissue factor (TF) is a cell surface glycoprotein expressed in a vast variety of cell types and essential for hemostasis. Upon exposure of the TF-rich subendothelium to the blood stream, Factor VII (FVII) can bind to TF. TF subsequently facilitates the activation of FVII into activated FVII (FVIIa) thereby initiating the extrinsic coagulation pathway followed by the activation of FX and thrombin formation. Besides its hemostatic role in the vasculature, the TF:FVIIa pathway is active in many other compartments and organs where it can take part and mediate different physiological and pathological processes. The so-called non-hemostatic functions of TF:VIIa play a role in diverse processes such as inflammation, atherosclerosis and vascular and cardiac remodeling. This narrative review aims to reassess the most important and recent findings regarding the complex signaling pathways initiated by the TF:FVIIa complex, with an emphasis on the heart and blood vessels. Understanding how the mechanisms of TF:FVIIa signaling contribute to both physiological and pathological processes, is one of the keys to the development of new treatment strategies in cardiovascular disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: The advancements in cancer treatment and detection of early cancer have resulted in steady increase of adult cancer survivors over the years. However, due to the long term toxic effects of chemotherapy and radiotherapy, the incidence of cardiovascular diseases (CVD) is increasing in survivors. Identifying risk factors and interventions to reduce the excess burden of CVD in this vulnerable population is urgently needed., Aim: To investigate the cardiovascular risk factors (CVRFs), inflammation and coagulation profile in cancer survivors from a large population-based study., Materials and Methods: Presence of CVRFs and laboratory markers have been compared in individuals with (n=1,359) and without (n=13,626) history of cancer. Standard laboratory profile, including blood glucose and lipid profile, has been evaluated in 15,010 individuals from the Gutenberg Health Study (GHS). Coagulation factors, D-dimer and von Willebrand factor (vWF) activity were available in N=4,993., Results: The individuals with history of cancer were older compared to no history of cancer with mean age of 61,5years and 54.4years, respectively (p<0.001). Traditional CVRFs as diabetes (14% vs 8.8%), dyslipidemia (49.6% vs 43.7%) and hypertension (60.3 vs 48.7%) were more frequent whereas smoking was less frequent (14.5% vs 19.9%) in cancer survivors (p<0.001). The standard laboratory profile showed cancer survivors with lower erythrocyte, platelet and white blood cell counts and higher C-reactive protein (CRP), glucose, HbA1c and triglycerides levels (p<0.001). Multivariable logistic regression analysis adjusted for age, sex and CVRFs demonstrated an independent association with diabetes (odds ratio, OR: 1.24, 1.02-1.50; p=0.027) and higher CRP (OR: 1.01, 1.01-1.02; p=0.00071). Fibrinogen, FV, FVII, FVIII and FXI, D-dimer and vWF activity were higher in cancer survivors (p<0.001). Multivariable logistic regression confirmed an independent association with higher fibrinogen (OR: 1.002, 1.000-1.003) and vWF activity (OR: 1.005, 1.001-1.008)., Conclusions: This is the first study investigating CVRFs, inflammation and coagulation profile in individuals with history of cancer from a well characterized population-representative adult sample. It gives evidence for higher prevalence of CVRFs, particularly diabetes in this vulnerable population. Markers of inflammation as CRP and fibrinogen and vWF activity were higher in cancer survivors independent of the cardiovascular risk profile. These results underline the increased risk of CVD and need for development of cardio-oncology programs offering cardiovascular prevention., (© 2016 Elsevier Ltd. All rights reserved.)

Objective: One of the contributing mechanisms in acute myocardial infarction (AMI) is plasma hypercoagulability. Recently, it was suggested that factor XI activation might play a role in atherothrombosis. To quantify factor XIa plasma levels, we developed a new thrombin generation based assay and hypothesized that in AMI patients factor XIa levels are increased during the acute thrombotic event., Methods: A prospective cohort study was performed including 56 patients with first AMI. Blood was collected upon admission and after 6 months. Reference blood samples were obtained from 30 apparently healthy control subjects. Plasma samples were diluted (1:5) in factor XI deficient plasma and factor XIa plasma levels were established using a reference curve (0-12.5 pM factor XIa) and an inhibitory anti-factor XIa antibody. The established FXIa concentrations were related to the 1-year outcome., Results: Factor XIa plasma concentrations were significantly increased in AMI patients on admission compared to 6 months after the event (3.7 pM [2.7-5.5] vs. 2.8 [1.9-4.3], median ± IQR; P=0.001) and compared to healthy controls (3.7 pM [2.7-5.5] vs. 2.7 [1.6-4.2], median ± IQR; P=0.004). However, a high factor FXIa level at baseline was not significantly associated with a recurrent cardiovascular event (OR 1.26, 95%CI 0.33-4.7)., Conclusions: This study presents the first application of a new thrombin generation based factor XIa assay, showing significantly increased factor XIa levels in AMI patients on admission compared to 6 months after the event and compared to healthy controls. The factor XIa concentration was not associated with the risk of recurrence., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: There is limited knowledge on the etiology of post thrombotic syndrome (PTS), although several mechanisms have been proposed. The objectives are to explore the role of different pathogenic mechanisms for PTS, through measurement of an elaborate panel of biomarkers in patients with and without PTS., Materials and Methods: Patients with a history of deep vein thrombosis (DVT) with PTS (cases) and without PTS after minimal 2years follow-up (controls), were selected from the outpatient clinic of two Dutch hospitals. As a reference to the normal population healthy individuals (HI) without a history of venous thromboembolism were invited to participate. The population consisted of: 26 cases, 27 controls, and 26 HI. A panel of predefined biomarkers was measured in venous blood., Results: D-dimer showed a decreasing trend from cases to controls to HI; p=0.010. Thrombin/antithrombin complex levels were significantly higher in cases than in controls; p=0.032, and HI; p=0.017. APC-ratio was significantly lower in cases compared to controls; p=0.032, and HI; p=0.011. A significant trend of increasing proTAFI from cases, to controls, and HI; p=0.002 was found. There were no differences in inflammatory markers (CRP, Interleukin-6, Interleukin-8). Thrombomodulin, tissue-plasminogen activator, and von Willebrand factor were higher in patients compared to HI. There was a significant trend of decreasing sVCAM, from cases, to controls, and HI; p=0.029., Conclusions: Patients with PTS displayed increased coagulation activity, an altered pattern of fibrinolytic marker expression, and increased endothelial activation. We found no evidence of systemic inflammation in patients with PTS at 63months since the last DVT., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The thrombin generation test (TGT) has demonstrated utility in evaluating overall hemostatic capacity both in bleeding and thrombotic disorders. Although the test is currently well accepted as a research tool, its role in clinical practice has not yet been defined through large prospective multicenter clinical studies. Such prospective studies have been limited by the lack of official standardization of the assay and its large inter-laboratory variability. This international study assessed the intra- and inter-assay imprecision of TGT as well as the inter-centre variability of results in one US and four European centres. Contact-inhibited plasmas from six healthy volunteers, one mild haemophilia A patient, and five patients with heterozygous prothrombin G20210 mutation were assayed. We demonstrated that, using identical equipement, standardized reagents, a carefully selected reference plasma for normalization of results and the same test procedure as described in our DVD, the assay variability was highly reduced compared to previously published data. Our results emphasize the importance of preheating on TGT results and the variability of the assay. In conclusion, our data demonstrated that the standardized TGT methodology evaluated in this study effectively reduces the variability of the assay to acceptable limits and may be used in clinical trials., (Copyright © 2012 Elsevier Ltd. All rights reserved.)