Plasma protein signatures for high on-treatment platelet reactivity to aspirin and clopidogrel in peripheral artery disease.

BACKGROUND: A significant proportion of patients with peripheral artery disease (PAD) displays a poor response to aspirin and/or the platelet P2Y12 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 P2Y12-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.

Inadequate response to antiplatelet therapy in patients with peripheral artery disease: a prospective cohort study.

BACKGROUND: Patients with peripheral artery disease (PAD) are treated with preventive strategies to improve the cardiovascular risk. The incidence of cardiovascular events and mortality however remains high in PAD populations. We therefore aimed to better characterize PAD patients suffering from cardiovascular events and mortality in order to tailor preventive treatment. METHODS: Between 2018 and 2020, 246 PAD outpatients (17 newly diagnosed, 229 with known PAD) were prospectively enrolled in this observational cohort study. Patient data and blood samples were collected after inclusion, and the primary composite endpoint (myocardial infarction, elective coronary revascularization, ischemic stroke, acute limb ischemia, mortality) was evaluated after one year. Secondary outcomes included platelet reactivity, measured using the VerifyNow assay, and medication adherence, assessed using the Morisky Medication Adherence Scale-8 (MMAS-8). Logistic regression models were used to identify associations between characteristics and the occurrence of events. RESULTS: The cohort comprised 207 patients with claudication and 39 with chronic limb threatening ischemia. Twenty-six (10.6%) patients suffered from an event during follow-up. Prior myocardial infarction (OR 3.3 [1.4-7.7]), prior ischemic stroke (OR 4.5 [1.8-10.9]), higher levels of creatinine (OR 5.2 [2.2-12.6]), lower levels of high-density lipoprotein (OR 4.2 [1.5-10.6]) and lower haemoglobin levels (OR 3.1 [1.3-7.1]) were associated with events. Patients with events had more often high on-treatment platelet reactivity (HTPR) on aspirin (OR 5.9 [1.4-25.1]) or clopidogrel (OR 4.3 [1-19.3]). High adherence to medication was associated with the occurrence of events (OR 4.1 [1-18]). CONCLUSIONS: Patients suffering from cardiovascular events and mortality were characterized by prior cardiovascular events as compared to patients who did not experience any events. Antiplatelet therapy was not optimally protective despite high medication adherence, and HTPR was independently associated with the occurrence of events. More research is needed on alternative treatment strategies such as dual antiplatelet therapy or combinations with anticoagulant drugs. TRIAL REGISTRATION: The Medical Ethics Committee (METC) of the MUMC+ approved the study (NL63235.068.17) and the study was registered in the Netherlands Trial Register ( NTR7250 ).

Translational insights into mechanisms underlying residual venous obstruction and the role of factor XI, P-selectin and GPVI in recurrent venous thromboembolism.

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:C1-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.

Discovery of four plasmatic biomarkers potentially predicting cardiovascular outcome in peripheral artery disease.

Peripheral artery disease (PAD) patients have an increased cardiovascular risk despite pharmacological treatment strategies. Biomarker research improving risk stratification only focused on known atherothrombotic pathways, but unexplored pathways might play more important roles. To explore the association between a broad cardiovascular biomarker set and cardiovascular risk in PAD. 120 PAD outpatients were enrolled in this observational cohort study. Patients were followed for one year in which the composite endpoint (myocardial infarction, coronary revascularization, stroke, acute limb ischemia and mortality) was assessed. Patient data and blood samples were collected upon inclusion, and citrated platelet-poor plasma was used to analyze 184 biomarkers in Olink Cardiovascular panel II and III using a proximity extension assay. Fifteen patients reached the composite endpoint. These patients had more prior strokes and higher serum creatinine levels. Multivariate analysis revealed increased plasma levels of protease-activated receptor 1 (PAR1), galectin-9 (Gal-9), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) and interleukin 6 (IL-6) to be most predictive for cardiovascular events and mortality. Positive regulation of acute inflammatory responses and leukocyte chemotaxis were identified as involved biological processes. This study identified IL-6, PAR1, Gal-9, TNFRSF11A as potent predictors for cardiovascular events and mortality in PAD, and potential drug development targets.

Predictive value of D-dimer testing for the diagnosis of venous thrombosis in unusual locations: A systematic review.

BACKGROUND: The value of D-dimer testing for the diagnosis of thrombosis in unusual sites is not properly established and evidence is scarce. We performed a systematic review of the literature. METHODS: The search was conducted in MEDLINE and Cochrane Library for papers published in the last 10 years including different presentations of thrombosis in unusual sites. Twenty-three articles were included, from January 1, 2008, to December 31, 2018, comprising 3378 patients with thrombosis in unusual sites (upper extremity deep vein thrombosis, cerebral vein thrombosis and splanchnic vein thrombosis). The Newcastle-Ottawa scale was used to assess the quality of the studies. RESULTS: Two articles were related to upper extremity thrombosis, showing a high sensitivity and negative predictive value for D-dimer testing. Twelve articles concerned cerebral vein thrombosis, concluding that the timing of D-dimer testing was important, and that patients with a shorter duration of symptoms showed higher D-dimer levels. Sensitivity and specificity in these patients ranged from 58% to 97% and from 77% to 97.5%, respectively. Nine articles were related to splanchnic vein thrombosis. One described a population of patients with mesenteric venous thrombosis, and the rest included patients with portal vein thrombosis. The D-dimer testing methods and the proposed cut-off levels were remarkably different among the included studies. CONCLUSION: D-dimer testing should not be currently recommended for the diagnosis of thrombosis in unusual sites as a first line diagnostic tool. The development of algorithms combining biomarkers such as D-dimer and clinical decision tools could improve the diagnosis.

Pleiotropic effects of the hemostatic system.

Atherothrombosis is characterized by the inflammatory process of atherosclerosis combined with a hypercoagulable state leading to superimposed thrombus formation. In atherosclerotic plaques, cell signaling can occur via protease-activated receptors (PARs), four of which have been identified so far (PAR1-PAR4). Proteases that are able to activate PARs can be produced systemically, but also at the sites of lesions, and they include thrombin and activated factor X. After PAR activation, downstream signaling can lead to both proinflammatory effects and a hypercoagulable state. Which specific effect occurs depends on the type of protease and activated PAR, and the site of activation. Hypercoagulable effects are mainly exerted through PAR1 and PAR4, whereas proinflammatory responses are mostly seen after PAR1 and PAR2 activation. PAR signaling pathways contribute to atherothrombosis, suggesting that inhibition of these pathways possibly prevents cardiovascular events based on this pathophysiological mechanism. In this review, we highlight the pathways by which PAR activation leads to proinflammatory responses and a hypercoagulable state. Furthermore, we give an overview of potential pharmacological treatment targets that promote vascular protection.