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 ).

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.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease.

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Design and synthesis of a multivalent catch-and-release assay to measure circulating FXIa.

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.

[Thigh muscle flaps for postoperative inguinal wound complications in vascular surgery : Sartorius muscle versus rectus femoris muscle]. / Oberschenkelmuskellappen bei inguinalen postoperativen Komplikationen in der Gefäßchirurgie : Musculus sartorius vs. Musculus rectus femoris.

BACKGROUND: Inguinal wound complications often cause postoperative morbidity and also mortality following vascular surgical interventions. The aim of this study was to report experiences and a comparison of the outcomes using rectus femoris muscle flaps (RFF) and sartorius muscle flaps (SMF). MATERIAL AND METHODS: A retrospective study was performed at two locations of a cross-border vascular center and all muscle flap interventions performed at the two centers within the vascular surgery department were reviewed. Primary outcomes were muscle flap survival, graft salvage and major amputations. RESULTS: A total of 44 RFFs were performed in 39 patients (mean age 67 years, 73% males) and 25 SMFs in 24 patients (mean age 64 years, 76% males). Wound infections were the most common indications for muscle flap reconstruction. At a mean follow-up of 24 months (±24) and 17 months (±20), respectively, comparable flap survival rates (91% vs. 84%), wound healing rates (72% vs. 83%), graft salvage (65% vs. 73%) and amputation rates (9% vs. 8%) were found. CONCLUSION: Muscle flap reconstruction is an effective way to cover groin defects resulting from deep wound infections after vascular surgery, achieving good results in a high-risk group of patients. No differences were found between SMF and RFF regarding amputation and graft loss. Both techniques can be safely performed, depending on the preference and experience of the surgical team. The RFF technique should be preferentially used to cover large tissue defects, whereas the SMF procedure can be preferred to cover smaller defects in the groin.

Radial artery bypass graft is a feasible and durable conduit for challenging infrainguinal revascularization: 17 years of Melbourne experience.

OBJECTIVES: The superiority of autogenous venous conduits in infrainguinal bypass surgery is well established. In the absence of suitable leg or arm veins the radial artery can be utilized as an alternative autogenous conduit. In contrast to cardiac surgery, experience with the radial artery as a conduit for infrainguinal bypass surgery is limited. The purpose of this study was to review the outcomes of our radial artery bypasses over the last 17 years. METHODS: All radial artery bypasses performed between 1995 and 2012 were identified from a prospective database. Patency, limb salvage, and survival were calculated using the Kaplan-Meier survival estimate method. RESULTS: Twenty-nine radial artery bypasses were performed in 28 patients. Median follow-up was 55 months (range 1-170). Twelve-month primary, assisted primary, and secondary patency rates were 49%, 62%, and 73% respectively; Both 3-year and 5-year primary, assisted primary, and secondary patency rates were 49%, 56% and 67% respectively. Limb salvage rate was 75% at 1- and 5-year follow-up. Patient survival at 1, 3, and 5 years was 96%, 88%, and 76%. CONCLUSIONS: For patients with need of challenging infrainguinal revascularization without suitable autogenous venous conduit, a radial artery bypass can be performed safely with favorable long-term patency and limb salvage rates.