Glycophorin C in carotid atherosclerotic plaque reflects intraplaque hemorrhage and pre-procedural neurological symptoms

Background and aims The accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. We therefore hypothesized, that glycophorin C is a marker for plaque vulnerability and may therefore reflect intraplaque hemorrhage (IPH), vulnerability of plaques and predict pre-procedural neurological symptoms. Methods We quantified glycophorin C in human atherosclerotic plaque samples from 1,819 consecutive asymptomatic and symptomatic patients undergoing carotid endarterectomy from the Athero-Express Biobank with the slideToolKit method. Results The prevalence of IPH was 62.4%, and the prevalence of pre-procedural neurological symptoms was 87.1%. The percentage glycophorin staining was significantly higher in male patients compared to female patients (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p<0.001). Quantified glycophorin C was associated with IPH (OR 1.90; 95% CI 1.63, 2.21; p=<0.001) when corrected for clinical confounders. Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p =0.005), and upon sex-stratified analyses, specifically in male patients (OR 1.37; 95%CI 1.12, 1.69; p=0.003), but not in female patients (OR 1.15; 95%CI 0.77, 1.73; p=0.27). Glycophorin was associated with classical features of a vulnerable plaque, such as a larger lipid core (OR 1.85; 95%CI 1.60, 2.15; p<0.001), a higher macrophage burden (OR 1.87; 95%CI 1.63, 2.14; p<0.001), less calcifications (OR 0.81; 95%CI 0.71, 0.91; p<0.001), a lower collagen and SMC content (OR 0.70; 95%CI 0.60, 0.82; p<0.001 and OR 0.60; 95%CI 0.52, 0.68; p<0.001, respectively). There were marked sex differences, in male patients glycophorin was associated with calcifications and collagen (OR 0.75; 95%CI 0.64, 0.87; p<0.001 and OR 0.65; 95%CI 0.53, 0.79; p<0.001, respectively) while these associations were not found in female patients. Conclusions Quantified glycophorin C was independently associated with the presence of IPH, symptomatic preprocedural symptoms in male, and with a more vulnerable plaque composition in both women and men. Which strengthens the hypothesize that plaque glycophorin C is a marker of plaque vulnerability. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial Not applicable. ### Funding Statement This work was supported by the Netherlands Cardiovascular Research Initiative: an initiative with support of the Dutch Heart Foundation (CVON-GENIUS-2 to GP and SWvdL). We are thankful for the support of the ERA-CVD program druggable-MI-targets (grant number: 01KL1802), the EU H2020 TO_AITION (grant number: 848146), EU H2020 Taxinomisis (grant number 755320 JM, GP, GJB DdK), and the Leducq Fondation PlaqOmics. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The current study was conducted in accordance with the Declaration of Helsinki. Both Medical Ethics Boards at the St. Antonius Hospital (Nieuwegein, The Netherlands) and University Medical Center Utrecht (Utrecht, The Netherlands) gave ethical approval for this study, and registered this decision under number TME/C-01.18 I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data as used here are available through DataverseNL. DOI: