Plasminogen Activator Inhibitor-1–Positive Platelet-Derived Extracellular Vesicles Predicts MACE and the Proinflammatory SMC Phenotype

Visual Abstract


SUMMARY
Patients with established coronary artery disease remain at elevated risk of major adverse cardiac events.
The goal of this study was to evaluate the utility of plasminogen activator inhibitor-1-positive platelet-derived extracellular vesicles as a biomarker for major adverse cardiac events and to explore potential underlying mechanisms. Our study suggests these extracellular vesicles as a potential biomarker to identify and a therapeutic target to ameliorate neointimal formation of high-risk patients. ( annualized adverse event rates as high as 8% to 12% in the following year. [3][4][5] Comparatively, the putative vulnerable plaque has an annualized event rate of 0.05%, making identification and treatment of patients after coronary stenting an unmet need. Two major complications resulting in target vessel failure are in-stent restenosis and stent thrombosis. 6 Plasminogen activator inhibitor (PAI)-1 is primarily an antifibrinolytic protein that inhibits fibrin clot degradation by preventing the action of tissue-type plasminogen activator. 7 Elevated levels of PAI-1 have been previously associated with type 2 diabetes, acute myocardial infarction, and unplanned revascularization. [8][9][10] Of note, the majority of plasma PAI-1 is derived from platelets. 7,9,11 Inhibition of PAI-1 has previously been shown to reduce vascular smooth muscle cell (VSMC) migration and prevent neointimal formation after vascular injury in mouse models. 12,13 However, in humans, few studies have evaluated or linked PAI-1 levels to outcomes in patients with CAD. Extracellular vesicles (EVs) are released from circulating blood and vascular cells at the time of vascular injury. [14][15][16] In addition to small studies suggesting a role as a biomarker, EVs potentially modulate stent failure in 2 ways: 1) EVs are abundant in surface-exposed negatively charged phosphatidylserine, which catalyzes thrombosis 17 ; and 2) EVs contain proinflammatory protein and genetic material that could potentially be transferred to recipient cells at sites of vascular injury to modulate a phenotypic response. [18][19][20][21][22] To date, no data exist on the relationship between PAI-1 and EVs in humans despite platelets being a common source; there are also no data on if EVs play a pathologic role in clinical events in patients with CAD.
Accordingly, we hypothesized that plateletderived EVs may represent a major source of pathologic PAI-1 (PAI-1-positive platelet-derived EVs [ie, PAI-1 þ PEVs]). We also evaluated if these EVs were associated with major adverse cardiac events (MACE). Finally, we sought to evaluate the role of PAI-1 þ PEVs in models of thrombus formation and in VSMC phenotypic switching for arterial remodeling post-revascularization.   Heart Institute is a regional tertiary center serving All patients included in the study completed their 1-year follow-up. Receiver-operating characteristic curves were generated, and the Youden index was identified to ascertain the optimal cutoff value to simultaneously maximize sensitivity and specificity of PAI-1 þ PEVs in the discovery cohort and validated in the validation cohort. 24 Kaplan-Meier curves were generated to evaluate time-to-event distributions and compared by using log-rank tests. HRs and corresponding 95% CIs were obtained from Cox proportional hazards models after adjustment by clinical characteristics (age, type 2 diabetes, and acute coronary syndrome) determined a priori based on clinical judgment of variables influencing MACE.

METHODS
All flow cytometry data were analyzed by using   Gates for Annexin V, CD41, and PAI-1 were based on the use of isotype and fluorescence minus one control where appropriate ( Figures 1A to 1I). Localization of PAI-1 on the surface of PEV was further validated by using electron microscopy by labeling with PAI-1 antibody and appropriate gold-linked immunoglobulin G secondary antibody ( Figure 1J). To determine the proportion of PAI-1 associated with EVs in human plasma, plasma was EV-depleted ( Figure 1K shows the relative decrease in PAI-1 levels), which demonstrates that PAI-1 þ PEV contributes to 19.1% AE 6.3% of PAI-1 levels in plasma.   (E) Time to onset of thrombus did not differ between PAI-1 þ PEV fractions (6.0 AE 0.3 minutes vs 6.0 AE 0.7 minutes for high vs low; n ¼ 10 and n ¼ 8; P ¼ 0.80).
(F) Time to thrombus occlusion was not different between PAI-1 þ PEV fractions (7.9 AE 0.2 minutes vs 8.2 AE 1.5 minutes for high vs low; n ¼ 10 and n ¼ 8; P ¼ 0.63).    When stratified according to MACE, differences in rates of previous PCI and myocardial infarction were observed (Supplemental Table 3 Figures 5C and 5D).

UTILITY OF PAI-1 D PEV AS A BIOMARKER OF MACE:
DISCOVERY AND VALIDATION COHORT. In an allcomer population, we sought to evaluate whether PAI-1 þ PEV levels were predictive of MACE, and the cohort was equally divided into a discovery and a validation cohort (Supplemental Table 4 (Figures 5G and 5H).

DISCUSSION
Patients with CAD continue to experience adverse events despite guideline-directed medical therapy and revascularization. Identification of novel markers to risk-stratify patients and/or to offer new therapeutic interventions remains a priority. Given the potential of EVs to behave as a biomarker and due to the common origin of PAI-1 and EV from platelets, our study sought to evaluate whether PAI-1 þ PEV predicts MACE and affects thrombus formation and the proinflammatory VSMC phenotype. Our study presents 5 novel findings regarding PAI-1 þ PEV: 1) the presence of a PAI-1 þ PEV complex in human plasma; 2) PAI-1 þ PEVs in standardized PEV fraction have no effect on thrombus formation; 3) PEV promotes the proinflammatory and pro-osteogenic VSMC phenotype; 4) inhibition of PAI-1 þ PEVs through TM5275 attenuates VSMC phenotypic switching but has no effect on thrombus formation; and 5) high PAI-1 þ PEV levels were predictive of MACE in both the discovery and the validation cohort. Overall, PAI-1 þ PEVs act as a promising biomarker and offer a novel target to modulate vessel biology in patients with CAD.
We hypothesized that circulating plasma PAI-1 was largely EV derived and would modulate event rates in patients with CAD by increasing thrombotic risk. We found that 19.1% of circulating PAI-1 is in PAI-1 þ PEVs, a newly identified subgroup of circulating PEVs. In our in vitro models, PAI-1 þ PEVs did not increase thrombogenicity compared with PEVs alone. Interestingly, PEVs were shown to interact with VSMCs,  PAI-1 mainly functions as an antiproteolytic protein but also appears to serve a ligand role affecting migration, proliferation, and apoptosis at sites of vascular injury. [26][27][28][29] The current study found that