Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease

Abstract

Background: Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear.

Objectives: The authors sought to investigate the role of platelets in a cohort of symptomatic PAD.

Methods: The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease.

Results: Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events.

Conclusions: The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103).

Keywords: macrophages; monocytes; peripheral artery disease; platelets; platelet–monocyte aggregates.

Figure 1. Platelet activity is increased in PAD

Platelet count (A), mean platelet volume (B), and reticulated platelets (C) in healthy and symptomatic PAD individuals. P-selectin (D) and PAC-1 (E) expression under basal or stimulated with thrombin, arachidonic acid (AA), ADP or epinephrine (Epi). Data are median ± IQR. *P < 0.05. (F) Representative platelet aggregation curve of healthy and PAD in response to Epi, (G) ADP and (H) collagen (Col). Data are mean ± SD. n= 25 for healthy and n= 54 for PAD. *P < 0.05.

Figure 2. Abundant expression of MRP-14 in PAD platelets

(A) Heatmap of platelet profiling by RNASeq from 3 healthy and 3 PAD individuals color-coded by increased intensity in red and reduced fold expression in blue. (B) Pathways analysis related to platelet physiopathology and atherothrombosis by DAVID software. (C) MRP-14 mRNA in platelets from healthy and PAD. (D) MRP-14 protein level in platelet releasates under basal (p=0.047) or thrombin activation (p=0.039). (E) Staining of MRP-14 (green) in CD61 (red) positive platelets in PAD. Scale bar=20μm. (F) Box plot of plasma and serum MRP-14 in healthy and PAD donors. *P < 0.05. (G) Correlation plots between serum and platelet MRP-14.

Figure 3. MRP-14 increases MPA in PAD

(A) Representative platelet aggregation profile in response to Epi and recombinant MRP-14. Flow cytometry quantitfication of PAC-1 (B) and P-selectin (C) at baseline or thrombin simulated in the presence of doses of MRP-14. Representative of 3 individual experiments (A–C). Flow cytometry density plots characterization of platelet-leukocyte aggregates (CD45⁺CD61⁺) (D) and platelet-monocyte clusters (CD14⁺CD61⁺) (E). Data are mean ± SD. *P < 0.05. Correlation plot between MRP-14 and CD14⁺CD61⁺ population (F). Platelet-CD14+ monocyte aggregates in healthy vs PAD in the presence of control IgG or anti P-selectin antibody (G). Flow cytometry density plots of platelet-THP-1 aggregates in the presence of MRP-14 and anti P-selectin antibody (H). PSGL-1 protein (I) and mRNA expression (J) in THP-1 stimulated with recombinant MRP-14. Data are mean ± sd, representative of experimental n=3. *P < 0.05.

Figure 4. MPA drive inflammation in PAD

(A) Scanning electron microscope images of a representative field depicting platelets (green) interacting with monocytes. (B) Magnified interactions of platelets: fusion (arrow), binding, projecting tubular structures. (C) Colocalisation (arrows) of CD68 (red) and platelet CD61 (green) in PAD lesion. (D) Detection and quantification of MRP-14 (green) in monocytes incubated with healthy or PAD PR in the presence of anti-MRP-14 or IgG (E). Bar 40 μm. Nuclei are counterstained with DAPI. Data are representative of n = 3 experiments. (F) RT-PCR of indicated mRNA in THP-1 stimulated with MRP-14, healthy or PAD PR (G) in the presence of anti MRP-14 or control IgG (H). Results are shown as mean ± sd, *P < 0.05.

Figure 5. MRP-14 promotes monocyte migration and is enriched in PAD lesions

(A) Automated migration profile of THP-1 towards healthy or PAD PR in the presence of anti-MRP-14 or IgG control antibody (B), or towards recombinant MRP-14 (C). Results are shown as mean ± sd, representative of 3 independent experiments. (D) Phalloidin staining to detect actin (red) in THP-1 treated with healthy or PAD PR. Scale bar, 20 μm. Right, cell surface area of monocytic cells. Data are mean ± SEM or SD. *P < 0.05. (E) Staining of PAD lesion with characteristic labelled layers. Magnified immunofluorescence MRP-14 (green; in the thrombus, box T) alone or double stained with macrophage marker CD68 (red) revealing their colocalization (yellow; arrows) in medial (G) and intimal/medial area (F). Nuclei are counterstained with DAPI (blue). Scale bars, 10 μm. Representative staining of 3 independent symptomatic sample.

Figure 6. MRP-14 correlates with PAD severity and is unassociated with carotid artery disease

MRP-14 levels measured by ELISA in serum of patients with PAD (n=68) versus carotid artery stenosis (CAS; n=10) (A) and in 68 PAD individuals without or with severe critical limb ischemia (CLI) (B). (C) Kaplan-Meier cumulative incidence plot in PAD patients undergoing lower extremity revascularization (MACLE; myocardial infarction, stroke, death or amputation; MRP-14 values above the median were associated with a 2.5 fold increase in MACLE (HR 2.5, 95% CI 1.1 to 5.9, P =0.03). *P=0.04; *P=0.004.

Central Illustration. Role of platelet-derived MRP-14 in PAD

Elevated MRP-14 was detected in activated platelets in PAD. MRP-14 increased the expression of P-selectin which fostered the formation of monocyte-platelet aggregates (MPA). MPR-14 from platelets was transferred to monocytes which induced their migration and inflammatory profile in PAD lesions. PAD = peripheral artery disease; MPA = monocyte-platelet aggregate; MCP-1 = monocyte chemoattractant protein-1; IL-1b = interleukin 1 beta; TNFa = Tumor necrosis factor alpha