Platelet hyaluronidase 2 enrichment in acute coronary syndromes: a conceivable role in monocyte-platelet aggregate formation

Abstract Acute Coronary Syndromes (ACS) with plaque erosion display dysregulated hyaluronan metabolism, with increased hyaluronidase-2 (HYAL2) expression. However, the expression and the role of this enzyme on platelets has never been explored. We evaluated the platelet’s HYAL2 (pltHYAL2) levels on I) stable angina (SA) and II) ACS patients, furtherly sub-grouped in Intact-Fibrous-Cap (IFC) and Ruptured-Fibrous-Cap (RFC), according to Optical Coherence Tomography. We assessed the HYAL2 role through an in vitro model setting of co-cultured monocytes and platelets, before and after treatment with low-molecular-weight hyaluronic acid (HA) as pro-inflammatory stimulus and with or without HYAL2-antibody to inhibit HYAL2 activity. ACS patients exhibit higher pltHYAL2 levels comparing to SA, with the higher expression for IFC group. The addition of HYAL2-antibody significantly reduced the percentage of monocyte-platelet binding, suggesting that pltHYAL2 enrichment at the site of the culprit lesion is a key mediator in the systemic thrombo-inflammatory status of ACS presenting with plaque erosion.


Introduction
Hyaluronic acid (HA) is an ubiquitous, natural, anionic glycosaminoglycan (GAG) of repeating b1,4-N-acetyl-D-glucosamine and b1,3-D-glucuronic acid units 1 with a biological function strictly dependent on its molecular weight 2 . The high molecular weight HA polymers (HMW-HA; MW >1000 kDa), physiological key components of vascular extra cellular matrix (ECM), maintain tissue homeostasis promoting anti-inflammatory and tissue repairing processes, while the low molecular weight counterparts (LMW-HA; MW <250 kDa) are involved in pro-inflammatory biological pathways and ECM derangement 2 .
Acute Coronary Syndrome (ACS) patients display dysregulated HA metabolism 3,4 that furthermore might promote the generation of monocyte-platelet complexes 4 , involved in increased platelet reactivity and in thrombus formation 5 . Systemic blood mononuclear cells from ACS presenting with Intact Fibrous Cap (IFC) atherosclerotic plaque at Optical Coherence Tomography (OCT) show increased levels of Hyaluronidase 2 (HYAL2), the key enzyme responsible of HA hydrolysis and LMW-HA generation 4 . Alongside mononuclear cells, platelets can bind HA as well; indeed, platelets and their precursors express HYAL2 and can stimulate mononuclear leukocytes to produce pro-inflammatory mediators 6 .
Aims of this study were to assess HYAL2 protein expression on platelets ( plt HYAL2) of patients presenting with ACS and to understand if the augmented cross-talk between platelets and immune cells depends on HYAL2, as a consequence of an altered HA metabolism.

Population study
We enrolled (1) patients with symptoms of stable effort angina (SA) (n ¼ 40) lasting more than 1 years, angiographically confirmed coronary artery disease, with no prior acute coronary events, and no ischaemic episodes during the last 48 h; (2) ACS patients at their first diagnosis admitted to our coronary care unit with non-ST-segment elevation myocardial infarction (NSTEMI) (n ¼ 40). The NSTEMI group was furtherly divided in two sub-groups, according to the Optical Coherence Tomography (OCT) analysis of the culprit plaque during coronary angiography: Intact Fibrous Cap (IFC) group and Ruptured Fibrous Cap (RFC) one.
All patients gave their written informed consent and the Ethics Committee of the Fondazione Policlinico Universitario "Agostino Gemelli" IRCCS-Universit a Cattolica del Sacro Cuore (UCSC) approved the study (Prt. N. 37077/19).

Platelet isolation and multicolour flow-cytometry analyses
Cellular analyses were performed on platelets isolated from peripheral whole blood collected in citrate dextrose tubes as follows. Platelet rich plasma (PRP) was obtained by centrifugation of fresh blood at 200 x g for 10 min at room temperature (RT). PRP was transferred into a new plastic tube, without disturbing the buffy coat layer, and centrifuged as above for 5 min to remove the residual erythrocytes/white blood cells. PRP supernatant was withdrawn and centrifuged in Acid-Citrate-Dextrose (ACD) solution (1 part ACD solution to 9 parts blood) (Sigma-Aldrich) at 800 x g for 20 min at RT. Platelet pellets were gently washed and resuspended in Tyrode's buffer (pH 7.8) and aliquoted according to the final analyses.
We performed a multicolour flow-cytometry for evaluating the surface protein expression of HYAL2 by staining isolated platelets with unconjugated HYAL2 antibody (ThermoFisher Scientific). The secondary antibody for the HYAL2 was Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000) (Invitrogen).We used CD45-ECD antibody (Beckman Coulter) for verifying the purity of our platelet samples and anti-CD62P-PE antibody (eBioscience, INC.) to determine platelet activation.

plt HYAL2 expression according to OCT investigation of coronary artery stenosis
We compared plt HYAL2 expression, as described above, within NSTEMI patients by sub-grouping them according to OCT investigation (C7-XR or ILUMIEN OPTIS, St. Jude Medical) in IFC plaques (n ¼ 6) and Ruptured Fibrous Cap (RFC) plaques (n ¼ 8). We classified as plaque erosion the presence of thrombus overlying a plaque with IFC, or the presence of luminal surface irregularity at the culprit lesion in the absence of thrombus, and as RFC the presence of fibrous cap discontinuity with a cavity formed inside the plaque or with a direct communication between lumen and inner core of a plaque 7 .

Isolation of human peripheral mononuclear cells
We isolated peripheral blood mononuclear cells (PBMCs) from whole blood Ethylene Diamine Tetraacetic Acid (EDTA) samples by density gradient centrifugation method (LympholyteV R -H Cell Separation Media, CEDARLANE). PBMC pellets were washed and resuspended in Dulbecco's phosphate-buffered saline (DPBS) (GIBCO, Invitrogen) and aliquoted according to the final analyses.

Enzyme-Linked immunosorbent assay (ELISA) of serum HA
We checked the circulating levels of total HA content performing an Enzyme-Linked Immunosorbent Assay (Hyaluronan Quantikine ELISA Kit, R&D Systems) on sera collected from SA patients and NSTEMI patients undergone to OCT, according to the manufacturer's recommendation.

Statistical analyses
Distribution of variables (described as Mean ± SD) were assessed by Shapiro Wilk test. We performed unpaired T-test and Mann-Whitney test (for nonparametric variables) to compare two groups, as appropriate. ANOVA for repeated measures, with Bonferroni's test were used for multiple comparisons. For all the experimental assays performed, a two-tailed p values 0.05 was considered statistically significant. We used multicolour flow-cytometry and Kaluza software for protein detection and analysis (FC 500 and Kaluza Software, Beckman Coulter, Brea, C.) and GraphPad Prism 8 Software (GraphPad Software, Inc., San Diego, CA, USA) for data analysis.

Results and discussion
The plt HYAL2-MFI analysis (Figure 1(A and B)) assessed that NSTEMI patients had a significant increase of plt HYAL2 expression with respect to SA patients (2.34 ± 1.01 vs 1.83 ± 0.71, respectively; p ¼ 0.022) (Figure 1(A)). Analyses of the NSTEMI sub-groups, according to OCT investigation, revealed an increased expression of plt HYAL2 in patients with IFC plaques compared to those ones with RFC plaques (2.63 ± 1.50 vs 1.40 ± 0.40, respectively; p ¼ 0.030) (Figure 1(C)). These results demonstrated, for the first time, that patients presenting with unstable plaques such as NSTEMI patients have a significant systemic increase of plt HYAL2 when compared to those ones with stable atherosclerotic lesions, and that, interestingly, this different expression is significantly driven by patients with IFC lesions.
A drug-depending analysis showed that anti-platelet therapies with clopidogrel or ticagrelor did not affect plt HYAL2 expression (Figure 1(D)) underling that plt HYAL2 expression seems to have a role independent of common treatment with P2Y12 receptor inhibitors, thus suggesting an alternative signalling route.
HA serum circulating levels were similar between SA patients and patients with IFC and RFC plaques (Figure 1(E)), remaining around the normal human concentration 8 . Since HA has a pathophysiological role that is strictly related to its molecular weight 2 , an explanation might be that the assay does not detect HA molecules with a size lower than 35 kDa, thereby excluding a relevant amount of hydrolysed glycosaminoglycan content.
Moreover, in NSTEMI patients, our in vitro model (Figure 2(A)) showed a significant reduction of monocyte-platelet binding (% CD14 þ CD42 þ ) following the incubation with anti-HYAL2 antibody, only for LMW-HA treated samples (p ¼ 0.032; Figure 2(B and C)). These in vitro setting corroborated that the synergic interaction between monocytes and platelets might play a role in ECM derangement and plaque destabilisation especially in presence of an altered HA catabolism, and that the HYAL2 surface ligation may negatively affect the monocyte-platelet aggregate formation.
Human platelets, whose count is significantly associated to all cause of mortality in patients with myocardial infarction, express bacterial receptors like TLRs that could trigger the thromboinflammatory response through activation of phosphoinositide 3kinase [9][10][11] . Indeed, as a direct consequence of plaque destabilization, platelets leave their quiescent state, adhere to the injured vascular endothelium and actively interact with the extracellular matrix (ECM), by releasing its aÀgranule content, including HYAL2 molecules 12 . Alongside, the innate immune system appears to be involved in ECM derangement and HA dyshomeostasis 13 through the commitment of molecule such as monocyte HYAL2 4 or HA synthase 3 (HAS3) 14 .
Thus, if on one side the profound perturbation of the immune system together with its residual inflammatory milieu might describe the mechanisms of plaque rupture 15,16 , on the opposite hand, the dysregulation of HA metabolism in ECM might explain the pathogenesis of plaque erosion (Graphical Abstract).
Clinicians should refine the current pharmacological treatments bearing in mind the different biological mechanisms underlying plaque erosion and plaque rupture 17,18 and our results, in concert with those ones obtained on systemic mononuclear cells 4 , might open the way for a novel and increasingly personalised medicine to supplement the classical anti-thrombotic therapies in patients with myocardial infarction.

Conclusions
HYAL2 might be the bridge between elements historically belonging to the immune system, such as monocytes, and elements belonging to the haemostasis and thrombotic system, such as platelets, representing a novel target to be reached in view of therapies increasingly personalised.

Disclosure statement
No potential conflict of interest was reported by the author(s).