Parkin Coordinates Platelet Stress Response in Diabetes Mellitus: A Big Role in a Small Cell

Increased platelet activation and apoptosis are characteristic of diabetic (DM) platelets, where a Parkin-dependent mitophagy serves a major endogenous protective role. We now demonstrate that Parkin is highly expressed in both healthy platelets and diabetic platelets, compared to other mitochondria-enriched tissues such as the heart, muscle, brain, and liver. Abundance of Parkin in a small, short-lived anucleate cell suggest significance in various key processes. Through proteomics we identified 127 Parkin-interacting proteins in DM platelets and compared them to healthy controls. We assessed the 11 highest covered proteins by individual IPs and confirmed seven proteins that interacted with Parkin; VCP/p97, LAMP1, HADHA, FREMT3, PDIA, ILK, and 14-3-3. Upon further STRING analysis using GO and KEGG, interactions were divided into two broad groups: targeting platelet activation through (1) actions on mitochondria and (2) actions on integrin signaling. Parkin plays an important role in mitochondrial protection through mitophagy (VCP/p97), recruiting phagophores, and targeting lysosomes (with LAMP1). Mitochondrial β-oxidation may also be regulated by the Parkin/HADHA interaction. Parkin may regulate platelet aggregation and activation through integrin signaling through interactions with proteins like FREMT3, PDIA, ILK, and 14-3-3. Thus, platelet Parkin may regulate the protection (mitophagy) and stress response (platelet activation) in DM platelets. This study identified new potential therapeutic targets for platelet mitochondrial dysfunction and hyperactivation in diabetes mellitus.


Introduction
Diabetes mellitus (DM) is a progressive and chronic metabolic disorder characterized by hyperglycemia arising from impaired insulin levels, insulin sensitivity, and/or insulin activity. Currently, over 19.7 million adults in the USA have diagnosed DM, and an estimated 8.2 million have undiagnosed DM [1]. Cardiovascular disease is the major cause of morbidity and mortality among DM patients with approximately 65% of deaths caused by thrombotic events like myocardial and cerebrovascular ischemia and infarction [2]. Platelets play key roles in thrombotic occlusions of major vessels and tissue death.

Results
We recently demonstrated that Parkin plays a key protective role against oxidative stress in platelets by inducing mitophagy [9,14]. The absence of Parkin during such stressors like DM and H 2 O 2 leads to platelet apoptosis [9,14].

Parkin Interacts with Various Key Platelet Proteins
We previously verified that mitophagy induction in DM platelets occurs through a Parkin-dependent mechanism [9]. Surprisingly, Parkin is highly expressed in human DM platelet ( Figure 1A) [9,14]. Although Parkin expression was only slightly increased in murine DM platelets, its expression was more than double that of other mitochondria-rich tissues like the heart, muscle, liver, and brain. ( Figure 1B) [14]. Electron microscopy demonstrated that Parkin is localized within mitochondria and granules (blue arrows), cytosol, and cell membrane (red arrows) in DM platelets, further elaborating on its potentially diverse functions ( Figure 1C). Mitochondrial Parkin also colocalizes with LC3, an autophagy marker ( Figure 1C). No other functions beyond mitophagy induction in platelets have been reported for Parkin [14].

Parkin Plays an Important Role in Mitochondrial Protection Through Mitophagy
Transitional Endoplasmic Reticulum ATPase (VCP/p97) Figure 3. Confirmation of interactions between Parkin and selected target proteins in human and murine platelets. (A) Immunoprecipitation of each specific antibody in pooled human DM platelets (4 HC, 5DM1, and 6DM2). We incubated 500 µg protein lysates incubated with specific antibodies overnight at 4 • C with 10% input as control. G1 represents interacting protein groups from LC-MS/MS results. G2 represents the non-interacting proteins group in LC-MS/MS results. G3 represents protein that interacted with Parkin and that were not found in the LC-MS/MS results. (B) Immunoprecipitation of each specific antibody in pooled murine platelets (3 WT and 5DM). We incubated 500 µg protein lysates incubated with specific antibodies overnight at 4 • C with 10% input as control.

Parkin Plays an Important Role in Mitochondrial Protection through Mitophagy
Transitional Endoplasmic Reticulum ATPase (VCP/p97) VCP/p97 is a hexameric protein of the AAA (ATPases associated with diverse cellular activities) family which generally utilizes energy from ATP hydrolysis [23]. VCP/p97 has been linked to various membrane trafficking processes, including Golgi reassembly post-mitosis [24] and control of lipid droplet biogenesis [25]. Emerging evidence has connected VCP/p97 to lysosomal protein degradation through its ability to facilitate cargo sorting via the endosomal pathway and autophagy [26][27][28]. One VCP/p97 mutation causes a rare multisystem disease, IBMPFD (inclusion body myopathy with Paget's disease and frontotemporal dementia) [28]. Several recent studies reported the involvement of VCP/p79 in mitophagy [29,30]. Here, we identified that VCP/p97 interacts with Parkin in DM platelets, possibly regulating the mitophagy process (VCP/p97: 78.5% coverage, Table 1).

Lysosomal-Associated Membrane Protein 1 (LAMP1)
LAMP1 is a well-known lysosomal protein that we previously confirmed to colocalize with Parkin and LC3 in DM platelets [9,14] and, again, in this study through IP (Figure 3). Although not identified in the LC-MS/MS, LAMP1 was used to confirm Parkin's role in autophagy activation (Tables 1-3). This highlights the potential deficiencies and false negatives of the LC-MS/MS, potentially due to the complex processing.

Mitochondrial Three Functional Protein A (TFPα, HADHA)
TFPα is a multienzyme mitochondrial complex harboring three major enzymes from the β-oxidation cycle of long-chain fatty acids. TFPα deficiencies present in neonates as a severe cardiac phenotype, often with death in the first weeks. Deficiency is related to maternal HELLP (hydrolysis, elevated liver enzyme and low platelets) syndrome and reduced birth weight [31]. HADHA is involved in long-chain fatty acid-induced autophagy of intestinal epithelial cells and is therefore proposed as a new therapeutic target for inflammatory bowel disease (IBD) [32]. A functional relationship may exist between Parkin and HADHA.

Prohibitin (PHB, Murine Only)
Two members of the prohibitin family, PHB1 and PHB2, are highly homologous proteins localized to the mitochondrial inner membrane [33,34]. The PHB complexes perform diverse functions in mitochondria, including regulation of membrane protein degradation, chaperones, regulation of oxidative phosphorylation, maintenance of mitochondrial genetic stability, and regulation of mitochondrial morphology [33,34]. PHB1 and 2 also function as autophagy receptors [35][36][37][38]. PHB2 directly interacts with LC3 potentially regulating the mitophagy process [37]. In platelets, prohibitin is expressed in membranes and is involved in PAR1-mediated platelet aggregation [39]. Here, we confirmed that prohibitin can interact with Parkin in murine platelets but not in human DM platelets ( Figure 3B).
Protein Disulfide-Isomerase (PDI, P4HB) Protein disulfide isomerase (PDI) was identified 20 years ago as an endoplasmic reticulum protein that facilitates the formation of correct disulfide bonds in nascent proteins [57]. There are more than 20 members of the PDI, seven containing a CGHC-active site [57]. Among CGHC-active site members, four are associated with platelet function and thrombosis (PDI, ERp57, ERp72, and ERp5) [57]. PDI was the first from this protein family identified in integrin-mediated platelet aggregation, adhesion, and thrombosis [58,59]. Here, in our LC-MS/MS results, we identified PDIA 1, 3, 5, and 6 as Parkin-interacting proteins (Tables 1-3). Parkin-associated integrin-mediated platelet function, homeostasis, and thrombosis are dependent on PDIs to form appropriate disulfide bonds.
Protein Disulfide-Isomerase (PDI, P4HB) Protein disulfide isomerase (PDI) was identified 20 years ago as an endoplasmic reticulum protein that facilitates the formation of correct disulfide bonds in nascent proteins [57]. There are more than 20 members of the PDI, seven containing a CGHC-active site [57]. Among CGHC-active site members, four are associated with platelet function and thrombosis (PDI, ERp57, ERp72, and ERp5) [57]. PDI was the first from this protein family identified in integrin-mediated platelet aggregation, adhesion, and thrombosis [58,59]. Here, in our LC-MS/MS results, we identified PDIA 1, 3, 5, and 6 as Parkin-interacting proteins (Tables 1-3). Parkin-associated integrin-mediated platelet function, homeostasis, and thrombosis are dependent on PDIs to form appropriate disulfide bonds.

Integrin Linked Kinase
Integrin-linked kinase was reported to interact with the cytoplasmic tail of β-integrin subunits and its serine/threonine kinase activity is upregulated through platelet stimulation [60][61][62]. ILKs have functions as adaptor proteins, interacting and regulating β1 and β3 integrin subunits [63]. ILK-deficient mice exhibit reduced platelet activation and aggregation and increased bleeding [60,62]. ILK regulates the rate of platelet activation rates and is essential for the formation of stable thrombi by controlling platelet response rates to collagen via GPVI [62]. From this, Parkin may regulate platelet functioning by binding with integrin and its related proteins like ILK (49.1% coverage, Table 1).

New Functions of Parkin and Parkin-Interacting Proteins in Platelets
Through GO and KEGG analysis in conjunction with literature reviews, we present and support additional Parkin functions through confirmed Parkin-interacting proteins. Key platelet functions including activation, aggregation, and mitochondrial functions appear to be regulated, at least partially, by Parkin. We previously verified Parkin-dependent mitophagy activation in a T2DM mouse model [9,14]. To confirm the importance of Parkin in platelets, we incorporated Parkin KO mice platelets. In Parkin KO mice platelets, there was increased cytochrome C and active caspase3 indicative of increased platelet apoptosis ( Figure 5A). The platelet activation maker, CD62P (pSelectin), was slightly decreased but not significantly. The LC-MS/MS analysis verified the functional relationship between platelet activation-associated proteins and Parkin (Tables 1-3), suggesting that Parkin regulates platelet activation. We then used a 14-3-3 inhibitor to interrupt the interaction with Parkin ( Figure 5B) which decreased platelet aggregation induced by collagen through 14-3-3 inhibitor treatment.

Discussion
We previously verified that Parkin expression levels were high in HC platelets, even more so in DM [9,14] and underwent post-translational modification (MetO) under oxidative stress [14]. Cysteine oxidation is significantly increased in PD and includes methionine oxidation [64]. We confirmed that the ubiquitylation and methionine oxidation that occurs in Parkin lead to mitophagy. In this study, we aimed to uncover and verify other key platelet functions regulated by Parkin through the identification of Parkin-interacting proteins using IP and LC-MS/MS. Through this proteomic approach, we provided experimental and literature evidence that Parkin may regulate various processes beyond mitophagy, including integrin-dependent signaling, mitochondrial energy metabolism, platelet activation/aggregation, and ER-mitochondrial cross-talk ( Figure 6). Furthermore, surface membrane receptor interactions with Parkin provide a possible link between external signaling and internal cellular processes. In conclusion, our results suggest that Parkin with its many actions on platelet mitochondria, activation, and aggregation may be a therapeutic target for antiplatelet treatment. However, further detailed molecular studies are required that focus on the individual binding partners of Parkin in DM platelets.
Parkin transfers the changes in the external environment, like diabetes, to the internal environment by interacting with membrane receptor proteins, like integrin, inducing mitophagy. Parkin also indirectly interacts with cytosolic proteins, F-actin functional ARP4/5, and directly with VCP/p97 associated with mitophagy activation. PINK1 is well-known to interact with Parkin and VCP/p97, after which this complex regulates dendritic arborization [65]. Based on these results, we hypothesize that Parkin regulates organelle cross-talk potentially transferring external signals to internal platelet environments and cell-cell cross-talk among other cells. Proteomic analyses also suggest that Parkin participates in diverse processes and mitophagy induction in DM platelets, like phagophore recruitment to damaged mitochondria through interactions with MsrB2 [14] and regulation of autolysosome formation through interactions with LAMP1. Parkin-deficient mice exhibit increased apoptotic platelets compared with healthy control mice ( Figure 5A) [9]. Parkin interacts with 14-3-3, PDIA, FREMT3, ILK, and F-actin-related proteins (well-known proteins associated with platelet activation and aggregation), and so we suggest that Parkin is also involved with platelet aggregation and activation ( Figure 6). In DM platelets, mitochondrial outer membrane is disrupted with mitochondrial protein release and exposed inner membrane proteins [14]. Consequently, the interaction between Parkin and HADHA and prohibitin may occur in human and murine DM platelets ( Table 2), suggesting that Parkin is likely to participate in platelet energy metabolism in DM ( Figure 6). Parkin transfers the changes in the external environment, like diabetes, to the internal environment by interacting with membrane receptor proteins, like integrin, inducing mitophagy. Parkin also indirectly interacts with cytosolic proteins, F-actin functional ARP4/5, and directly with VCP/p97 associated with mitophagy activation. PINK1 is well-known to interact with Parkin and VCP/p97, after which this complex regulates dendritic arborization [65]. Based on these results, we hypothesize that Parkin regulates organelle cross-talk potentially transferring external signals to internal platelet environments and cell-cell cross-talk among other cells. Proteomic analyses also suggest that Parkin participates in diverse processes and mitophagy induction in DM platelets, like phagophore recruitment to damaged mitochondria through interactions with MsrB2 [14] and regulation of autolysosome formation through interactions with LAMP1. Parkin-deficient mice exhibit increased apoptotic platelets compared with healthy control mice ( Figure 5A) [9]. Parkin interacts with 14-3-3, PDIA, FREMT3, ILK, and F-actin-related proteins (well-known proteins associated with platelet activation and aggregation), and so we suggest that Parkin is also involved with platelet aggregation and activation ( Figure 6). In DM platelets, mitochondrial outer membrane is disrupted with mitochondrial protein release and exposed inner membrane proteins [14]. Consequently, the interaction between Parkin and HADHA and prohibitin may occur in human and murine DM platelets ( Table 2), suggesting that Parkin is likely to participate in platelet energy metabolism in DM ( Figure 6).
In conclusion, our results suggest that Parkin with its many actions on platelet mitochondria, activation, and aggregation may be a therapeutic target for antiplatelet treatment. However, further detailed molecular studies are required that focus on the individual binding partners of Parkin in DM platelets.

Preparation of Human Platelet
Venous blood was drawn from healthy and patients at Yale University School of Medicine (HIC#1005006865) from multiple outpatient clinics including the cardiovascular, diabetes, and neurology clinics. Informed consent was obtained from all subjects, and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report (IRB#1006006865, 5/11/2017). All healthy subjects were free from medication or diseases known to interfere with platelet function [5,6,9]. Upon informed consent, a venous blood sample (approximately 20 cc) was drawn by standard venipuncture and collected into tubes containing 3.8% trisodium citrate (w/v). Blood samples were prepared as previously described [66]. Platelet-rich plasma (PRP) was obtained by differential centrifugation. Purity of platelet preparation was determined by Western blot analysis using platelet markers (CD41), monocyte markers (CD14), and red blood cell markers (CD235a) [9].

Preparation of Mice Platelets
Blood (0.7-1 mL) was directly aspirated from the right cardiac ventricle into 1.8% sodium citrate (pH 7.4) in WT (C57Bl/6) and diabetic mice (mice were 8 weeks of age; STZ injected for 5 days followed by high-fat diet for 12 weeks). Citrated blood from several mice with identical genotype was pooled and diluted with equal volume of HEPES/Tyrode's buffer. PRP was prepared by centrifugation at 100 g for 10 min and then used for Immunoprecipitation and Western blotting. All mice were of

Western Blotting
Standard Western blot analysis protocols were used. A 10% Input of IP lysates was loaded in each well as loading control. We used specific individual antibodies and dilutions (Parkin: abcam #ab15954

Immunoprecipitation
The 500 µg pulled healthy/DM platelet lysates and cell lysates (after transient transfection) were mixed with 1 µg specific target antibodies and the same species IgG control with HC) and incubated overnight at 4 • C. Then 50% slurry protein A sepharose beads and 50% slurry protein G sepharose beads were mixed 1:1. Next, 30 µL of the 50% slurry washed A/G beads with lysates/antibodies mixture was incubated for 1 h at 4 • C. After 3 more washes with lysis buffer, we used 1-10% lysates were used for the input. We pooled 4 healthy subjects and 11 human DM platelets (5 pooled in DM1 and 6 pooled in DM2) for Parkin immunoprecipitation ( Figure 3A) and pooled 3 WT and 5 DM mice platelets ( Figure 3B).

Silver Staining and LC-Mass Analysis
Mass spectrometry was performed according to the manufacturer's protocols by Pierce silver stain, and individual bands were excised for LC-MS/MS after silver staining. In-gel digestion, LC-MS/MS, and peptide identification were performed by Yale MS & Proteomics Resource.
In-Gel Digestion: Silver-stained gel bands were treated with 5% acetic acid for 10 min with rocking. The acid was removed, and the bands were covered with freshly prepared destaining solution (made fresh by mixing in a 1:1 ratio stock solutions of 30 mM potassium ferricyanide in water and 100 mM sodium thiosulfate in water) until the brownish color disappeared. The bands were then rinsed three times with 0.5 mL of water for 5 min to remove the acid and chemical reducing agents. The gel bands were cut into small pieces and washed for 30 min on a tilt-table with 450 µL 50% acetonitrile/100 mM NH 4 HCO 3 (ammonium bicarbonate) followed by a 30 min wash with 50% acetonitrile/12.5 mM NH 4 HCO 3 . The gel bands were shrunk by the brief addition then removal of acetonitrile, and then dried by speed vacuum. Each sample was resuspended in 100 µL of 25 mM NH 4 HCO 3 containing 0.5 µg of digestion grade trypsin (Promega, V5111) and incubated at 37 • C for 16 h. Supernatants containing tryptic peptides were transferred to new Eppendorf tubes and the gel bands were extracted with 300 µL of 80% acetonitrile/0.1% trifluoroacetic acid for 15 min. Supernatants were combined and dried by speed vacuum. Peptides were dissolved in 24 µL MS loading buffer (2% acetonitrile, 0.2% trifluoroacetic acid) with 5 µL injected for LC-MS/MS analysis.
LC-MS/MS on the Thermo Scientific Q Exactive Plus: LC-MS/MS analysis was performed on a Thermo Scientific Q Exactive Plus equipped with a Waters nanoAcquity UPLC system utilizing a binary solvent system (A: 100% water, 0.1% formic acid; B: 100% acetonitrile, 0.1% formic acid). Trapping was performed at 5 µL/min, 97% Buffer A for 3 min using a Waters Symmetry ® C18 180 µm and 20 mm trap column (Waters, USA). Peptides were separated using an ACQUITY UPLC PST [67] C18 nanoACQUITY Column 1.7 µm, 75 µm × 250 mm (37 • C) and eluted at 300 nL/min with the following gradient: 3% buffer B at initial conditions; 5% B at 1 min; 35% B at 50 min; 50% B at 60 min; 90% B at 65 min; 90% B at 70 min; return to initial conditions at 71 min. MS was acquired in profile mode over the 300-1700 m/z range using 1 microscan, 70,000 resolution, AGC target of 3E6, and a maximum injection time of 45 ms. Data dependent MS/MS were acquired in centroid mode on the top 20 precursors per MS scan using 1 microscan, 17,500 resolution, AGC target of 1E5, maximum injection time of 100 ms, and an isolation window of 1.7 m/z. Precursors were fragmented by HCD activation with collision energy of 28%. MS/MS were collected on species with an intensity threshold of 2E4, charge states 2-6, and peptide match preferred. Dynamic exclusion was set to 20 s.
Peptide Identification: Tandem mass spectra were extracted by Proteome Discoverer software (version 1.3, Thermo Scientific) and searched in-house using the Mascot algorithm (version 2.6.0, Matrix Science). The data were searched against the SwissProt database (version 2017_01) with taxonomy restricted to Homo sapiens (20,172 sequences). Search parameters included trypsin digestion up to 2 missed cleavages, peptide mass tolerance of 10 ppm, MS/MS fragment tolerance of 0.02 Da, and methionine oxidation and propionamide adduct to cysteine as variable modifications. Normal and decoy database searches were run with the confidence level set to 95% (p < 0.05).