endogenous negative modulators of platelet function · endogenous negative modulators of platelet...

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Abstract. – Platelets are megakaryocyte-de-rived nuclear-free fragments that participate in cardiovascular diseases including acute myo-cardial infarction, ischemic stroke, hyperten-sion, and atherosclerosis. At the endothelium damage site, platelets interact with sub-endo-thelial matrix proteins such as glycoprotein VI/Fc receptor γ-chain (GPVI/FcRγ), G protein-cou-pled receptor/phospholipase Cγ(β) (GPCR/PLCγ(β)), Rho/RhoK and integrin. The activa-tion of these signaling pathways triggers intra-cellular calcium increase and causes platelet adhesion, aggregation, granule release and fi-nally thrombus formation. Some endogenous platelet modulators reported to negatively reg-ulate this process are: (1) platelet surface inhib-itory receptors: carcinoembryonic antigen cell adhesion molecule 1, 2 (CEACAM 1, 2), plate-let endothelial cell adhesion molecule-1 (PE-CAM-1), and G6b-B; (2) nuclear receptors: ret-inoic X receptor (RXRs), glucocorticoid recep-tor (GR), peroxisome proliferator-activated re-ceptors (PPARs) and liver X receptors (LXRs); (3) intracellular adaptor proteins: CLP36, paxil-lin, downstream of tyrosine kinase (Dok), c-Ca-sitas B-lineage lymphoma (c-Cbl), protein ki-nase Cδ (PKCδ), glycogen synthase kinase(G-SK)-3β, phospholipase D2 (PLD2), peroxiredox-in II (PrxII), T-cell ubiquitin ligand-2 (TULA-2); (4) extracellular modulators released from platelet granules: adapter protein disabled-2 (DAB2) and diadenosine 5,5-P1, P2-diphosphate (Ap2A). The discovery of biological or endogenous modula-tors of platelet activation is regarded as a poten-tial therapeutic target for thrombotic disease. This review highlights the recent findings on the endogenous negative regulatory molecules re-leased from platelets and their impact on plate-let thrombus formation.

Key Words:Platelet, Adhesion molecules, Nuclear receptor,

Adaptor proteins, Thrombosis.

Introduction

Platelet activation, adhesion, and aggregation on the sub-endothelial collagen matrix is an important process in normal hemostasis. How-ever, abnormal platelet activation under patho-logical conditions may lead to thrombosis and ischemia1,2. Designing of drugs targeting the platelet function is an effective strategy for the treatment of thrombotic diseases. Sodium nitrite can inhibit platelet aggregation3. In patients with type 2 diabetes (TD2M), pioglitazone may be more effective in inhibiting activation of plate-lets than glipizide4, although there are concerns regarding drug resistance. Therefore, it is crucial to explore the endogenous molecules that inhibit platelet function. Under normal circumstances, platelet homeostasis is regulated by activation and inhibitory signals. Inhibitory signals domi-nate over activation signals under physiological conditions keeping platelets in blood circulation in stable state. When platelets encounter lesion sites, abnormal platelet activation is initiated, which might cause the development of thrombo-sis. The contribution of platelets to hemostasis and thrombosis may, therefore, be considered like a rheostat, instead of an “on/off switch”5. Activated platelets release molecules such as ADP, thromboxane (TX) A2, serotonin, epi-nephrine, and tachykinins, which are regarded as pro-thrombotic factors. Several researches have demonstrated the existence of endogenous inhibitory molecules and regulatory mechanisms of platelet function. Moreover, these molecules are well-recognized anti-thrombotic targets. Healthy vascular endothelium produces and re-leases active molecules into the circulating blood of which, nitric oxide (NO) and prostacyclin I2 (PGI2), are the most potent inhibitors of platelet

European Review for Medical and Pharmacological Sciences 2017; 21: 3146-3158

Y.-J. LI1, H.-X. ZHU2, D. ZHANG1, H.-C. LI1, P. MA1,3, L.-Y. HUANG1

1School of Medical Technology, Xuzhou Medical University, Xuzhou, Jiangsu Province, China2Clinical Medicine Department, Xuzhou Medical University, Xuzhou, Jiangsu Province, China3Department of Medical Laboratory, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu Province, China

Corresponding Author: Linyan Huang, Ph.D; e-mail: [email protected] Ma, MD; e-mail: [email protected]

Novel endogenous negative modulators of platelet function as potential anti-thrombotic targets

Endogenous negative modulators of platelet function

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activation6. However, when the integrity of the vascular endothelium is compromised by blood vessel damage or vascular disease, roles of NO and PGI2 on platelet activation are weakened or even suppressed. So NO and PGI2 are not includ-ed into our consideration for this purpose, mean-while some exogenous chemicals are also exclud-ed. Platelet surface inhibitory receptors contain-ing platelet endothelial cell adhesion molecule-1 (PECAM-1), carcinoembryonic antigen cell ad-hesion molecule 1, 2 (CEACAM 1, 2) and G6b-B have been studied for decades. Furthermore, nuclear receptors and intracellular adaptor pro-teins, such as LIM domain family, downstream of tyrosine kinase (Dok) and c-Casitas B-lineage lymphoma (c-Cbl), are involved in assembling downstream signaling complexes after platelet activation by agonists especially collagen and collagen-related peptide (CRP) upon binding to GPVI-FcRg chain. Some signal molecules, such as protein kinase Cδ (PKCδ), glycogen synthase kinase (GSK)-3β, (GSK3β) and phospholipase D2 (PLD2) also facilitate platelet inactivation. Besides, once activated, platelets release active molecules from its granular stores into the blood, of which adapter protein disabled-2 (DAB2) and diadenosine 5,5-P1,P2-diphosphate (AP2A) are typically known to negatively modulate platelet function. Therefore, in this review we highlight several important negative modulators of platelet function during platelet activation.

Inhibitory Receptors on the Surface of Platelets

At the vascular injury site, the sub-endothelial matrix such as collagen is exposed and resting platelets are immediately activated to interact with the collagen. This process is called early platelet activation6. The interaction of platelets and collagen is mediated by integrin α2β1

7 and the GPVI/FcRγ-chain complex8, whereas platelet activation by laminin is mediated by the related integrin α6β1

7, and similarly requires signaling via GPVI/FcR γ-chain8. Hence, GPVI/FcR γ-chain signal largely dominates collagen-mediated plate-let response. At least five immunoreceptors ty-rosine-based inhibitory motif (ITIM)-bearing receptors namely recruit protein tyrosine phos-phatases SHP-1 and SHP-29 to down-modulate platelet-collagen interactions PECAM-1, CEA-CAM-1/2, G6B, TREM-like transcript 1(TLT1) which are expressed on platelets via Src homol-ogy-2 (SH2) domain10-15. Although individually these receptors exert moderate inhibition of colla-

gen or thrombin signaling in platelets, collective-ly they regulate the cell-contact signaling events involved in the initiation of platelet thrombus for-mation to limit occlusive pathological thrombi. In contrast to other ITIM receptors, TLT1 enhanced rather than inhibited FcζR I-mediated calcium signaling in rat basophilic leukemia cells16.

CEACAM Super Family

CEACAM1 CEACAM1 is expressed on the surface and in

intracellular pools of resting murine and human platelets8, which has two isoforms: long ITIM isoform (CEACAM1-L; 70-73 amino acids) and short ITIM-less isoform (CEACAM1-S; 10-12 amino acids)17. CEACAM1-L protein has two ITIMs that bind SHP-1 and SHP-2 to dephosphor-ylate Src family kinases (SFKs). Inactive SFKs cannot phosphorylate collagen-mediated GPVI/FcR γ-chain downstream signals. Therefore, the CEACAM1 inhibits platelet activation. As demonstrated, CEACAM1-/- mice were more sus-ceptible to thrombosis, and CEACAM1-/- platelets displayed enhanced type I collagen and CRP-me-diated platelet aggregation, adhesion and dense granule secretion8.

CEACAM2CEACAM2 negatively regulate collagen-medi-

ated GPVI/FcRγ-chain and the C-type lectin-like receptor 2 (CLEC-2) signaling15. Compared with wild-type platelets, CEACAM2-/- platelets had elevated platelet aggregation, adhesion, and dense granule release. Furthermore, thrombi formed in CEACAM2-/- mice were larger and more stable than in wild-type controls in vivo. Unlike CEA-CAM1, CEACAM2 can negatively regulate both collagen GPVI and CLEC-2 ITAM signaling pathways in platelets15. In a physiological arterial shear flow analysis in vitro, CEACAM2 defect increased the surface coverage of platelets on immobilized type I collagen, but not thrombin or PAR-4 agonist peptide. This platelet aggregation reaction was different from that of CEACAM1-/- platelets15. CEACAM1-/- platelets had compara-ble amplitude and slope of thrombin-mediated aggregation response compared with wild-type platelets8. These data demonstrate that platelet re-sponses are orchestrated by both CEACAM1 and CEACAM2 through the GPVI-FcR-γ chain-inte-grin α2β1. However, both CEACAM1 and CEA-CAM2 have no effect on G-protein-coupled re-

Y.-J. Li, H.-X. Zhu, D. Zhang, H.-C. Li, P. Ma, L.-Y. Huang

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ceptor (GPCR) signaling. Despite these findings, recent observations have revealed controversial questions that CEACAM1 and CEACAM2 were negative modulators in GPVI/FcRγ-chain sig-naling on platelet activation. CEACAM1/2 is essential for normal integrin αIIbβ3-mediated platelet function. Disruption of mouse Ceacam1/2 induced integrin αIIbβ3-mediated functional defects18,19. In resting platelets, CEACAM1/2 is constitutively associated with integrin αIIbβ3. CEACAM1-/- mice had prolonged tail- bleeding time and high blood loss, which was rescued by supplementation with CEACAM1/2 platelets18. CEACAM1/2-/- platelets exhibited kinetic defects in platelet fibrinogen spreading and impaired re-traction of the fibrin clot in vitro. However, CEA-CAM1/2-/- platelets displayed normal “inside-out” signaling properties19. Therefore, CEACAM1/2 is essential for normal integrin αIIbβ3-mediated platelet function.

PECAM-1PECAM-1 has been shown to be expressed

on platelets and hematopoietic cells20,21. The PE-CAM-1 protein contains a 574 amino acid extra-cellular domain, a 19 amino acid transmembrane domain, and a cytoplasmic domain. In addition, PECAM-1 also possesses two ITIMs, which re-cruits Src homology 2 (SH2) domain-containing proteins such as protein tyrosine phosphatase 2 (SHP-2), during inflammatory response22. Follow-

ing platelets adhesion to immobilized laminin, PECAM-1 is tyrosine phosphorylated on its cy-toplasmic ITIM-motifs. At the same time, the GPVI-FcRγ-chain signaling is activated. Both of these processes lead to the activation of integrin α6β1. However, laminin-induced tyrosine phos-phorylation of PECAM-1, Syk and the FcR-γ chain was observed after 15-30 min while PECAM-1 inhibited thrombus formation in vivo 5-10 min af-ter vascular injury7. Stimulation of the PECAM-1 signaling at high concentrations of agonists (colla-gen, CRP and convulins) reduces platelet aggre-gation23. Consequently, this leads to decrease in platelet secretion, calcium mobilization, inositol phosphates production, and total protein tyrosine phosphorylation. Consistent with the inhibition of PECAM-1 in vitro, PECAM-1-deficient platelets exhibited hyper-reactivity to collagen or CRP. In addition, platelet aggregation was enhanced at low agonist concentrations and the threshold for the release of serotonin or ATP from dense gran-ules was reduced. This led to increased thrombus formation in vitro on immobilized Type I colla-gen under arterial flow conditions11,12. Taken to-gether, these observations indicate that the three types of adhesion molecules present on the plate-let surface and intracellular pools (CEACAM1/2 and PECAM-1), once bound to their correspond-ing ligands, result in the inhibition of platelet adhesion and aggregation and release particles by their ITIMs (Figure 1).

Figure 1. The platelet surface inhibitory receptors PECAM-1, G6b-B and CEACAM-1 inhibit integrin inside-out signaling. These three receptors contain ITIM/ITSM in the cytoplasmic tails, which bind the SH2 domain containing non-transmembrane PTPs, SHP-1 and SHP-2. Then SHP-1/2 dephosphorylates src family kinases, which prevent SFKs from activating integrin αIIbβ3. The conformation change from inactive to active provides binding sites for fibrinogen or vWF factor through RGD domains.

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G6b-BG6b also belongs to the immunoglobulin su-

perfamily and has two ITIMs, which under-go alternative splicing to produce G6b-A and G6b-B isoforms24. Tyrosine phosphorylation of GPVI-FcRγ-chain and CLEC-2 is critical for the interaction of G6b with SHP-1 and SHP25. G6b-B is closely related to platelet production and func-tion. G6b-B gene knockout in mice was associat-ed with a bleeding tendency. Megakaryocytes in G6b-B-deficient mice showed enhanced metallo-proteinase production, which led to increased hy-drolysis of cell-surface receptors, including GPVI and GPIbα. In addition, G6b-B-deficient mega-karyocytes displayed reduced integrin-mediated functions and defective formation of proplate-lets. Thus, G6b-B is a major inhibitory receptor regulating megakaryocyte activation and platelet production26.

Nuclear Receptors

RXRsRetinoid X receptors (RXRs) consist of three

nuclear receptor isoforms (α, β, and γ) that are activated by 9-cis-retinoic acid (9cRA)27-29. 9cRA inhibit ADP-induced platelet aggregation, but its isomer all-trans-retinoic acid, which is not an activator of RXRs, has no effect on platelet ag-gregation30. This demonstrates that RXRs have a pharmacological activity that inhibits the positive feedback of additional ADP and TXA2 produced by the activated platelets. ADP and TXA2 re-ceptors in platelets link predominantly to Gq, Gi, and Gq/G12/13 G proteins31-33. Human Gq contains a single LXXLL motif, usually located on coactivators interacting with nuclear receptors in its n-terminal region34,35. When incubated with 9cRA, RXRs rapidly binds Gq in a ligand-de-pendent manner and inhibits Gq-induced Rac ac-tivation and intracellular calcium release36. Since RXRs ligands are involved in the anti-atheroscle-rotic effects associated with inhibition of platelet activation, RXRs ligands have significant effects in reducing the progression of atherosclerosis in apoE knockout mice37 (Figure 2).

GRThe glucocorticoid receptor (GR) belongs to

the nuclear hormone receptor superfamily. Glu-cocorticoids bind to the GR and exert anti-in-flammatory effects through nuclear transloca-tion or transrepression of transcription factors

and rapid inhibitory action on nuclear factor κB activation38,39. GR in platelets forms het-erodimeric complexes with mineralocorticoid receptor, which is activated by its specific li-gands40. Platelets acts like pools of various pro-inflammatory mediators, which are rapidly released at sites of endothelial injury41,42. After addition of prednisolone, a corticosteroid, to platelet rich plasma, decreased platelet aggrega-tion significantly, while dexamethasone had no significant effect on reduction of aggregation43. Platelet aggregation is inhibited by thromboxane B2 (TXB2), 12(S)-hydroxyeicosatetraenoic acid (12-(S)-HETE), and 12(R)-hydroxyeicosatetrae-noic acid (12-(R)-HETE). Furthermore, human experiments confirmed that this phenomenon was related to the production of TXB2, an in-hibitor of platelet aggregation. These results indicate that prednisolone-mediated produc-tion of TXB2 was stronger than dexametha-sone-mediated production of 12-S-HETE and 12-R-HETE43. Therefore, platelet GR is not only a target for anti-thrombosis, but also a poten-tial therapeutic target of vascular inflammation. Numerous studies have also reported that pred-nisolone and dexamethasone can suppress mito-chondrial oxidative phosphorylation by sodium azide through the reduction of ROS production in platelets44. Whether the reduction of ROS in platelets is another mechanism that may orches-trate the glucocorticoids-mediated decrease in platelet function is a matter that warrants further investigation.

PPARsPeroxisome proliferator-activated receptors

(PPARs) is a family of three nuclear recep-tor isoforms (α, β/δ, and γ) that modulate tran-scription of the target genes45. Rosiglitazone and prostaglandin, 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2), are synthetic ligands for PPARγ that have clinical use46. Rosiglitazone and 15d-PGJ2 inhibited collagen-stimulated platelet aggrega-tion and thrombus formation under arterial flow conditions, and this effect was mediated, at least in part, through binding to PPARγ in platelets. Furthermore, PPARγ was found to associate with Syk and LAT after platelet activation. Moreover, PPARγ ligands inhibit tyrosine phosphorylation levels of multiple components of the GPVI sig-naling pathway. Besides, PPARγ ligands have been reported to inhibit platelet aggregation in response to ADP accompanied by a reduction in markers of platelet activation such as P-selectin


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exposure, TXA2 synthesis and sCD40L release47. In conclusion, these findings indicate that PPAR ligands inhibit platelet activation through attenu-ation of GPVI and GPCR signals.

LXRsLiver X receptors (LXRs) is another member of

the nuclear receptor superfamily, whose natural ligands, oxysterols, are cholesterol derivatives48. LXRs maintain cholesterol homeostasis by reg-ulating the transcription of genes associated with lipid metabolism, such as cytochrome P4507 al-pha-hydroxylase 1 (Cyp7a1) and apolipoprotein E (ApoE)49. LXRs ligands, GW3965 and T0901317, modulate a series of agonist-stimulated non-ge-nomic platelet aggregation by acting on LXR-β50. These data illustrate that GW3965 caused LXR to associate with the signaling components prox-imal to the collagen receptor, GPVI, suggesting a potential mechanism of LXRs action in platelets

that leads to diminish platelet responses. These results indicate that GW3965 has antithrombotic effects and reduce the size and stability of throm-bi51. These atheroprotective effects of GW3965, together with its novel anti-platelet/thrombotic effects, indicate that LXRs is a potential target for prevention of athero-thrombotic disease51.

Intracellular Adaptor Proteins

LIM-Domain Family Members Several LIM-domain family members (such

as CLP36 and Paxillin) communicate with plate-let activation. The CLP36 is an adaptor protein containing PDZ domain at the N-terminus, a short linker region, and the LIM-domain at the C-terminus52. Paxillin on the other hand, contains two conserved structural domains, the N-terminus and C-terminus, which consists of

Figure 2. The platelet nuclear receptors RXR, GR, PPAR and LXR inhibit platelet response. Once ligand specifically activates RXR, RXR interacts with Gq to hinder Rac phosphorylation and activation of downstream Rho pathway. GR binds to transcription factor controlling the expression of anti-inflammatory factors including TXB2 and ROS to prevent platelet activation. PPAR and LXR both bind with SFK to impede SFK activation and subsequent platelet response.


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four LIM-domains53. Leupaxin and Hic-5 have been identified as variants of paxillin on murine platelets54. However, only Hic-5 has been shown to be predominantly expressed in human plate-lets55.

CLP36CLP36, the only PDZ-LIM family mem-

ber expressed in platelets, negatively regulat-ed platelets granule secretion, aggregation, integrin activation, and thrombus formation56. CLP36-defecient platelets were sensitive to the agonists of CRP, collagen and convulxin rather than ADP and thrombin. This was associat-ed with hyperphosphorylation of GPVI down-stream signaling proteins and enhanced Ca2+ mobilization, granule secretion, and integrin activation. Moreover, GPVI-induced tyrosine phosphorylation of PLCγ2, Src family kinas-es (including Yes, Fyn, Lyn), and FcRγ chain, as well as inositol-1,4,5-trisphosphate produc-tion, and Ca2+ mobilization were increased in CLP36-defecient function platelets56.

Paxillin Paxillin, another LIM-domain family mem-

ber, is localized in platelets57. Paxillin-/- platelets were slightly enlarged and had elevated integrin αIIbβ3 activation following stimulation of both GPVI and GPCRs, which was different to CLP36-

/- platelets58. Thromboxane A2 biosynthesis and the release of α and dense granules following platelet adhesion and thrombus formation in vivo were also augmented in paxillin-/- mice. Hic-5-de-ficient mice exhibited prolonged tail bleeding times. In addition, thrombin rather than con-vulxin, U46619, and ADP slightly impaired the activation of integrin αIIbβ358. It is not yet known whether these platelet responses are similar in human (Figure 2).

Dok Dok adaptor proteins are expressed on both

human platelets (Dok-1, Dok-2 and Dok-3) and mouse platelets (Dok-1 and Dok-2)59-61. Dok-1 is a negative modulator of platelet integrin αIIbβ3 that involved in the ‘outside-out’ signaling61. It was shown to enhance platelet spreading on fi-brinogen and clot retraction, increase phosphory-lation of PLCγ2, and accelerate arterial thrombo-sis in Dok-1-deficient mice, but did not affect the ‘inside-out’ signaling, which displayed normal aggregation, P-selectin expression and soluble fibrinogen binding62.

c-Cblc-Cbl is a universally expressed adaptor pro-

tein that function in hematopoietic cells63. It can be phosphorylated at tyrosine residues and interact with proteins that contain the SH2 do-main64,65. C-Cbl is phosphorylated in activated human platelets, whereas its phosphorylation is decreased in Fyn- and Lyn-deficient platelets65. In c-Cbl mutant platelets, both CRP and throm-bin-induced platelet aggregation in the GpVI sig-nal pathway were increased66. This confirms that c-Cbl is an important negative modulator of the GpVI signaling in regulating platelet activation (Figure 3).

Inhibitory Molecules Involved in the Platelet Activation Pathway

PKCδThe protein kinase C (PKC) family plays a

role in the regulation of exocytosis and cell adhe-sion67,68. Based on the previous research, various PKC isoforms have been shown to act as positive regulators of platelet cytoskeleton reorganization and aggregation69. However, Pula et al70 proposed that PKCδ may have a negative role in platelet filopodia formation, actin polymerization, and platelet aggregation. Using PKC-selective inhib-itor, they showed that the vasodilator-stimulated phosphoprotein (VASP), a marker of platelet skel-eton organization, was increased70. Moreover, PKCδ has also been shown to negatively influence platelet formation in vitro71. Hence, PKCδ plays a crucial role in the formation and activation of platelets, as well as in stabilization of thrombus.

GSK-3βGlycogen synthase kinase (GSK3β) is a serine/

threonine kinase regulated by Akt phosphoryla-tion on ser 972. It has been shown to possess an-ti-melanogenic effects of Aster spathulifolius and to suppress metastasis of hepatocellular carcino-ma cells73. ADP and thrombin regulate platelet function through the GPCR complex and phos-phoinositide 3-kinase( PI3K)/Akt pathways74. In PI3K and Akt deficient mice, platelet adhesion and aggregation were reduced, suggesting that PI3K and Akt are positive regulators of platelet function75. Considering that the kinase GSK3β is a downstream effector of Akt, whether it is a positive or negative regulator of platelet function is not clear. However, Akt negatively regulated the GSK3β activity by phosphorylating GSk3β


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ser9 site activity73,74. This might be due to sus-tained suppression of GSK3β substrates release76. This hypothesis was confirmed by in vivo and in vitro experiments. In fact, GSK3β+/- platelet had enhanced agonist-induced platelet aggregation, dense granule release and fibrinogen binding. GSK inhibitors also confirmed this phenomenon. Indeed, an in vivo model of thrombosis proved that GSK3β+/- mice were more sensitive to throm-bosis. Therefore, GSK3β acts as a negative reg-ulator of platelet function in both in vitro and in vivo75,76.

PLD2Phospholipase D (PLD) catalyzes the hydroly-

sis of phosphatidylcholine into phosphatidic acid (PA) and choline, which participates in platelet activation77. The PLD has two isoforms, PLD1 and PLD2. PLD1 has low basal activity and is activated by PKC and small GTPases (adenos-ine diphosphate (ADP)-ribosylation factor (ARF) and Rho family)78. On the other hand, PLD2 has high basal activity and is activated by a vari-ety of agonists such as collagen, thrombin, and the TXA2-mimetic U4661979. Lack of PLD1 in platelets impaired integrin αIIbβ3 activation and shear dependent thrombus formation. This led to

protection against arterial thrombosis and isch-emic brain infarction while degranulation and aggregation were unaffected80. PLD inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), enhanced dense granule secretion and aggrega-tion on human and mouse wild-type and PLD1-

/- platelets following stimulation with high con-centrations of agonists that activate Gα12/13 and Gαq signaling in platelets81. It seems that PLD1 and PLD2 have different platelet functions in vivo. The enhanced aggregation and secretion in FIPI treatment of PLD1-/- platelets closely related to the high basal activity of PLD2. Hence, PLD2 might be a potential negative regulator of platelet sensitivity81.

PrxIIPeroxiredoxin II (PrxII) is a cellular perox-

idase that breaks down endogenous hydrogen peroxide (H2O2) produced upon platelet-derived growth factor or epidermal growth factor activa-tion82. H2O2 functions to promote GPVI signaling activation. PrxII deficiency enhanced GPVI sig-naling pathway during platelet activation, which correlated with the accumulation of H2O2 in the body. Reduction of H2O2 increases the oxida-tive inactivation of SHP-2, which results in en-

Figure 3. The intracellular inhibitory signal molecules mainly aim at GPVI-FcR g and GPCR signaling Pathway. The deficiency of CLP36, TULA2, c-Cbl and PrxII universally improve the phosphorylation of GPVI downstream signaling proteins and calcium release (green arrow), while Paxillin defect both affect GPVI and GPCR signaling pathway (green arrow and red arrow). PKC and PI3K/Akt positively regulate integrin activation; however, PKCδ and GSK3β (PI3K/Akt downstream) defect negatively regulate integrin activation.


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hanced tyrosine phosphorylation of key compo-nents of the GPVI signaling cascade83. The PrxII/SHP-2-mediated inhibition of platelet activation appears to occur in lipid rafts. PrxII-deficient platelets showed a marked increase in adhe-sion and aggregation activity in vitro84. The an-ti-thrombotic activity of PrxII was also verified in arterial injury in vivo model 84. Hence, PrxII down-regulates the GPVI signaling and throm-bosis and is recognized as a potential target for anti-platelet and anti-thrombotic therapy.

TULA-2T-cell ubiquitin ligand-2 (TULA-2), is a histi-

dine tyrosine phosphatase, that occurs as the sole family member of TULA in platelets. It is encoded by the ubiquitin associated and SH3 domain-con-taining protein B (UBASH3B) gene85. TULA-2 de-phosphorylated the spleen tyrosine kinase (Syk)86. Ablation of TULA-2 resulted in hyperphosphory-lation of Syk and its downstream effector phospho-lipase C-γ2 as well as enhanced GPVI-mediated platelet responses87. Therefore, TULA-2 functions as a negative regulator of GPVI signaling in plate-lets88. Not only does it negatively regulate murine platelet activation via glycoprotein VI (GPVI)/Fc receptor γ-chain (FcRγ), but also negatively mod-ulates FcγRIIA, which is the sole IgG -receptor present in human platelets89. Interference of en-dogenous murine mmu-miR-148a-3p with locked nucleic acids (anti-miR-148a-3p LNA), upregulated platelet TULA-2. This leads to hypophosphoryla-tion of Syk and protection of thrombocytopenia (HIT), a disorder characterized by low platelet count and thrombosis90.

Extracellular Modulators Released fromPlatelet Granules

DAB2Adapter protein disabled-2 (DAB2) contains

three domains: the N-terminal phosphotyrosine binding (PTB) domain, the aspartic-acid-pro-line-phenylalanine (DPF) motif, and the C-ter-minal proline-rich region91. DAB2 is a modulator released from platelet granules in a PKC-depen-dent manner during platelet activation92. DAB2 negatively regulate the interaction of fibrinogen with integrin αIIbβ3 located in the extracellular region of the platelet surface93. Biochemical and mutational analysis revealed that the DAB2 con-tains the Arg-Gly-Asp (RGD) motif (with amino acid residues 64-66). The binding of fibrinogen

to integrin αIIbβ3 occurs through the RGD mo-tif (amino acid residues 171-464)94. Therefore, the RGD motif of DAB2 competitively occupies the binding region on the integrin αIIbβ3, there-by preventing integrin and fibrinogen binding, which forms the basis of DAB2 inhibition of platelet aggregation.

Ap2AThere are three types of new growth-promoting

extracellular mediators in platelet secretory gran-ules: diadenosine 5,5-P1,P2-diphosphate (Ap2A) , adenosine guanosine diphosphate (Ap2G), and diguanosine diphosphate (Gp2G)95. The ADP-ri-bosyl cyclase (CD38) enzyme synthesizes Ap2A and its two isomers, P18 and P24 from cyclic ADP-ribose and adenine (Ade). The Ap2As are characterized by an unusual N-glycosidic bond between one adenine and the ribose (C1-N1 in P18 and C1-N3 in P24)96,97. Furthermore, it was shown that CD38 was expressed on the human platelet. Presently, it has been proven that Ap2A influences platelet aggregation. Additionally, Ap2A and its isomers were expressed in resting human platelets and were released during throm-bin-induced platelet activation98. They are also involved in the inhibition of platelets aggregation, which indicates a feedback mechanism that limits platelet aggregation through a generation of ade-nylic dinucleotides. Ap2A, P18, and P24 bind to the purinergic receptor P2Y11, thereby increasing intracellular cAMP concentration and production of nitric oxide99. These results suggest a role for Ap2A, P18, and P24 as negative endogenous reg-ulators of platelet aggregation98 (Figure 4).

Conclusions

Small molecules and chemically designed drugs have been widely used to treat cardiovas-cular diseases and malignant tumors. However, limited experiments are carried out in humans and the detrimental effects of long-term use of these drugs have greatly hindered their applica-tion. Therefore, the endogenous regulatory mole-cules present an excellent choice to research. This paper demonstrates a clear role of endogenous negative modulators of platelets. Furthermore, we explore their therapeutic potential especially for their use in thrombosis and atherosclerosis. Further research is advocated to advance and exploit their value as prospective anti-platelet and anticoagulant drugs.


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AcknowledgementsThe author is grateful to the National Natural Science Foun-dation of China (No: 81402918) and the Natural Science Foundation of Jiangsu Province (No: BK20140228) for its assistance, and the support of the China Postdoctoral Sci-ence Foundation Committee (No: 2016M591924).

Conflict of InterestThe Authors declare that they have no conflict of interests.

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Figure 4. The extracellular modulators released from activated platelet granules. Various active substances stored in platelet particles, once the platelet activation happens, will be released from platelets regulated by PI3K /AKT signaling pathway. AP2A binds to P2Y11-coupled Gai receptor, blocks the AC activity, reduces cAMP production but increases NO production, which is the potent inhibitor of platelet aggregation.


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