Platelets at the Vessel Wall in Non-Thrombotic Disease

Abstract

Platelets are small, anucleate entities that bud from megakaryocytes (MKs) in the bone marrow. Among circulating cells, platelets are the most abundant cell, traditionally involved in regulating the balance between thrombosis (the terminal event of platelet activation) and hemostasis (a protective response to tissue injury). While platelets lack the precise cellular control offered by nucleate cells, they are in fact very dynamic cells, enriched in pre-formed ribonucleic acid (RNA) that allows them the capability of de novo protein synthesis which alters the platelet phenotype and responses in physiological and pathological events. Antiplatelet medications have significantly reduced the morbidity and mortality for patients afflicted with thrombotic diseases, including stroke and myocardial infarction (MI). However, it has become apparent in the last few years that platelets play a critical role beyond thrombosis and hemostasis. For example, platelet-derived proteins by constitutive and regulated exocytosis can be found in the plasma and may educate distant tissue including blood vessels. Firstly, platelets are enriched in inflammatory and anti-inflammatory molecules that may regulate vascular remodeling. Secondly, platelet-derived microparticles released into the circulation can be acquired by vascular endothelial cells through the process of endocytosis. Thirdly, platelets are highly enriched in mitochondria that may contribute to the local reactive oxygen species (ROS) pool and remodel phospholipids in the plasma membrane of blood vessels. Lastly, platelets are enriched in proteins and phosphoproteins which can be secreted independent of stimulation by surface receptor agonists in conditions of disturbed blood flow (D-flow). This so-called biomechanical platelet activation occurs in regions of pathologically narrowed (atherosclerotic) or dilated (aneurysmal) vessels. Emerging evidence suggests platelets may regulate the process of angiogenesis and blood flow to tumors as well as education of distant organs for the purposes of allograft health following transplantation. This review will illustrate the potential of platelets to remodel blood vessels in various diseases with a focus on the aforementioned mechanisms.

Insight from platelet formation and removal: where do they come from, where do they go?

Human platelets are small (2 to 4 μm), anucleate cells that bud from the mature megakaryocytes (MKs) in the bone marrow. During megakaryopoiesis, MKs transfer their cytoplasmic organelles, such as ribosomes, granules, and mitochondria at the time of mature platelet budding. Each platelet contains 5–8 functional mitochondria required to generate the chemical energy for the impressive array of biochemical processing required to drive splicosomal processing, protein synthesis and post-translational modification, granular synthesis and packaging, vesicle exocytosis, and protein degradation using the ubiquitin and proteasomal pathways.

Production:

Every day, humans make approximately 10¹¹ new platelets¹ with a circulating lifespan of approximately 8 days. The process of platelet production (thrombopoiesis) requires hepatic and renal secretion of the glycoprotein thrombopoietin (TPO) which is then cleared by platelets and its precursors. TPO receptors (c-Mpl) are found on human adult bone marrow hematopoietic stem/progenitor cells (HSC/PC).^2,3 TPO receptor activation induces differentiation of hematopoietic cells to MKs that will eventually bud platelets. The process by which platelets bud from MKs requires biomechanical stimulation of the MK plasma membrane through putative membrane mechanosensors. Known platelet membrane mechanoreceptors include the ion channel Piezo1 which may be important for the process of platelet budding.⁴ Most interestingly, it was demonstrated that blood-flow induced hemodynamic shear stress enhanced proplatelet production from differentiating MKs in murine bone marrow sinusoids ^{5, 6}. Also, using an immortalized MK cell line, the enzyme nardilysin (NRDC) appears to be required for thrombopoiesis and is activation D—flow conditions. Nardilysin may adhere extracellularly to elongated proplatelets, facilitating shear stress-induced platelet shedding through its metallopeptidase activity.⁶

Removal:

Platelets have a plentiful number of antibody FCγRIIa receptors on their surface membrane. The fixed domain of circulating immunoglobulins can ligate the platelet FCγRIIa receptors and engage cells in the reticuloendothelial system, mostly macrophages.^7,8 Of interest, another platelet-specific receptor of mechanotransduction, the glycoprotein IIb/IIIa receptor (GPIIb/IIIa), appears to interact with FCγRIIa ⁹ and so may have important functions by which platelets transduce signals internally after contacting the blood vessel wall. Furthermore, platelet surface mechanosensors may include platelet surface glycoprotein 1bα (GPIbα) which plays a pivotal role in the kinetics of platelet clearance from the circulation.¹⁰ Platelet dense granules store an abundance of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) that are released when vascular damage occurs. ATP and ADP released by dense granule exocytosis then binds to P2Y₁R and P2Y₁₂R receptors expressed on platelets, amplifying the process of platelet degranulation. Although it is not explicitly stated in the literature, efferocytosis may be a factor in platelet removal. The activation of P2YR initiates efferocytosis. Activation of P2Y₁R increases the expression of phagocytic receptors and an abundance of P2Y₆R and P2Y₁₂R initiate the “eat-me” signal of efferocytosis.¹¹

Interaction of the platelets with the vessel wall:

Circulating platelets contain granules filled with proteins mediating transient interactions of platelets with the blood vessel wall. Platelet α-granules are the most abundant with a plethora of proteins like adhesion proteins (integrin αIIb, α6, β3, GPVI, GPIb-IX-V complex, P-selectin), clotting factors (Factor V, von Willebrand factor (VWF), Fibrinogen), protease inhibitors (matrix metalloproteinases [MMPs], especially MMP2, and MMP9), growth factors (PDGF, bFGF, SDF1α), cytokines, and chemokines. Dense granules store ADP, serotonin, Ca²⁺, and lysosomal hydrolytic enzymes that, upon secretion, may modify the structure and function of blood vessels walls.¹² During vessel wall injury, rapid and complex interactions occur between circulating platelets and exposed sub-endothelial structures resulting in platelet adhesion to the damaged endothelium.¹³ The nature by which platelets interact with the vascular wall was once believed to be an irreversible event, and granule exocytosis was typically hypothesized to be a terminal event. Biomechanical platelet activation through surface shear stress induces circulating VWF to unfold, allowing platelets to roll along the endothelium through GP Ib/IX/V ligation which in turn initiates GPIIb/IIIa-mediated platelet aggregation at the blood/endothelial boundary (Figure 1). Stimuli for platelet granule release include surface agonists exposure via ADP, Thromboxane A₂ (released from activated platelets) and thrombin (produced on the surface of activated platelets).¹³ Thrombin is a strong platelet-activating peptide produced during vascular injury. Thrombin synthesis occurs through complex proteolytic events that are initiated by the exposure of tissue factor (TF) upon proteins in the coagulation cascade that circulate in blood.¹⁴ In addition, platelets were also demonstrated to contain the gap junction proteins connexin (Cx) 37, Cx40, and Cx62^15,16,17 that could potentially secrete molecules directly into the microvasculature or even transiently ‘dock’ against endothelial surface gap junctions to transfer small signaling molecules of < 1KDa and therefore regulate endothelial function in a manner distinct from thrombosis.

Figure 1: Non-thrombotic remodeling of blood vessels by platelets.

Schematic of transient interactions between blood vessels and platelets and platelet-mediated release of granules and microparticles that can influence vascular function and remodeling. Top: Platelet release of microparticles or granules with MMPs, galectin, TGFβ, PF4 and TIMP1 can alter remodeling the vascular intimal endothelial cells (EC). Bottom: platelet-mediated phosphoprotein activation and ROS production from dysfunctional mitochondrial influences signaling of vascular intimal endothelial cells (EC). ROS=Reactive Oxygen Species. MMP=matrix metalloproteases. TIMP1=Tissue Inhibitor of MMP 1. PF4=Platelet Factor 4. TGFβ=Transforming Growth Factor β.

Platelets and vascular remodeling

Release of mediators from activated platelets may contribute to negative remodeling of the vasculature that may include narrowing of the vessel wall, or destruction of proteins in the tunica media extracellular matrix such as collagen and elastin.¹⁸ Platelets are among the first responders to sites of vascular injury and work to stop bleeding through the process hemostasis.¹⁹ Platelets also secrete pro-inflammatory cytokines that mediate platelet-leukocyte interactions in chronic inflammatory diseases.^20,21

(a). MMPs play an important function in vascular remodeling:

MMPs including gelatinase A (MMP2) and gelatinase B (MMP9) are well-described as causative in aneurysmal progression of the thoracic as well as the infrarenal aorta. The origination of MMPs includes secretion from activated circulating macrophages, fibroblasts as well as paracrine release from phenotypically-altered vascular smooth muscle cells (VSMCs).^22,23,24 Under healthy conditions, human platelets have negligible quantities of MMP9. However, under pathological conditions such as acute MI, MMP9 content and activity inside the precursor MK and platelet are increased coincident with increased MMP activity in the blood and subsequent remodeling of the heart at the time of MI.^{25,26, 27,28} It is potentially revealing that the use of antiplatelet drugs including aspirin or clopidogrel prevents rupture of advanced AAAs (abdominal aortic aneurysm) in a murine model coincident with attenuating arterial wall MMP activity. The lesser accumulation of platelets and macrophages in the aorta of mice treated with platelet inhibitors results in less proteolytic destruction of the blood vessel architecture, presumably from MMPs. Antiplatelet medications decrease MMP2 and MMP9 in platelets, macrophages, as well as in aorta.²⁹ In another study, the P2Y₁₂ receptor antagonist AZD6140 inhibited ADP-induced platelet aggregation while limiting growth of AAA in a rat model. Furthermore, MMP-9 expression in the aortic wall correlates with mural thrombus area - an association that is lost after treatment with AZD6140.³⁰ In atherosclerosis, secretion of MMP2 and MMP9 from platelets results in degradation of extracellular matrix promoting plaque instability and rupture.^31,32,33 Wang et al. reported MMP12 (macrophage metalloelastase) in platelets. MMP12 is a 54 kDa proenzyme with two proteolytically active forms (45 kDa and 22 kDa) and can cleave the CEACAM1 (carcinoembryonic antigen related cell adhesion molecule1) exodomain into several short peptides. CEACAM1 is a type I transmembrane protein that acts as a negative regulator of platelet-collagen interactions. Therefore, MMP12 facilitated type I collagen-induced platelet aggregation, adhesion and alpha granule secretion through shedding of CEACAM1.³⁴ Proteolytic activity of MMP12 was also reported in the wall of the aorta and in blood samples from patients with aortic dissection.³⁵

(b). Cytokines secreted by platelets and their role in vascular remodeling:

Vascular inflammation is caused by small molecules secreted from circulating cells. Platelets are enriched in inflammatory molecules including members of the cytokine family.³⁶ For example, interleukin 1β (IL-1β) is a well-established cytokine secreted from leukocytes and platelets and leads to accelerated atherosclerotic changes in the blood vessel wall.³⁷ The CANTOS trial showed a benefit in using the IL-1β receptor antagonist Canakinumab.³⁸ Just as cytokines are known to contribute to vascular inflammation, suppressors of cytokine signaling (SOCS) proteins including SOCS-3 are also resident in platelets and may regulate vascular inflammation. In a study by Barret et al. platelet SOCS-3 promotes an atherosclerotic phenotype through induction of platelet IL-1β, IL-6, and Tissue Necrosis Factor α (TNFα) secretion.³⁹

Atherosclerotic plaque formation at level of the vascular intima relies on monocyte recruitment and differentiation into macrophages from the sub-endothelium.³⁹ Macrophages may undergo phenotypic switching to both M1 and M2 subtypes depending on their immediate environmental conditions. Mechanism of plaque formation in the vessel wall by M1 macrophages includes the release of inflammatory cytokines.³⁹ Again, recruitment of monocytes into atherosclerotic plaques of the blood vessel wall may involve an intricate balance between secreted cytokines and cytokine suppressor molecules from the platelet. For example, SOCS-3 upregulates nuclear factor kB (NF-kB) and suppresses signal transducer and activators of transcription 3 (STAT3). In other models in which the SOCS-1: SOCS-3 ratio is decreased, the macrophage phenotype favors M1, leading to the increase of IL-6 by blocking STAT1 and STAT3 signal transduction pathways. For this macrophage polarization to occur, Carestia et al. proposed that direct cell-cell interactions are necessary, suggesting another plausible mechanism by which transient interactions between platelets and the blood vessel wall are feasible. In addition, the authors report platelet stimulation by lipopolysaccharides (LPS) did not alter anti-inflammatory or pro-inflammatory pathways but, rather, platelet-macrophage interactions are required for a local inflammatory tissue response. Surprisingly, well-described platelet surface receptors for adhesion and thrombosis including GP1bα may also regulate vascular inflammation through distinct signal transduction pathways. Interactions between GP1bα on platelets and the integrin MAC-1 on macrophages promotes bacteria engulfment by platelets and bacterial clearance in systemic inflammatory diseases such as sepsis.⁴⁰ Similar to the GP1bα/MAC-1 interaction, C-type lectin-receptor 2 (CLEC-2 also known as CLEC-1b) plays a role in inflammatory processes. CLEC-2 is a type II transmembrane protein expressed as a dimer that can be found on the surface of platelets, MKs, and dendritic cells.^{41, 42} CLEC-2 contains an immunoreceptor tyrosine-based activation motif (ITAM) that allows for signaling through syk.⁴¹ Podoplanin (PDPN), type I single transmembrane protein is an endogenous ligand of CLEC-2 that causes platelet activation and aggregation on binding to CLEC-2.⁴¹ Rayes et al. found that the CLEC-2/podoplanin interaction has a negligible effect on hemostasis but instead does play a role in inflammation.⁴² These interactions have been found to occur in cancers, specifically tumor proliferation due to the immunosuppressive environment produced by CLEC-2/podoplanin interaction, rheumatoid arthritis, as well as bacterial and viral infections.⁴² In this way, platelets have a dual pro-inflammatory role by releasing cytokines while simultaneously fighting infection.

(c). Galectins and their role in platelet activation and vascular remodeling:

Galectins (gals) are a class of carbohydrate-binding proteins with a multitude of roles regulating physiologic processes essential to both immunologic and pathologic conditions.⁴³ Gals are found in a wide variety of tissues and aid in cell adhesion, apoptosis, differentiation, proliferation, as well as promoting healing at sites of tissue injury.⁴⁴ Of the 15 known gals identified in mammals, gal-1, gal-3, gal-8 and gal-9 have been shown to interact with platelet surface receptors to cause negative remodeling of the vessel wall through inflammation and atherosclerosis.^{45,46,44, 47} It has been shown that structurally divergent galectins (gal-1, -3 and -8), can initiate platelet responses including adhesion, spreading, aggregation, release of granular content and expression of P-selectin through their interaction with carbohydrate part of platelet receptors GPIb/IX/V complex and integrin α_IIbβ_3.^{45, 48} During vascular injury, platelets adhere to the extracellular matrix (ECM) and gals interact with GP Ib/IX/V to promote platelet-ECM adhesion.⁴⁴ Given the expression of gals in the platelet transcriptome, it is possible that direct platelet synthesis and secretion of gals occurs locally. Gal-1, gal-2, and gal-3 were found to be expressed in human and murine platelets to a greater or lesser extent (Figure 2).⁴⁹ Despite the structural and sequence similarity in the galectins, they exert different responses in the platelets. Gal-3 is approximately 30kDa and mainly released into circulation from immune cells and cardiomyocytes.⁴⁶ Until recently, gal-3 was primarily studied as a biomarker in association with heart failure, it is now being further evaluated for its role in vascular inflammation and atherosclerosis.⁵⁰ A study by Nachtigal et al. revealed the increased expression of gal-3 in atherosclerotic plaques correlates linearly with atherosclerosis progression.⁵¹ It was demonstrated that gal-3 positively correlates with platelet function and directly interacts with dectin-1, a c-type lectin receptor expressed on the surface of platelets.⁴⁶ The binding of gal-3 to dectin-1 initiates the spleen tyrosine kinase (Syk) pathway to mount a local inflammatory tissue response in the platelets.⁴⁶ The dectin-1/Syk signaling cascade in platelets promotes phospholipase C-gamma (PLCγ) activation, causing an increase in platelet cytosolic Ca²⁺ and protein kinase C (PKC) phosphorylation. The terminal event in this signal transduction pathway in platelets was ROS generation and platelet activation, a pathological event that was abolished in dectin-1^−/− platelets following gal-3 stimulation.⁴⁶ In a recent study, Gal-9 has also been shown as an important platelet agonist that activates platelet functions via interaction with the platelet ITAM receptor GPVI and CLEC-2 in human and murine platelets causing platelet spreading, secretion and aggregation. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins downstream of GPVI and CLEC-2 including syk and linker of activated T cells (LAT) in human platelets. Gal-9 effect on platelet aggregation and spreading suggests its potential role in thrombus formation. In addition, the interaction between P-selectin expressed on platelets in response to Gal-9 and PSGL-1 (P-selectin glycoprotein ligand-1, CD162) expressed on leukocytes potentially facilitates platelet–leukocyte aggregation causing leukocyte recruitment and inflammation.⁴⁷

Figure 2: Galectin expression in platelets.

Platelet Galectin1, Galectin 2, TIMP1, and TIMP2 transcript expression in human and murine platelets with level of expression noted in parentheses. Data available through the following publicly-available search engine www.plateletomics.com.

(d). CXCL4 released by activated platelets and its role in vascular remodeling :

CXCL4 or Platelet Factor 4 (PF4) is a platelet specific cytokine, stored in platelet α-granules and released into the plasma in micromolar concentrations following platelet activation.⁵² CXCL4 affects all nucleated cells of the vasculature, controlling important regulatory processes such as apoptosis, cell differentiation, survival, proliferation⁵³ and circulating monocyte to tissue macrophage differentiation. CXCL4 binds to a 200 kDa chondroitin sulfate proteoglycan on the surface of human neutrophils and bind to CXCR3B on the surface of microvascular endothelial cells (ECs). Like members of the galectin family, CXCL4 is also detected in atherosclerosis lesions and correlates with atherosclerotic lesion severity, suggesting the role of platelets in vascular remodeling.⁵⁴ CXCL4 knockout ApoE^−/− mice have reduced atherosclerotic lesions size, indicating a crucial role for PF4/CXCL4 in atherogenesis in vivo.⁵⁵ Macrophage culture experiments have shown that platelet derived CXCL4 promotes atherogenesis by suppressing macrophage CD163 which then blocks atheroprotective heme oxygenase-1. CD163 is reported to be atheroprotective hemoglobin receptor that helps in scavenging hemoglobin and hemoglobin–haptoglobin (Hb-Hp) complexes.⁵⁶ CXCL4 released by activated platelets might be involved in the accumulation of the Low-density lipoprotein (LDL) by inhibiting LDL receptor which may enhance amount of esterified oxidized (ox)-LDL in macrophages, and promote foam cell formation. This offers a potential mechanism by which platelet CXCL4 secretion at sites of vascular injury may promote the accumulation of deleterious lipoproteins.⁵⁷

Platelets and vascular repair

Platelet-derived molecule, of which several are enzymes, may interact with protein constituents of the blood vessel wall and regulate remodeling. Similar to cytokines, secreted proteins from platelets may prevent further damage or reinforce structural integrity. An example is local delivery of platelet-derived Nitric Oxide (NO), platelet-derived CXCL12, platelet-derived tissue inhibitor of metalloproteinases (TIMP1), and transforming growth factor (TGF-β).

(a). Nitric Oxide:

NO is produced by platelets in small quantities from the substrate L-Arginine. L-Arginine to L-Citrulline conversion is catalyzed by the enzyme Nitric oxide synthase (NOS).⁵⁸ Two NOS isoforms (NOS2 and NOS3) are reported to be expressed in platelets.⁵⁹ NOS3 is the predominant NOS isoform in platelets.⁶⁰ NO is produced under resting basal conditions in platelets, and may be augmented in a sex-dependent manner by platelet uptake of L-arginine.⁶¹ NO produced by platelets stimulates soluble guanylyl cyclase (sGC) activity to hydrolyze cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) and decrease intracellular Ca²⁺ mobilization in the platelet which limits degranulation and platelet reactivity. Cyclic GMP also activates cGMP-dependent protein kinase G (PKG) and phosphorylates downstream proteins, like a vasodilator-stimulated phosphoprotein (VASP) to limit platelet adhesion to extracellular proteins including GPIIb/IIIa and P-selectin.⁶² Platelet β₂-adrenoceptors are capable of stimulating NOS3 and PKG activity.⁶³ Stimulation of platelets by different aggregating agents, leads to an increase in intracellular Ca²⁺ that activates platelet NOS3 and produces NO. This may operates in a negative feedback mechanism regulating platelet activation.⁵⁸ Platelet derived NO inhibits not only platelet aggregation but also may influence the tunica media of the blood vessel wall by preventing ECs inflammation and degranulation of pro-thrombotic vesicles. Platelet-derived NO also has an anti-inflammatory and anti-proliferative effect on vascular smooth muscle cells (VSMCs) and decreases VSMC tone to decrease systemic vascular resistance.⁶⁴ Systemic arterial hypertension, conversely, may limit platelet-derived NO. The L-arginine analogues asymmetric dimethyl-arginine (ADMA) and N G-monomethyl- L -arginine (L -NMMA) are endogenous inhibitors of NOS. It has been shown experimentally in hypertensive patients that platelet L-arginine influx is altered in a concentration dependent manner using ADMA and L-NMMA. This suggests the possibility of competitive inhibition of L-arginine and inhibition of the synthesis of NO, which may be involved in the platelet activation and pathogenesis of sustained hypertension.⁶⁵Proton pump inhibitors (PPIs) are gastric acid-suppressing agents widely prescribed for the treatment of gastroesophageal reflux disease (GERD). Sustained use of PPIs may pose risk of cardiovascular diseases in the patients. Ghebremariam et. al discovered that PPIs increase intracellular Asymmetric dimethylarginine (ADMA) in both ex vivo human tissue and murine models, likely via inhibition of the enzyme responsible for ADMA degradation, dimethylarginine dimethylaminohydrolase (DDAH).⁶⁶ Despite these findings, human studies in 1298 subjects failed to show increased plasma ADMA in patients taking PPIs.^{67, 68} Nolde et. al, confirmed that while plasma ADMA did not increase in patients taking PPIs, citrulline concentration (a byproduct of ADMA metabolism) and vascular endothelial functions were lower in those patients. ⁶⁷ As such, this implies long term use of PPIs might alter DDAH activity that can impair vascular tone by altering NO production.⁶⁷

(b). Stromal cell-derived factor 1a (SDF-1a):

Platelet-derived SDF-1a, also known as CXCL12, modulates paracrine mechanisms such as chemotaxis, adhesion, proliferation and differentiation of nucleated cells, including progenitor cells. CXCL12 is a ubiquitously-expressed and highly conserved protein stored in platelet α granules and plays an important autocrine role in cell homeostasis, recruitment, and arrest through binding to chemokine receptors CXCR4 and ACKR3 (CXCR7). CXCR4 is expressed in MKs and may regulate megakaryopoiesis.⁶⁹ At the site of vascular injury, platelets adhere to the exposed sub-endothelial matrix and release stored CXCL12. Damaged endothelium leads to platelet activation through the secretion of known platelet agonists including ADP, thrombin, and sub-intimal collagen. This causes release of platelet CXCL12 in the microcirculatory bed, further driving progenitor cell (PC) migration from the bone marrow to the site of injury for the recovery of the damaged endothelium. CXCL12 transmits signals via CXCR4 and activates platelets through PI3Kβ and other small tyrosine kinases including Syk and Btk, mediating intracellular calcium release, and MAPK.⁷⁰ CXCL12 is present in atherosclerotic lesions and may play a role in the formation of a platelet-rich thrombus after plaque disruption.⁷¹ Hypoxia-inducible factor (HIF)-1α induces CXCL12 expression in hypoxic conditions and controls the recruitment of multipotent progenitor cells that leads to tissue repair.⁷² Wire-induced injury of the left carotid artery was performed in ApoE^−/− mice and reveled that CXCL12 regulates neointima formation after arterial injury by attracting Smooth muscle progenitor cells (SPCs) at the site of injury.⁷³ The arterial lesion in ApoE^−/− mice treated with CXCL12, showed reduced macrophage content, increased number of smooth muscle cells, increased fibrous thickness and increased collagen content leading to stability of the atherosclerotic lesions. ⁷⁴ Upregulation of SDF-1α (and CXCR4) leads to the recruitment of bone marrow-derived inflammatory cells in the injured aorta in mice. Supporting these observations, the blockage of SDF-1α signaling using AMD3100 decreases macrophage infiltration into the tunica adventitia in AAA.⁷⁵

(c). TGF-β:

Platelets are an enriched source of circulating TGF-β and contribute most of the blood TGF-β content that blood vessels are exposed to.⁷⁶ TGF-β has several protective effects on the vasculature, including protection against inflammatory and fibrotic remodeling in murine models AAA.^77,78,79 Blocking TGF-β signal transduction in vascular smooth muscle cells in experimental murine models of AAA or in humans who harbor genetic variants of TGF-β or the TGF-β receptors display a clearly-remodeled vascular phenotype with a predisposition to arterial aneurysm formation, dissection, and rupture of conduit vessels including the aorta.⁸⁰

(d). Tissue Inhibitors of Matrix Metalloproteinases (TIMPs):

MMPs, as already stated, contribute significantly to remodeling of arteries by degrading collagen in the tunica media of the blood vessel. TIMP 1–4 are naturally occurring inhibitors of MMP by inhibiting enzymatic activity.⁸¹ Platelet α-granules are enriched in TIMP 1–4 and so conceivably released by constitutive and regulated exocytosis.⁸² Therefore, the local release of TIMP by platelets into the blood vessel wall could play a protective role by preventing MMP activity and negative vascular remodeling. Interestingly, TIMPs are expressed in human platelets more than murine platelets, with TIMP1 expression in the top 2% of expressed proteins in the human platelet proteome (Figure 2).

Platelet-Derived microparticle regulation of blood vessel wall

Platelet-derived microparticles (PDPs) are 100–1000 nm sized fragments. PDPs are distinct from platelets granules which are packaged in the megakaryocyte. PDPs are derived from the plasma membrane of mature platelets during platelet activation by physiological agonists such as thrombin or collagen or in response to high shear stress. Particles of <100 nm in diameter are called exosomes, that are secreted from the multivesicular alpha granule population. Whereas, the particles >1000 nm in diameter are termed apoptotic bodies derived from the plasma membrane and are remnants of dead cells.⁸³ PDPs microparticles constitute (70–90%) of circulating microparticles making parent platelets the most abundant source.^{84,85, 86} During platelet activation, increased Ca²⁺ concentration activates cytosolic enzymes and prompts cytoskeleton disassembly, causing flopping of phosphatidylserine (PS) from the inner layer of the platelet membrane to cause membrane blebbing and shedding of microparticles. As a consequence of membrane blebbing, these PDPs carry parental antigens and surface adhesion proteins including P-selectin, GPIIb/IIIa and GPIb.^85,84,87,88 Furthermore, PDPs are rich in PS and Tissue Factor (TF) which may alter signaling in ECs.⁸⁹ Studies have shown bidirectional communication between endothelial and platelet co-cultures via PDPs which is a likely model of what occurs naturally in vivo.⁹⁰ PDPs, like platelets, also control non-thrombotic mechanisms of blood vessel remodeling including inflammation, oxidative stress, apoptosis, angiogenesis, and tumor formation.^85,84,91,88 Like platelets, PDPs also contain cytokines, mRNAs, and microRNAs which mediate intercellular signaling, cell activation, cell reprogramming, and overall contribute to vascular inflammation and negative remodeling.^92,85 PDPs can also be internalized by ECs via endocytosis and by macrophages that ultimately adversely remodel the blood vessel wall.⁹³ Faille et. al demonstrated that human brain endothelial cells (HBECs) actively internalize PDPs primarily via phagocytosis and macropinocytosis uptake. ⁹³ Both phagocytosis and micropinocytosis are characterized by extensive membrane reorganization. Upon treating HBECs with cytochalasin D—an inhibitor of microfilament formation vital for phagocytosis and macropinocytosis—PDP uptake was greatly diminished.⁹³ Treatment of HEBCs with monoclonal antibodies against ICAM-1 and VCAM-1 as well as the introduction of TNFα, did not impact PDP endocytosis, revealing that PMP internalization is independent on adhesion receptors from endothelial cells.⁹³ The authors further revealed that PDP uptake dramatically increased in the presence of decomplemented serum, supporting a role for PDP opsonin-dependent phagocytosis.⁹³ In addition, PDPs may also reach VSMCs and fibroblasts in the tunica media by endothelial cell transcytosis.^94,93,86 PDPs facilitate atherosclerosis through inflammation, increased smooth muscle cell proliferation, and upregulation of cellular adhesion molecules as well as by lipid deposition.⁹⁵ Endothelial damage in response to mechanical injury, hypercholesterolemia and smoking in atherosclerosis promotes PDP adherence to the vessel wall via GPIIb/GPIIIa interactions and promotes repair of damaged vessel wall.⁹⁶ Mause et. al demonstrated that PDPs contain substantial amounts of RANTES/CCL5, which recruits monocytes to the damaged vessel wall and also increases the expression of endothelial adhesive molecules such as intercellular adhesive molecular-1 (ICAM-1) that further promote leukocyte adhesion and transmigration into the vascular wall.^97,96. Recruited monocytes become engorged with cholesterol and cholesterol esters, resulting in foam cells accumulating within arterial plaque.⁹⁸ PDPs reduce endothelial NOS activity and reduces NO production. This process can enhance the damage of endothelial function, leading to atherosclerosis.⁹⁶ Similar to platelets, PDPs are rich in thromboxane A₂ (TBA₂), a potent mediator of tone. Additionally, PDPs have been shown to impair endothelium-dependent relaxation by downregulating NO and by promoting caveolin-1 overexpression.⁹⁹

Overall, the inflammatory and atherosclerotic processes of PDPs are clearly linked to the severity of conditions such as coronary artery disease (CAD) and peripheral artery disease (PAD).^95,88

Finally, PDPs are elevated in both abdominal and thoracic aortic aneurysms, and worsen pathology through their atherosclerotic impact and upregulation of proteolytic and inflammatory mediators.¹⁰⁰ Thoracic aortic aneurysms (TAA) involve the aortic root, the aortic arch, and descending supra-renal aorta. TAA are more commonly associated with cystic medial degeneration leading to dilation of the aortic root that is common in inherited aortopathies such as Marfan’s Syndrome and Ehlers Danlos Syndrome.¹⁰¹ Among aortopathies, PDPs were detected in large (> 45mm) aneurysms ¹⁰⁰ and were likely to provide the PS-enriched membranes that favor thrombin generation and platelet activation.¹⁰²

AAA are pathological dilations of the abdominal aorta below the level of the renal arteries. AAA are more likely to occur due to loss of integrity of structural proteins.¹⁰³ MMPs cleave elastin and collagen which are essential structural proteins in the blood vessel, and contribute to both thoracic and abdominal aortic aneurysmal pathology.^22,23,24 PDPs upregulate MMP-2 and MMP-9 expression in Human umbilical vein endothelial cells (HUVECs).¹⁰⁴ MMPs contribute to the loss of smooth muscle cells and the overall destruction of the extracellular matrix.¹⁰³ Upregulation of MMP activity leads to proteolytic remodeling of the basement membrane, especially type IV collagenase activity by MMP2.¹⁰⁴ Remodeling of the aortic wall initiates vessel dilation and subsequent rupture. Another mechanism by which PDPs play a role in AAA pathology involves the upregulation of a lectin pathway recognition molecule, Ficolin-3. Ficolin-3 is typically secreted from the liver, but may be extra-hepatically expressed during inflammatory responses. In AAA tissue-conditioned medium, PDPs were increased that released Ficolin-3.¹⁰⁵ Ficolin-3 content was associated with both the presence and progression of AAA, suggesting a potential role in the immune response contributing to AAA.¹⁰⁵ PDPs contribution to atherosclerosis likely furthers the risk factors for AAA. The Tromsø study revealed that atherosclerosis is an independent risk factor for AAA, but did not show a causal relationship. The authors also demonstrated that atherosclerosis was more common in abdominal aortic diameters ≥ 27 mm.¹⁰⁶ Atherosclerosis has been associated with abdominal and descending thoracic aortic aneurysms than with ascending aortic aneurysms.¹⁰¹

Platelet-derived Reactive Oxygen Species

During megakaryopoiesis, MKs transfer mitochondria to the platelets prior to platelets budding. In the absence of a nucleus, platelets have prepackaged RNA with translational machinery.^{107,108, 109} Mitochondria produces adenosine triphosphates (ATP) through oxidative phosphorylation (OXPHOS) and tricarboxylic cycle (TCA) which arecritical for the cellular processes including differentiation and autophagy.¹¹⁰ Studies have shown that mitochondria are not only limited to homeostasis, but also regulates cell signaling and vascular wall responses through the production of ROS.^111,112 ROS are highly reactive radicals and non-radical oxygen species such as singlet oxygen (O₂), superoxide anion (O₂⁻), hydroxyl radical (HO•), hydroxyl ion (OH⁻), peroxide (O²⁻) and hydrogen peroxide (H₂O₂). Mitochondria appear to play a critical role in the activation of the platelets. Exposure of platelets to hydrogen peroxide leads to their activation through intracellular ROS generation and, as such, has been implicated in collagen-induced activation of platelets which leads to secretion of small molecules by exocytosis. Overall, platelets are both source and target of ROS.^{113,114, 115}

Platelet-derived ROS plays an important role in the initiation and progression of cardiovascular diseases¹¹⁶ and can influence vascular endothelial function and remodeling of blood vessels. In addition, secretion of ROS from platelets may influence the tone of the microvasculature as well as subsequent tissue perfusion. A mechanistic link between platelets and mitochondria was found in Sickle cell disease (SCD). SCD is a monogenetic disorder due to a single base-pair point mutation in the β-globin gene of adult hemoglobin (HbS). In SCD there is hypoxic polymerization of HbS, which leads to diminished red blood cell deformability and impaired microvascular blood flow. A few high quality mechanistic studies have linked vascular remodeling and vascular dysfunction with dysregulated platelet activity in SCD patients posing a thrombotic risk that leads to mortality and morbidity.^{117, 118,119} Sickle cell patients showed platelet inhibition of complex V in the electron transport system and subsequent mitochondrial dysfunction, mitochondrial membrane hyperpolarization, and augmented ROS production in. Platelet mitochondrial dysfunction in SCD correlates with platelet activation and hemolysis which could in part be mitigated by mitochondrial-targeted therapeutics.¹²⁰ A summary of platelet-derived mediators that regulate vascular remodeling or function with appropriate literature citations is indicated in Table 1.

Table 1:

Platelet-derived mediators that induce vascular remodeling or dysfunction.

Releasate	Origin	Effector	Effect	Reference
MMP2 MMP9	Platelets	Vessel wall	Extracellular matrix degradation;	Li T et al., PMID: 33082709; Loftus IM et al., PMID: 10625713; Wågsäter et al., PMID: 21567073
MMP12	Platelets	Platelets	Platelets adhesion, aggregation and granule secretion	Wang J et al., 2017, PMID: 28385529; Song Y et al., 2013, PMID: 23642232
IL-1β	Platelets and Leukocytes	Macrophage	Inflammatory response through monocyte/macrophage activation	Barrett T et al., 2019, PMID: 31694925
Galectins (1,3,8)	Endothelial Cells	Platelets	Increase platelet hyperactivity	Chen Y et al., 2022, PMID: 35165707
Galectin-9	Endothelial cells	Platelets	Platelet activation, spreading and aggregation	Zhi Z et al., 2022, PMID: 34936188
Dectin-1	Platelet Receptor	Galectins	Mediator of inflammatory response through Syk pathway	Chen Y et al., 2022, PMID: 35165707
CXCL4	Platelets	Nucleated vascular cells	Macrophage differentiation from monocytes and atherogenesis	Pitsilos S et al., 2003, PMID: 14652645
NO	Platelets	Endothelium	Relaxation of VSMC; inhibition of platelet aggregation; inflammation/degranulation of prothrombotic vesicles	Zhou Q, 1995, PMID:7701481
CXCL12	Platelets	Endothelium	Platelet activation through PI3Kβ, Syk Btk, MAPK and mediates Ca2+i release	Leberzammer J et al., 2022, PMID: 35313337
TIMPs (1–4)	Platelets	Vessel wall	Prevents MMP activity and negative vascular remodeling	Villeneuve J et al., 2009, PMID: 19410025
TGF-β	Platelets	Vasculature	Protects against inflammatory and fibrotic remodeling	Wang Y et al., 2010, PMID: 20101093 Raffort J et al., 2019, PMID: 30792060; Lareyre F et al., 2017, PMID: 28912363
ROS (O2, O2−, HO• OH−, O2−, H2O2)	Platelets	Endothelium, Platelet	Collagen-induced platelets activation; small molecule exocytosis	Principe DD et al., 1985, PMID: 3996592; Principe DD et al., 1991, PMID: 1896957 Praticó D et al., 1999, PMID: 10377074
(GP) Ib-V-IX	Platelet surface receptor	Vessel wall	Platelet adhesion	Hansen CE et al., 2018, PMID: 29865873; Sadler JE, 1998, PMID: 9759493
GPIb	(GP) Ib-V-IX complex on platelets	VWF; gals	Platelet recruitment under shear stress; platelet adhesion to extracellular membrane	Hansen CE et al., 2018, PMID: 29865873; Sadler JE, 1998, PMID: 9759493; Schattner M, 2014, PMID: 25405160
GPIIb/IIIa	Platelets	VWF; fibrinogen	Stabilization of platelet aggregates	Chen Y et al., 2019, PMID: 30911119
GPVI	platelets	vasculature	Collagen induced platelet activation	Moroi M, 2004, PMID: 15381385
VWF	Endothelial cells and platelets	Injured sub-endothelium	Under shear stress, facilitates platelet adhesion; triggers mechano-signaling; influx of Ca2+ in platelet leading to platelet aggregate stabilization	Chen Y et al., 2019, PMID: 30911119
PECAM-1; CLEC-2	Platelets	Endothelial cells	Platelet adhesion to Endothelial cells	Meza D et al., 2017, PMID: 28013181; Stevens HY et al., 2008, PMID: 19048083; Tang C et al., 2021, PMID: 34815786
BMK1	Platelet	Arterial vasculature	Inhibit angiogenesis under ischemic conditions	Cameron SJ et al., 2015 PMID: 25934838

Biomechanical Platelet Activation and Platelets as a mechanosensors:

Platelets are subjected to hemodynamic and shear forces in the circulation, which may lead to the secretion of granular contents that can remodel distant vasculature.¹²¹ In regions of vasculature discontinuity, including stenosis, atherosclerosis, and ectasia, platelet may secrete molecules directly onto cells within the blood vessel wall to alter their structure and function. We recently showed that platelets are biomechanically-activated in regions of Disturbed-flow (D-flow) in patients with AAA and in murine models of AAA, causing platelet to secrete MMPs directly into the aortic wall to promote aneurysmal develoment.²³ Additional reports acknowledge the existence of platelet surface mechanosensors including (GP) Ib-V-IX receptor complex, GPIb, GPIIb/IIIa (integrin αIIb/β3), GPVI, and plasma levels of VWF to mediate shear-dependent platelet adhesion, spreading and aggregation.^{122,123,124,125} Biomechanical platelet activation depends on both the magnitude and duration of surface stress.¹²⁶ At shear rates >1000 s⁻¹, platelet adhesion at injured vessels is strongly dependent on VWF. Most VWF is released from the ECs (stored in Weibel Palade Bodies) and platelet α-granules \ into the circulation.¹²⁷ VWF is mechanosensitive, undergoing conformational changes following mechanical shear. The globular Ultralarge von Willebrand Factor (ULVWF, molecular weight above 20,000 kDa) binds to the exposed subendothelial collagen (types I and III) at sites of injury through the A3 domain. After adhesion, the disturbed blood flow elongates the ULVWF to expose the A1 and A2 domains. Then the circulating platelets bind to VWF through interactions between the VWF-A1 domain and the GPIb–IX–V receptor complex on the outer platelet surface membrane. This shows the absolute requirement of GPIb in the recruitment of the platelets in the shear stress conditions.^128,129 VWF-GPIb interaction not only mediates platelet adhesion but also triggers the other mechanosignals leading to intra-platelet calcium flux and activation of GPIIb/IIIa which stabilizes the platelet aggregates during high shear rates.¹³⁰ Under shear stress, platelet endothelial cell adhesion molecule-1 (PECAM-1) and CLEC-2 also mediate platelet adhesion to ECs^131,132,133. In the study by Ilkan et al, the authors showed the presence of Piezo1 mechanosensitive ion channel in human platelets and MKs. Disturbed flow in the arteries stimulates platelets and Meg-01 (MKs cell line) Ca²⁺ proving the presence of mechanosensitive cation channels. Thrombus formation under shear stress is inhibited after using the mechanosensitive channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) further signifying the potential role of mechanosensitive ion channels in platelet function.¹³⁴ Another study also showed shear stress induced activation of Piezo1 in platelets is required to mediate Ca²⁺ entry and facilitate thrombus formation.¹³⁵

The stiffness of the underlying fibrin/fibrinogen substrate also govern the differential platelet adhesion and spreading through mechanotransduction. The number of adherent platelets and the average spreading area of platelets positively correlates to the substrate stiffness. The polyacrylamide (PA) gels stiffer than 5 kPa caused more platelet activation, namely increased GPIIb/IIIa activation, P-selectin secretion, and PS exposure, compared with softer gels. Shear stress led platelet activation activates Rac1-Rap1 which acts downstream of GPIIb/IIIa and mediates platelet mechanosensing of substrate stiffness.¹³⁶ D- Flow causes platelet activation which has an important role in underlying diseases:

(a). Change in hemodynamics and platelets in the atherosclerotic lesions:

Atherosclerosis develops preferentially at arterial branches and curvatures which are typically areas of low shear and D-flow. Changes in hemodynamics due to plaque formation in atherosclerosis, intensifies the propensity to increase plaque in an arterial stenosis environment, making it unstable and causing rupture.¹³⁷ Platelets as well as inflamed ECs in atherosclerosis, can sense the change in the hemodynamic forces through different receptors expressed under such conditions. Under d-flow, inflamed ECs express high levels of adhesion molecules (e.g., P-selectin, ICAM-1, vascular cell adhesion molecule 1 (VCAM-1)) and adhesion proteins (e.g., vWF, Fibrin).^138,139 These molecules stimulate platelets to adhere and roll to damage sites via platelet receptors, such as GPIbα, GPVI, or GPIIb/IIIa along with vWF.¹³⁹ These adhered platelets under shear stress can expose flow-induced protrusions (FLIPRs, long negatively charged membrane strands) and capture the circulating leukocytes and form platelet-leukocyte microparticle complexes which further progresses the inflammation in atherosclerosis.¹⁴⁰ Platelets in atherosclerosis also showed the expression of receptors like PECAM-1 and CLEC-2 mediating their accumulation in sub-endothelium and ameliorating plaque formation in mice models.^132,133

(b). Change in hemodynamics and platelets in abdominal aortic aneurysm:

In aneurysmal arteries, blood transitions from steady laminar flow (S-flow) to D-flow and this change in hemodynamics, may mechanically activate the circulating platelets.¹³⁰ Hemodynamic changes in the blood around aneurysm sac leads to an increase in the wall shear stress which results in the platelet activation and deposition at the site of disrupted endothelium and promotes the development of the intraluminal thrombus (ILT).¹⁴¹ AAA is characterized by the development of the intraluminal thrombus (ILT) in the aneurysmal arteries which is linked to both progression and rupture of AAA.^142,143,144 In a randomized clinical trial in 144 patients with small AAA, treatment with the platelet P2Y₁₂ receptor antagonist ticagrelor did not affect AAA growth, indicating that the size of aortic aneurysms (and so the magnitude of D-flow) as well as the platelet surface receptor all may play a role in aortic remodeling and AAA progression.¹⁴⁵

Bidirectional communication between platelets and blood vessel walls

Our group reported in a murine model of MI that the platelet proteome changes and platelet-derived MMP2 increases coincident with MMP activity in the heart and remodeling of the coronary circulation that is enriched in ROS²⁷ (Figure 3). We also observed that platelet-surface thrombin receptor sensitivity is augmented immediately in the post MI environment.¹⁴⁶ Similarly, a primary vascular disorder might also affect platelet function. For example, in a murine model of critical limb ischemia, platelet reactivity is augmented coincident with increased Big Mitogen-Activated Protein Kinase 1 (BMK1) activity and platelet-specific BMK1-deficient mice showed accelerated angiogenesis in the ischemic hind limb.²⁷ These observations clearly suggest bidirectional communication between platelets and the arterial vasculature (Figure 4). Furthermore, in patients with AAA, the platelet transcriptome and platelet-surface receptor sensitivity were divergent from health individuals and inhibiting platelets in a murine models of AAA resulted in a decrease in AAA progression and rupture.^23,29 These findings support the presence of unidentified platelet mechanosensors that may alter platelet responsiveness and secretory pathways in conditions of vascular pathology where there is d-flow. Endothelial Cells (ECs) and MKs influence one another through cytokine and growth-factor mediated signaling.^{147, 148} ECs play an important role in the MK maturation and thrombopoiesis by secreting CXCL12 and Fibroblast Growth Factor 4 (FGF4).^{147, 148} Lefrançais and Ortiz-Muñoz imaged murine lung microcirculation and demonstrated extra-medullary lung MK pools which repopulated platelet numbers during thrombocytopenia and relative stem-cell deficiencies.¹⁴⁸ Platelet biogenesis via lung involvement was proposed to arise from two key processes: 1) proplatelets and MKs within the lung vasculature could promote formation and release of platelets and 2) MKs and hematopoietic progenitors in the lung interstitium could migrate out and restore marrow deficiencies.¹⁴⁸ These findings suggest that blood vessels relay feedback to drive MK formation and platelets budding.¹⁴⁸ Blood vessel feedback can additionally inhibit MKs as destruction of endothelial cell integrity impairs MK platelet formation.¹⁴⁹ Conversely, MKs also influence ECs through a variety of interactions including production of Vascular endothelial growth factor (VEGF) and Fibroblast Growth Factor (FGF), and anti-angiogenic factors (thrombospondin and PF4).¹⁴⁷

Figure 3. Platelet ERK5 deletion remodels myocardial vasculature.

Platelet reprogramming and ROS production after myocardial infarction leads to remodeling of the vasculature in the mouse myocardium. Top: gross micrograph of the heart and methylene blue-injected heart vasculature at the time of MI (blue=normal perfusion). Middle and bottom: cross section of the mouse left ventricle shows myocardial remodeling that is in part dependent on platelet phosphoprotein ERK5 signaling.

Adapted from Fig. 5 Cameron SJ, Ture SK, Mickelsen D, Chakrabarti E, Modjeski KL, McNitt S, Seaberry M, Field DJ, Le NT, Abe J, Morrell CN. Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion. Circulation. 2015 Jul 7;132(1):47–58. doi:10.1161/CIRCULATIONAHA.115.015656. Epub 2015 May 1. PMID: 25934838; PMCID: PMC4532543.

Figure 4. Platelet ERK5 deletion after critical limb ischemia alters muscle angiogenesis.

Thermal Doppler color imaging showed more rapid return of blood flow in platelet ERK5^−/− mouse limbs and quantification (mean ratio in the ischemic/nonischemic limb ± SEM *P<0.001 Sham WT vs WT HLI, **P=0.013 WT HLI vs ERK5 ^−/− HLI, ***P=0.003 WT HLI vs ERK5 ^−/−.

Adapted from Fig. 6 Cameron SJ, Mix DS, Ture SK, Schmidt RA, Mohan A, Pariser D, Stoner MC, Shah P, Chen L, Zhang H, Field DJ, Modjeski KL, Toth S, Morrell CN. Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype. Arterioscler Thromb Vase Biol. 2018 Jul;38(7):1594–1606. doi: 10.1161/ATVBAHA.118.311186. Epub 2018 May 3. PMID: 29724818; PMCID: PMC6023774.

Closing thoughts: is there a feedback mechanism from the blood vessel wall to MKs?

Our initial observation several years ago in a murine model of MI and then in a second murine mode of critical limb ischemia (CLI) that timed blood draws following acute arterial occlusion in the heart and in the leg, respectively, revealed a divergent population of platelets.^150,27. While this observation suggests the mature circulating platelet is where the phenotype shift occurs, the possibility remains that a different platelet, as observed in the circulation of these animals with vascular disease, was being released from reprogrammed MKs in the bone marrow. Future work should therefore focus on circulatory signaling mechanisms and feedback that determine the fate of bone-marrow-derived MKs from which mature circulating platelets are derived.

Non-Standard Abbreviations:

D-flow

Disturbed Blood Flow

ROS

Reactive Oxygen Species

MKs

Megakaryocytes

GPIbα

Glycoprotein 1bα

GPIIb/IIIa

Glycoprotein IIb/IIIa receptor

ECs

Endothelial Cells

AAA

abdominal aortic aneurysm