Novel Roles and Therapeutic Approaches Linking Platelets and Megakaryocytes to Non-Hemostatic and Thrombotic Disease

Abstract

Historically, pharmacological interventions aimed at platelets have targeted their canonical hemostatic and thrombotic roles. The therapeutic vision, however, has minimally embraced alternate mechanisms by which anucleate platelets, their parent cells, megakaryocytes, and cellular derivatives may be utilized to yield novel and effective therapies. Platelets contain storage granules rich in a wide variety of proteins, chemicals, growth factors, and lipid particles that can modulate the fate and activity of diverse cell types, and impact diseases not previously thought to have a platelet component. In this article, we will address unconventional platelet contributions to health and disease development. Recent studies indicate extensive platelet roles in neurodegeneration, insulin secretion, and bone marrow fibrosis, along with a recognition of platelets as immune cells in their own right, partially based on the presence of surface MHC, Toll-like receptors, and stored immunomodulatory molecules. Recent technological advances have produced iPS-derived gene-editable megakaryocytes (MKs) that have been differentiated to clinical-grade platelets for transfusion; however, such successes are still rare. Continued improvements in the standardization of cell isolation, iPS differentiation protocols, technology for the utilization of platelet derivatives, and platelet Omics will expand our understanding of underlying platelet and MK heterogeneity and direct novel therapeutic applications. Furthermore, additional roles for these cells as microniche sensors that monitor systemic pathology by endocytosing shed particles as they circulate through the vasculature will be explored. Taken together, novel insights into the many exciting potential uses of platelets outside of their canonical roles are on the horizon, and continued amelioration of existing protocols and enhanced understanding of communication pathways between platelets and specific cells will help expand opportunities for platelet-related clinical trials to yield improved health outcomes.

1. Introduction

Platelets are anucleate fragments derived from parent megakaryocyte (MK) cells. MKs are primarily produced in the bone marrow by nuclear replication without cytokinesis (endomitosis), thereby generating very large cells of 30–100 μm. Recent evidence from mouse and human lung tissue proposes the lung as a secondary site of hematopoietic stem cells producing MKs [1,2,3]. Future platelet regions, designated by demarcation membranes, are systematically populated with receptors, cytoskeletal and α granules, and dense granule components, delivered on tubulin tracks [4]. As progeny of MKs, platelets have a full range of cellular organelles including mitochondria, a dense tubular system, the Golgi apparatus, and an invaginated open canalicular system that is a source of membrane for cell diameter expansion upon platelet activation (Figure 1). Platelet α and dense granules are astonishing sources of bioactive reagents that serve as paracrine and endocrine stimulants and repressors, antibacterials, and inflammatory and anti-inflammatory modulators to direct platelet function and for heterocellular communication. Platelet activation promotes exocytosis of granule contents and release of microvesicles. Although platelets cannot replicate, they nevertheless are equipped with translational and splicing machinery [5] and host a range of MK-derived RNA populations [6], including mRNA and long and small non-coding RNAs. The RNA repertoire of platelets is increasingly investigated as part of the search for reliable liquid biopsy targets (see Section 3.2). MKs exhibit heterogeneity throughout organs and the lifespan, as revealed through proteomics and transcriptomics, with MKs from embryonic yolk sac and adult bone marrow or lung showing significant differences and lung MKs exhibiting an immature morphology at all stages [7,8,9]. Furthermore, the existence of platelet-biased and platelet-restricted HSC pathways that may be activated in case of thrombocytopenia [10] was discovered. Together, they likely influence how platelet production is maintained at adequate levels to sustain normal function. This review briefly summarizes basic platelet mechanisms of hemostatic and thrombotic function, but the greater goal is to provide an overview of multiple non-traditional roles for platelets as modulators of disease, as sensors for disease diagnostics, and as a source for platelet derivatives, and to consider how these additional functions may be mined for clinical practice.

2. Conventional and Non-Conventional Roles of Platelets in Disease

2.1. Platelets in Hemostasis and Thrombosis

Platelets are best known for their canonical role in hemostasis and thrombosis. While many of the principal pathways and signaling intermediates in the regulation of platelet activity have been identified [11], the quest for new players is still ongoing. This is due to the narrow therapeutic window separating thrombosis and bleeding, processes that share many signaling pathways. Thus, the holy grail is to identify antithrombotics that leave hemostasis unperturbed.

Platelet surfaces are richly endowed with diverse ion channels and transmembrane receptors. Several classes of receptors mediate initial capture and stable adhesion to exposed matrix or neighboring platelets: integrin receptors αvβ3, α2β1, α6β1, α5β1, and αIIbβ3, the platelet-specific integrin essential for stable platelet adhesion to fibrinogen and VWF for hemostasis; and immunoreceptors such as GPIb-IX-V, GPVI, and Fc receptors. In addition to thrombotic and hemostatic functions, these receptors and additional immune-related surface receptors such as Toll-like receptors (TLR)s, NOD receptors, and complement receptors serve as intermediaries for heterocellular interactions in immune function, inflammation, and organ pathology [12,13]. Platelet activation may be initiated through a multitude of canonical and non-canonical agonist receptors that induce signaling cascades, with principal signal-pathway intermediates including protein kinase C, PI-3K, calcium, and a variety of other tyrosine and serine kinases, phosphatases, and post-translational modification enzymes. Platelets also have an extensive cytoskeletal network that is amplified through complex actin-regulatory molecules, producing filopodial and lamellipodia protrusions and stress fibers, which help support firm adhesion despite mechanical stresses of shear flow.

2.2. Role of Platelets as Immune Cells in Non-Hemostatic Disease

Apart from their hemostatic functions, platelets are rich in receptors and granule proteins that can directly alter immune and inflammatory outcomes and non-hemostatic disease. The role of platelets as immune cells in their own right, and in inflammation, is becoming increasingly recognized, and only a brief discussion is presented here, with more detailed references provided. Platelets contribute to the immune response to pathogens both positively and negatively. Platelets can facilitate bacterial and viral infection through pathogen recognition of their surface integrins and immunoreceptors [14,15,16], promoting internalization, cloaking, and infection of distant organs [17,18]. Platelets also express TLRs including TLRs 2, 4, 6, 7, and 9 that recognize structurally conserved molecules derived from microbes, and are important in initiating innate immunity and promoting antigen-specific adaptive immunity [19]. Furthermore, platelets secrete immune mediators such as defensins, kinocidins, e.g., PF4/CXCL4 and RANTES/CCL5, and platelet microbicidal proteins (PMPs) from alpha granules [15].

Table 1. Select platelet molecules and their immune functions.

Molecule	Function	Platelet Connection	Disease Relevance	References
P-selectin (CD62P)	Adhesion molecule that binds leukocyte PSGL-1 and possibly Mac-1	Expressed on activated platelets; stored in α-granules; mediates platelet–leukocyte co-aggregates	Seen in SLE, rheumatoid arthritis, and antiphospholipid syndrome; marker of platelet activation in infections	[21]
CD40/CD40L	Immune co-stimulatory dyad; regulates inflammatory and humoral immune responses	Expressed and released by activated platelets; source of sCD40L; promotes endothelial activation and leukocyte recruitment	Involved in bacterial/viral clearance; contributes to vascular and neuronal inflammation in chronic inflammatory diseases	[15,22,23,24,25,26]
MHC Class I	Antigen presentation to CD8+ T cells	Platelets and megakaryocytes (MKs) present antigens via MHC I; link to adaptive immune activation	Triggers antimicrobial responses and interferon production	[27]
MHC Class II, CD11c	Antigen presentation to CD4+ T cells	More highly expressed in lung-resident MKs, suggesting APC-like phenotype	Suggests enhanced immune surveillance role for lung MKs	[28]
CFTR	Ion channel involved in epithelial fluid transport	Hyperactivation shown by CFTR-deficient platelets; impact on inflammatory lung response	Aggravates inflammation in CF; potential marker and modulator target in CFTR therapy	[29]
TRPC6	Cation channel implicated in calcium signaling and platelet activation	Platelet activation and lung injury reduced by inhibition in CF models	Target for reducing CF-related lung damage and platelet hyperactivation	[29]
Thrombopoietin receptor (mpl)	Regulator of platelet production	Impaired platelet regulation and increased susceptibility to lung injury in mpl-/- mice	Impaired defense in Pseudomonas infection; relevance in infection control and platelet homeostasis	[30]
β-defensins	Antimicrobial peptides that disrupt microbial membranes	β-defensin-1 and β-defensin-3 expressed and released by platelets, contributing to antimicrobial activity	Role in innate immunity and defense against pathogens	[31,32,33]
CXCL4 (PF4)	Chemokine that attracts monocytes, modulates T cell function, and can have antimicrobial properties	Abundantly stored in α-granules; released upon activation	Implicated in inflammatory diseases, thrombosis, and infection-related immune responses	[34,35]
CCL5 (RANTES)	Recruits leukocytes such as T cells and monocytes to sites of inflammation	Released by activated platelets; synergizes with CXCL4	Elevated in autoimmune diseases, cardiovascular disease, and infections	[36]
CXCL12 (SDF-1α)	Attracts hematopoietic and immune cells; supports vascular repair	Released from platelet granules; promotes leukocyte recruitment	Key in inflammation, cancer metastasis, and vascular diseases	[37]
IL-1β	Proinflammatory cytokine that initiates and amplifies inflammatory responses	Released by activated platelets and MKs	Found in platelet-driven inflammation in cardiovascular and neuroinflammatory diseases, and Kawasaki disease	[38,39,40]
Serotonin (5-HT)	Monoamine neurotransmitter with vasoconstrictive and proinflammatory properties	Stored in dense granules; released during platelet activation	Contributes to vascular tone, platelet aggregation, and inflammation in pulmonary and cardiovascular diseases	[41,42]

lists several key platelet-associated immune molecules. Thromboinflammation is the dysregulation of two often-coordinated processes, inflammation and thrombosis, and has been recognized as a pathological condition where thrombosis and inflammation occur together, often secondary to initial triggering events such as ischemia reperfusion injury. Treatment is complicated and may vary depending on the initiating factors. Antithrombotics have shown some benefit, although they entail bleeding risks, while anti-inflammatory drugs have shown more limited benefit and are potentially immunosuppressive [20].

2.3. Role of Platelets in Non-Hemostatic, Nonimmune Disorders

Our understanding of how platelets may contribute to the development, resolution, or aggravation of non-thrombotic, nonimmune disease is still incomplete, while the list of diseases in which platelets are implicated is constantly growing. Interestingly, platelet activation often occurs secondary to initial disease development. Platelet signaling pathways can selectively promote the progression of distinct diseases, and the resolution of platelet-related inflammation or thrombosis may allay disease advancement. Proteomic and transcriptomic studies are being mined with the aim of identifying new molecular players, in particular, in neurodegenerative disease and the triad of fibrosis, inflammation, and cancer (Figure 2).

2.4. Role of Platelets in the Nervous System

Although platelets and neuronal cells arise from different germ layers, mesoderm and ectoderm respectively, parallels exist between their cellular storage compartments and release mechanisms. Membrane fusion occurs via similar docking molecules: SNARES, syntaxins, and VAMPS [43,44]—however, neurotransmitters from neuronal synaptic vesicles are released into the synaptic cleft for neuron–neuron communication, whereas platelet granules are exocytosed directly into the blood circulation to reach distant sites. Platelet granules contain bioactive neuromodulators such as serotonin, epinephrine, dopamine, histamine, brain-derived neurotrophic factor (BDNF) [45], amyloid-beta precursor protein (APP), the inhibitory transmitter γ-aminobutyric acid (GABA), and the excitatory transmitter glutamate, through which platelets can modulate neural precursor proliferation and neurogenesis [46]. Additionally, the two ATP-gated P2X receptor family members in platelets, P2X1 and P2X7, have been proposed as candidate treatment targets to control neuroinflammatory disease, but more studies specifically linking platelet P2X receptors to neurodegenerative disease are needed [47]. The evidence for platelet contributions to neuropathology and neuroinflammation is strong.

Although platelets and neuronal cells arise from different germ layers, mesoderm and ectoderm respectively, parallels exist between their cellular storage compartments and release mechanisms. Membrane fusion occurs via similar docking molecules: SNARES, syntaxins, and VAMPS [43,44]—however, neurotransmitters from neuronal synaptic vesicles are released into the synaptic cleft for neuron–neuron communication, whereas platelet granules are exocytosed directly into the blood circulation to reach distant sites. Platelet granules contain bioactive neuromodulators such as serotonin, epinephrine, dopamine, histamine, brain-derived neurotrophic factor (BDNF) [45], amyloid-beta precursor protein (APP), the inhibitory transmitter γ-aminobutyric acid (GABA), and the excitatory transmitter glutamate, through which platelets can modulate neural precursor proliferation and neurogenesis [46]. Additionally, the two ATP-gated P2X receptor family members in platelets, P2X1 and P2X7, have been proposed as candidate treatment targets to control neuroinflammatory disease, but more studies specifically linking platelet P2X receptors to neurodegenerative disease are needed [47]. The evidence for platelet contributions to neuropathology and neuroinflammation is strong.

Alzheimer’s Disease (AD) neuropathology is marked by the accumulation of amyloid peptide (Aβ) and neurofibrillary tangles (NFTs). Aβ is derived from the processing of amyloid-beta precursor protein (APP) by beta-secretase 1 (BACE1) and γ-secretase, through an amyloidogenic pathway [48,49]. Platelets are the primary peripheral source of amyloid precursor protein (>90% of circulating APP and ≤90% Aβ) [50], with APP release upon platelet activation [51]. Conversely, Aβ peptides can activate platelets and promote their aggregation. In an AD mouse model, APP/PS1 transgenic mice form Aβ plaques starting at three months of age that increase in severity over time, with higher numbers of activated, surface CD62P positive platelets found within the brain parenchyma in close contact with astrocytes compared with WT mice. Platelet lysate from patients with AD revealed altered APP isoform ratios (APPrs) compared to healthy controls [52], which appeared to correlate with cognitive decline [49]. Thus, the APPr has been considered as a potential diagnostic biomarker. The observation that Aβ40 can bind platelet receptors GPVI and αIIbβ3 and the ligand fibrinogen, together with granule-secreted clusterin, to collectively form amyloid clusters at the platelet surface suggested a possible platelet-mediated mechanism contributing to Aβ aggregation in cerebral vessels. Indeed, blockade of α_IIbβ₃ or GPVI reduced platelet-associated amyloid aggregates, indicating a therapeutic benefit of antiplatelet regimens to ameliorate amyloid plaque formation in patient cerebral vessels and brain parenchyma [53]. Two other platelet molecules may be of interest in neuronal modulation: brain-derived neurotrophic factor (BDNF; >90% platelet-derived in blood) and reelin. BDNF is a member of the nerve growth factor family that may prevent neuronal death during development or after lesions [54]. Humanized mice with MK and platelet expression of BDNF were used to show neuroprotective effects on the dendrite complexity of retinal ganglion cells [55]. Reelin is an extracellular matrix glycoprotein produced by Cajal–Retzius neurons acting through canonical receptors VLDL and ApoER2 [56,57] that normally controls neuronal positioning and migration during brain development. As reelin operates within an optimal expression range, the perturbed expression and glycosylation found in patients with AD are significant [58,59,60]. Secreted platelet reelin can rebind platelet receptors GPIb, ApoER2, and APP [61] to activate platelets but may also undergo binding to the latter two receptors on neuronal cells, thereby providing another platelet link to AD. Furthermore, platelet loss of reelin in chimeric mice beneficially reduced infarct volumes in a cerebral ischemia model, indicating a separate platelet reelin–brain mode of action [61].

Parkinson’s Disease (PD) affects about 10 million people worldwide, involves the progressive loss of dopaminergic neurons within the substantia nigra, and entails motor and non-motor dysfunction and eventual death. α-synuclein mutation and the amassed Lewy bodies containing intracellular aggregates of α-synuclein protein [62] accumulate in the brain and peripheral organs and are considered a “hallmark” of the disease. α-synuclein is highly abundant in both the brain and platelets. Platelets additionally contain Parkin, and PTEN-induced kinase 1 (PINK1), tyrosine hydroxylase (TH), and DAT, all contributors to PD development, thus positioning blood platelets as a valuable peripheral model for PD. Similarly to AD, platelet shape and function are altered in PD, with greater adhesion, activation, and degranulation [63], which may drive thrombotic events. In view of the increased cardiovascular risk in patients with PD, platelet antagonists, particularly against platelet P2X receptors, are being investigated as a potential therapeutic option in PD and in other neurological disorders [47,64]. Assays of humanized platelet anti-GPVI Fab-fragment-inhibitor EMA601, encouragingly, have also shown profound protection against arterial thrombosis as well as cerebral infarct growth in tMCAO (transient middle cerebral artery occlusion), without endangering hemostasis [65].

Glutamate, an important neurotransmitter in the CNS, acts on a wide range of receptors and is important for synaptic transmission, learning, memory, and neural plasticity regulation. Platelets aid in clearing glutamate from the bloodstream through the excitatory amino acid transporter (EAAT) receptors 1, 2, and 3 [66], and contribute to maintaining steady-state values in the blood. In both patients with AD and PD, elevated blood glutamate levels are found along with decreased glutamate uptake in platelets, suggesting systemic dysfunction in glutamate metabolism [67] that could contribute to exacerbating excitotoxicity and neuronal death. Further studies could help elucidate links between platelet regulation of glutamate and neurodegenerative diseases.

In EAE mouse models of Amyotrophic lateral Sclerosis (ALS), platelets have been found to drive neuroinflammation in the spinal cord [68] and display mitochondrial dysfunction that includes changes in morphology and mitochondrial membrane potential [69]. The transactive response DNA-binding protein 43 (TDP-43) is a protein that, in nuclear cells, plays a role in RNA metabolism and gene regulation and, in non-nuclear cells, may bind RNA, regulate mRNA translation, miRNA processing, and the formation of intracellular RNA/stress granules, and regulate the transport of macromolecules in and out of mitochondria. TDP-43 was recently found to be present at high levels in platelets, along with many of its known RNA targets, and represents the main source in PRP. A molecular weight variant of TDP-43 was detected in ALS platelet cytosol samples, which was absent in samples from healthy controls [70]. The recent discovery of an interaction of TDP-43 with Optic atrophy 1 (OPA1), a GTPase involved in mitochondrial fusion, cristae integrity, and mtDNA maintenance, furthermore suggests a link between TDP-43 and platelet mitochondrial dysfunction in ALS and neurodegenerative disease [71]. In Huntington’s disease, platelets have increased levels of mutant Htt and changes in aspartate and glycine levels, that may promote Huntington’s [69,72]. Thus, controlling platelet levels of neuromodulatory proteins may offer a therapeutic strategy for treating neurological diseases [69].

2.5. Role of Platelets in Diabetes

In Type II diabetes (T2D), a complex metabolic disorder encompassing hyperglycemia and insulin resistance, many systemic processes are fundamentally disrupted, and may affect wound healing, nephropathy, retinopathy, and bone homeostasis. Normally, platelets may contribute to insulin homeostasis through releasate-mediated support of insulin secretion from pancreatic β cells [73,74]. Platelets in a decreased-insulin environment exhibit elevated platelet count and baseline activation, which, together with endothelial dysfunction, may contribute to diabetic complications, particularly in relation to cardiovascular events [75,76]. One potential mechanism of platelet activation in T2D may be an increased N-linked glycosylation of CD36 at Asn^408,417 that can stimulate the cellular uptake of long-chain fatty acids and promote activation by way of Lyn-JNK signaling [77]. Hyperglycemia may also induce platelet elevation of stromal interaction molecule 1 (STIM1), a transmembrane protein that, together with Orai-1, forms a calcium channel that regulates calcium influx. Accordingly, an inhibitor of store-operated calcium entry in mouse models and in patients, CM4620, attenuated agonist-induced platelet aggregation and other activation markers [78]. Thus, targeting hyperglycemia-induced platelet activation and canonical platelet receptors such as GPIb-V-IX [79] may help reduce atherosclerosis and potentially other platelet-related contributions to T2D.

2.6. Role of Platelets in Fibrotic Disease and Cancer

Fibrotic disease, a disorder with cell- and tissue-specific underlying biology, may cause tissue stiffening and compromised function, or may be a prelude to cancer, particularly in a setting of inflammation. Two fibrosing tissues with associated platelet involvement are the liver [80] and the lung.

The liver is a source of several coagulation enzymes, plus the adhesion proteins fibrinogen and VWF, and liver disease can alter their levels, leading to microthrombosis and increased platelet consumption [81]. Thrombocytopenia is a common side effect and can further aggravate cholestasis-induced liver fibrosis through reduced platelet HGF release and the c-Met receptor pathway [82,83,84]. In a mouse model of liver fibrosis induced by chronic treatment with CCl4, platelet-specific overexpression or PF4-C3GKO-mediated knockdown of C3G (aka RapGEF1), a molecule involved in platelet activation and secretion, were, respectively, protective against fibrosis or pathogenic with an increased proinflammatory phenotype and larger liver tumors than in treated control mice [85]. Thus, at normal expression levels, platelet C3G may be part of a regulatory mechanism to prevent liver fibrosis and cancer development. In systemic sclerosis, marked by progressive fibrosis of multiple tissues, platelets together with neutrophils may exacerbate symptoms, since platelet depletion reduced neutrophil activation and fibrosis that were in part dependent on a GPVI-mediated interaction with neutrophils [86].

A role for platelets in idiopathic pulmonary fibrosis (IPF) was also hypothesized, since in patients with IPF, an increased platelet count was correlated with higher mortality. While TGFβ1 is a known driver of lung fibrosis, TGFβ1^fl/fl PF4-Cre-mediated MK- and platelet-specific deletion did not significantly promote inflammation or fibrosis in a bleomycin model of acute fibrosis, despite increased overall platelet reactivity [87]. Nevertheless, in a similar bleomycin model, platelet depletion ameliorated histologically confirmed loss of pulmonary function, and attenuated the development of fibrotic lung lesions, suggesting that platelet factors other than TGFβ1 may help drive fibrosis [88]. In studies of human patients with IPF, coadministration of antiplatelet drugs and anti-fibrotic drugs nintedanib and pirfenidone failed to slow IPF progression and was even associated with poorer survival [89,90]. Thus, while the preponderance of evidence supports a role for platelets in fibrosis, much work remains to be done to identify specific molecular pathways that would be aided by perfecting disease models, e.g., for IPF [91,92], to better recapitulate the complex, progressive nature of fibrotic disease and to optimize combination therapeutics.

Lastly, whether through contributions to inflammation, fibrosis, or tumor angiogenesis, platelets have been shown to play a significant role in cancer development and metastasis [93,94], and in turn, cancer elicits changes in platelet counts, activation, and tissue infiltration [95,96]. In vitro platelet co-culture with tumor cells or in vivo platelet-specific knockout models have provided evidence for a direct platelet role in inducing EMT, proliferation, and metastasis [97,98,99], reducing mitochondrial membrane potential, and increasing resistance to apoptosis [100]. Consistently, platelet depletion reduced tumor size and improved the efficacy of chemotherapy in a mouse breast cancer model, while thrombocytosis was correlated with ovarian cancer progression, with increased tumor size and decreased drug efficacy observed in patients transfused with platelets [101,102]. Exploration of growth factors using immunoassays or proteomic profiling will aid in identifying key platelet-associated targets that support tumor cell proliferation and survival.

3. Platelet Derivatives for Potential Therapeutic Application

The canonical functions of MKs and platelets are well documented, and in Section 2, novel roles for these cells and molecular players in the development of disease were discussed. However, there remain new creative ways that platelets and MKs and their derivatives may be exploited. The following section explores the potential of novel applications related to these cells that may be harnessed therapeutically and also discusses some advances and concerns related to realizing these goals.

3.1. Potential of Stem Cell-Differentiated MKs and Platelets for Translational Medicine

To fulfill a need for augmenting platelet blood levels in chemotherapy-treated patients [103] and in various thrombocytopenic disease settings such as bone marrow deficiencies, platelet depletion in autoimmune thrombocytopenia ITP) [104], and microangiopathies [105], platelet counts must be raised endogenously or exogenously. In chronic diseases, therapeutics focus on boosting thrombopoiesis by regulation of thrombopoietin or its receptor, mpl. For acute treatment, the infusion of platelet concentrates may transiently boost platelet counts; however, supplies are often limited, and concentrates may carry risks for allergic reactions, infection, and the development of alloimmunization to human leukocyte antigens (HLAs). Hence, the motivation to produce autologous or off-the-shelf stem cell-derived platelets, primarily from human-induced pluripotent stem (iPS) cells, or alternatively, from CD34+ umbilical cord blood cells [106], is strong and has been buoyed by recent upgrades to protocols for hIPSC differentiation to MKs and platelets. Furthermore, updated gene editing techniques [107,108] have allowed selective modification of the MK genome and of downstream-generated platelets, and prompted the promise of future correctable targets.

While at present, reasonably good-quality, functional platelets may be produced at a small scale, unfortunately, the long-term goal of delivering clinical-grade platelets as an adjunct to platelet transfusion at a large scale is still hampered by production limitations and economic feasibility. In recent years, specific molecular pathways and mechanical approaches have been identified that enhance MK and platelet yield from differentiating stem cells—in particular, transient expression of c-Myc [109,110,111]; miRNA let-7a-5p and let-7g-5p contribution to lineage determination in immature MKs [112]; recognition of the inverse relationship of epigenetic regulator SET domain-containing 2 (SETD2) with platelet count [113]; antagonism of the aryl hydrocarbon receptor transcription factor, known to maintain CD34 expression of progenitor cells [114]; and use of turbulent flow reactors [111]. The hope is that gene-edited iPSC-derived platelets, or even off-the-shelf universal MKs and platelets, may eventually be incorporated into clinical applications [115]. With the aim of treating alloimmunized patients with thrombocytopenia, an exciting proof-of-principle study from Koji Eto’s group in Japan resulted in the iPLAT1 study, the first in-human Phase 1 autologous transfusion clinical trial, which successfully modified a patient’s peripheral blood mononuclear cells, reprogramming them to iPSCs and differentiating them to platelets, to circumvent the absence of a suitable donor. The cells produced were immune-matched and avoided alloimmune platelet transfusion refractoriness (allo-PTR) [116]. In another iPS-MK success, albeit in vitro, MKs and platelets from WAS protein-induced pluripotent stem cell (iPSC) lines established from patients with Wiskott–Aldrich Syndrome (WAS), a severe X-linked disorder, with characteristically small platelets and defects in proplatelet formation, showed improved platelet size and proplatelet morphology [117]. In contrast, in a preclinical model of familial platelet disorder with associated myeloid malignancies (FPDMM) driven by RUNX1 heterozygous mutations in rhesus macaques, attempts to transplant autologous RUNX1-edited HSPCCs to correct germ-line loss of function did not show amelioration and failed to rescue low platelet counts and megakaryoblastic dysplasia [118], thus highlighting potential challenges to this approach that may be disease dependent.

3.2. Role of Platelets as Diagnostic Sensors

As discussed above, MKs are mostly restricted to their niches of origin, surrounded by endothelial cells and multipotent mesenchymal stromal cells [119]. In contrast, platelets circulate freely and endocytose foreign particles—proteins, chemicals, mRNA, and miRNA [120]—from encountered environments and thus have the capacity to report on systemic changes. Foreign endocytosed material may remain cytoplasmic, be stored for release, or translated (in the case of mRNA). AI-aided high-resolution microscopy [121] and proteomic studies of platelets have been helpful in this regard, but it is the advent of platelet transcriptomics that is advancing the quest for platelet-based diagnostics for both cancer and non-cancer diseases, and the characterization of platelets as sentinels of the body and overall organismal health. A central hypothesis is that platelets, upon contact with tumor cells, become “tumor-educated platelets” (TEPs), acquiring tumor-specific RNA and demonstrating altered platelet transcriptomic profiles [122,123]. This has paved the way for the use of liquid biopsies to source platelets and analyze their transcriptomes to semi-quantitatively assess tumor biomarkers and offer non-invasive diagnostics compared with conventional tissue biopsies.

Platelets offer a unique opportunity to report on systemic changes due to (a) the ease of platelet isolation; (b) widespread circulation and access to diverse cell types; (c) the limitation of nuclear signature changes to newly acquired inputs, since platelets are anucleate; (d) RNA stability over 48 h [124] and the protection of ingested RNA from circulating RNAse; (e) the short platelet lifespan (7 days) ensuring relatively recent molecular profiles; and (f) platelet presence of a functional spliceosome (unlike erythrocytes) that can splice pre-mRNA upon platelet activation signals [125]. Thus, their transcriptomic signature makes them ideal candidates for detecting and profiling chronic or progressive malignancies, or even other diseases such as AD that may affect platelets [126]. The combination of large data cohorts available for reanalysis through the publicly available Gene Expression Omnibus (GEO) and other databases and machine learning algorithms further allow querying specific markers and potential prognostication.

In multiple studies across different tumor types, platelet transcriptomic alterations have yielded insights into systemic cancer change. In non-small cell lung cancer (NSCLC), distinct platelet subtypes were identified and platelet-derived chloride channel gene Best 3 and cytoskeletal protein, Filamin A, expression levels could be used to identify High-Risk Populations that were correlated with tumorigenesis, metastasis, and poor prognosis [127,128]. Alternatively, miRNAs [129] or gene clusters may be useful for early screening in NSCLC analysis of RNAseq data [130]. Platelet sensors have been used in glioblastoma, breast cancer, and ovarian cancer [131], amongst other malignancies, for detection and potential prognosis [132,133,134,135,136]. Spliced RNA profiles of TEPs from glioblastoma and multiple sclerosis and asymptomatic controls were able to reliably distinguish false-positive cancer progression from true progression via liquid biopsy [133]. Unfortunately, however, some transcriptomic studies showed limited platelet diagnostic ability in esophageal cancer and mixed results in pancreatic cancer. Whether this is a function of variations in preparation protocols, detection limitations, or tissue-based differences in particle shedding or other factors remains to be determined. Other platelet-related parameters proposed for diagnostics have included platelet count, platelet lymphocyte ratio and preoperative thrombocytosis; however, they have not consistently tracked with cancer-free survival or cancer progression [137,138].

While overall there is widespread enthusiasm for platelet-based liquid biopsies as a non-invasive way to track tumor progression in humans, one cautionary study using blood samples from patients with glioblastoma suggested that tumor transcripts in platelets may have been indirectly derived from endocytosed, contaminating leukocytes interacting with tumors [139]. However, a recently developed photo-cleavable mass-tagged self-assembled (SAMT) nanoprobe conclusively tracked the transfer of PD-L1 from cancer cells to TEPs and found increased TEP PD-L1 correlated with higher tumor stages in patients with NSCLC [140]. As an alternative to platelet-based liquid biopsy, cancer presence may be monitored by assaying circulating tumor DNA (ctDNA) in blood [141,142]. Although this is a rapid, qPCR-based method, ctDNA is somewhat unstable in blood (t_1/2 < 2.5 h) and is limited to detecting specific pre-identified tumor mutations. Advantageously, platelets provide a more comprehensive picture of changes due to tumors, incorporating protein, RNA, and other molecular snapshots, and may distinguish cancers earlier than ctDNA. Thus, despite some hiccups, TEPs will continue to be investigated as prognostic sentinels of cancer, with ongoing optimization and development of novel analytical techniques.

3.3. Challenges to Implementation of Platelet-Based Diagnostics

What then are the challenges to implementing platelet-based Omics diagnostics that enable comparisons between experimental groups and between disease states as they vary over time or tissues? There are a few key areas to address: (a) standardization of platelet preparation protocols that minimize activation, avoid the loss of nuclear material, and prevent leukocyte contamination [143], the latter potentially circumvented by single-cell RNAseq [144]; (b) optimization of RNA purification to preserve sample integrity and stability without transcript loss; (c) development of user-friendly Omics analysis algorithms that preserve data accuracy, objectivity, and complexity [145] and allow inter-disease and intra-disease stage or subtype delineation; (d) cost reduction for Omics analyses, particularly for large case–control cohorts; (e) incorporation of classifiers including age, pathology, gender, and ethnic background. Relying solely on platelet transcriptomes may ignore supplemental information gleaned from proteomics and under-represent the following: (i) membrane receptors, channels, and other low-abundance proteins; (ii) imported transcription/translation proteins; and (iii) thus-far-uncharacterized proteins [146]. For clinical utility, additional sources of variability should be factored in [145] such as disease classification accuracy and sampling time for examining early versus late disease progression, especially for challenging cancers where patients may be largely asymptomatic at early stages. Omics preparations of nucleated MKs now allow data integration between proteomics, genomics, and transcriptomics, to bolster the specificity of analytic data obtained [147]. Additionally, MK DNA exhibits unique genome-wide methylation patterns [147] that may be transmitted to platelets and used to distinguish them from erythrocytes. In special cases with low-abundance transcripts, where increasing read depth is inadequate [148], more sensitive methods such as RT-qPCR may be recommended to validate tumor-derived transcripts.

3.4. Platelet-Rich Plasma for Regenerative Medicine

In addition to their utility as sensors, platelets can also provide therapeutic benefit if they are transported in platelet-rich plasma (PRP), “peeled” to isolate platelet membranes with integral receptors and ion channels, or shed as microvesicles containing mRNAs, assorted cytokines, chemokines, and other factors.

Low-speed centrifugation of whole blood allows for relatively easy, abundant separation of PRP from the buffy coat layer containing leukocytes and from the red blood cell layer. PRP is a nonhomogeneous mixture of plasma and granule secretome rich in growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and hepatic growth factor (HGF), along with matrix metalloproteinases, various chemokines and cytokines, and microvesicles, that may be individually isolated and quantified. A role for PRP in therapeutics has been postulated for over forty years, and what was regarded with great skepticism initially is now more widely accepted and actively being tested in clinical trials in regenerative medicine across fields. The benefit of PRP injections into ailing tissues has been investigated as treatment for bone degeneration in patients [149], diabetes-associated-wound healing [150], skin cosmetic rejuvenation and for application in sports medicine and orthopedic surgery [151], albeit with some inconsistencies in clinical outcomes. As there are some excellent comprehensive reviews [152,153,154,155,156,157], only a few recent results and techniques are briefly summarized here.

Of interest, some modifications to existing protocols are intended to lengthen PRP residence time and better guide PRP to desired sites. To improve bone regeneration and mineralization in osteoblast cells, in one study, a collagen-based matrix with PRP was implanted to deliver an osteo-inductive BMP-2 gene plasmid to an area of bone damage [158], while in another, a platelet-rich fibrin (PRF) matrix was enriched with denatured albumin (Alb-CGF) [159]. These modified PRP formulations induced bone formation and increased proliferation, cell density, and mineralization over time, as indicated by micro-CT analysis and alizarin-red histological staining. Alternatively, the dispensing of platelet lysate alone, extracted from platelet concentrate, is also being tested due to the lack of a size barrier in accessing injured sites. Surprisingly, platelet lysate has shown therapeutic benefit even when administered by intranasal route in an intracerebral hemorrhage model in mice [160], and in patients with traumatic brain injury. Cognitive abilities and motor functions were enhanced relative to the control group, and decreased levels of cortical neuroinflammation and oxidative stress were found [161].

3.5. Platelet and MK Extracellular Microvesicles (EVs)

Platelet EVs are released from stimulated or apoptotic platelets and are the most prevalent of the plasma EVs. They may be isolated from PRP or stored platelets. Their sufficiently small size (100–1000 nm for membrane-derived microvesicles and 30–100 nm for endosome-derived exosomes) permits access to blood and lymph and synovial fluid [162]. Additionally, EVs contain heterogeneous cargo that may vary with microenvironmental conditions and the mode of platelet activation or release [163], that may lead to variations in adhesion receptor levels [164]. As expected, platelet EVs are effective procoagulant and hemostatic agents, can attenuate the vascular permeability of endothelial cells [165], and, in recent studies, have shown some success in hemorrhagic shock and wound healing models as well [163,166,167,168]. EVs derived from MKs or MK cell lines are sufficiently larger to accommodate synthetic cargo, as demonstrated by bone marrow-targeted expression of fluorescent reporter plasmids [169], and were able to boost platelet counts. The potential for expression of other genetically engineered proteins in MK EVs is tantalizing but requires further extensive optimization.

3.6. Platelet Membranes in Nanorobotic Therapeutics

There are ongoing efforts to use cellular membranes of platelets and other cells for the coating of nanovesicles that can hold therapeutic cargo in order to preserve the surface complexity and biocompatibility of biological cells. Platelet-coated nanoparticles are being investigated in applications from cancer [170,171] to infectious disease [172], ischemia [173], and pre-natal drug delivery [174]. Isolation of membranes involves lysis by chemical or mechanical means such as Triton X-100 detergent or ultrasonic treatment [175], isolation, and purification, and binding to loaded nanoparticles, or “nanorobots”. The functional properties of cell membranes improve the blood residence time and tumor targeting and may help enhance blood brain penetration [176,177]. Optimization efforts are aimed toward improving delivery modalities, nanoparticle size: cargo ratio, disease-dependent cargo formulation and tissue targeting, and pharmacokinetics.

There are a variety of nanoparticle configurations that are in development and, indeed, depending on the intended focus, may demand size, composition, and cargo modifications. Some interesting delivery modes and applications [175] include: (1) biomimetic magnetic nanorobots, loaded with PD-L1, encapsulated with hybrid membranes from platelet and M1 macrophages that, enabled by their 50 nm diameter, could enhance BBB penetration and reach glioblastoma tissue to deliver immunotherapy and photothermal therapy [176]; (2) platelet-membrane coated Poly lactic-co-glycolic acid (PLGA) nanoparticles with encapsulated hepatic growth factor that were used to enhance angiogenesis and restore tissue perfusion in ischemic conditions [173]; (3) inhalable platelet-membrane-coated algae-based microrobots functionalized with polymeric nanoparticles loaded with vancomycin that were used to access lungs in a mouse model of acute methicillin-resistant Staphylococcus aureus pneumonia [178] and reduce bacterial burden; (4) hyperechogenic agarose-based “nanobubbles”, coated with a thrombotically inert platelet membrane with intact integrin receptors, that were used to view thrombi by diagnostic ultrasound imaging in vitro and in live mice in vivo [179].

For platelet derivatives to be of clinical utility, they must be subjected to stringent standards compliant with regulations to ensure pathogen-free delivery, adaptability for large-scale production with appropriate permitted production facilities, and an established tracking system that covers the journey from blood collection to the end of production and delivery to ensure a non-toxic, nonimmunogenic product. Success in therapeutic utilization of platelet derivatives may also vary with the patient’s conditions, the combination of medication or surgical treatments, and with variations amongst human platelets sourced for derivatives.

4. Future Perspectives

With many new exciting developments in our understanding of platelet biology in various disease settings and the benefits of platelet derivatives, the challenge remains as to how to best extract molecular specificity for eventual therapeutic control, without impinging on key platelet functionalities. Thus, any new molecules identified for potential non-thrombotic applications will need to be assessed in the context of the very broad range of cells, tissues, and diseases touched by platelets. As discussed, the advent of platelet-friendly transcriptomic techniques supported by better in vitro and in vivo mouse models, and eventually human patient trials, will help establish disease-relevant platelet networks and steer valuable future discoveries.