# Defining and Harnessing the Megakaryocyte/Platelet Checkpoint

Alexandra Mazharian, Yotis A Senis

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Alexandra Mazharian, Yotis A Senis. Defining and Harnessing the Megakaryocyte/Platelet Checkpoint. Molecular and Cellular Biology, 2025, 45 (3), pp.116-128. 10.1080/10985549.2025.2451279. hal-05536391

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# Defining and Harnessing the Megakaryocyte/Platelet Checkpoint

Alexandra Mazharian & Yotis A. Senis

**To cite this article:** Alexandra Mazharian & Yotis A. Senis (2025) Defining and Harnessing the Megakaryocyte/Platelet Checkpoint, Molecular and Cellular Biology, 45:3, 116-128, DOI: 10.1080/10985549.2025.2451279

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# Defining and Harnessing the Megakaryocyte/Platelet Checkpoint

Alexandra Mazharian ID and Yotis A. Senis ID

Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche-S 1255, Etablissement Français du Sang Grand Est, Université de Strasbourg, Strasbourg, France

## ABSTRACT

Platelets, or thrombocytes are anucleate cell fragments of megakaryocytes (MKs) that are highly reactive to sites of vascular injury and implicated in many pathologies. However, the molecular mechanisms regulating the number and activity of platelets in the circulation remain undefined. The primary out- standing question remains what is the triggering mechanism of platelet production, or thrombopoiesis? Putative stimulatory factors and mechanical forces are thought to drive this process, but none induce physiological levels of thrombopoiesis. Intrinsic inhibitory mechanisms that maintain MKs in a refractory state in sites of thrombopoiesis are conspicuously overlooked, as well as extrinsic cues that release this brake system, allowing asymmetric platelet production to proceed toward the vascular lumen. Here we introduce the novel concept of a MK/platelet checkpoint, putative components and a working model of how it may be regulated. We postulate that the co-inhibitory receptor G6b-B and the non-trans- membrane protein-tyrosine phosphatases (PTPs) Shp1 and Shp2 form an inhibitory complex that is the primary gatekeeper of this checkpoint, which is spatiotemporally regulated by the receptor-type PTP CD148 and vascular heparan sulfate proteoglycans. By advancing this alternative model of thrombopoie- sis, we hope to stimulate discourse and a shift in how we conceptualize and address this fundamental question.

## ARTICLE HISTORY

Received 12 August 2024
Revised 18 December 2024
Accepted 18 December 2024

## KEYWORDS

Megakaryocytes; platelets; checkpoint; G6b-B; CD148

## The MK/Platelet Checkpoint and Asymmetric Platelet Production

Platelets, or thrombocytes are anucleate cell fragments of megakaryocytes (MKs) that are highly reactive to sites of vascular injury. They have been implicated in many pathophysiological processes, the foremost being thrombosis and hemostasis, and also maintenance of vascular integrity and wound repair, immunity and inflammation, cancer metastasis and age-related cognition (Figure 1). Common to all pathological outcomes in which they play a role is the number and biological activity of platelets in the circulation, however, the molecular mechanisms regulating these parameters remain undefined. The primary driver of MK development, proliferation and maturation, collectively referred to as megakaryopoiesis is the cytokine thrombopoietin (Tpo) produced by the liver, kidneys and bone marrow (BM), the biological effects of which are transmitted by the myeloproliferative leukemia protein (Mpl) receptor expressed on hematopoietic stem and progenitor cells (HSPCs), MKs and platelets (Figure 2).¹ Plasma Tpo levels are inversely related to platelet counts, but Tpo does not induce MKs to undergo platelet production, or thrombopoiesis. Decades of research have yet to reveal the hypothetical stimulatory factor and molecular mechanism regulating when and how MKs produce platelets.

Existence of an extrinsic thrombopoietic factor is plausible, but no conventional candidate investigated thus far yields physiological levels of thrombopoiesis, including cytokines, chemokines and components of the extracellular matrix (ECM). The prevailing thinking is that mechanosensing is the primary driver of thrombopoiesis,²⁻⁵ but this is inconsistent with physiological conditions. First, the biophysical properties and cellularity of primary sites of thrombopoiesis vary greatly, from the high pressure, rigid BM, where the majority of MKs reside and platelets are produced, to the low pressure, malleable environments of the spleen and lungs. MKs nonetheless generate proplatelets and release preplatelet fragments in all of three sites.⁶⁻⁸ What makes the BM the primary source of platelets is the number of resident MKs, not the biophysical parameters of the organ or rate of platelet production per se. Second, neither tissue resident MKs nor their proplatelet extensions experience high shear, turbulent blood flow in sinusoidal blood vessels. Such mechanical forces exist in downstream regions of the vasculature, but capturing and defining the exact moment of platelet biogenesis in the fluid microenvironments of the circulation is challenging. It appears an intrinsic auto-regulatory mechanism drives the process of proplatelet fragmentation and platelet release once set in motion, involving molecular motors, cytoskeletal rearrangements and cleavage enzymes, which is augmented by external mechanical forces.⁹,¹⁰ Notably, a significant

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<figure><img src="image_19.png" /><figcaption>**Figure 1.** The duality of platelets in health and disease. Platelets play a fundamental role in a variety of pathophysiological processes and are implicated in leading causes of morbidity and mortality globally. There is an urgent medical need for improved platelet therapeutics and an unlimited supply of transfusable platelets.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 2.** Triggering mechanism of thrombopoiesis. The cytokine thrombopoietin (Tpo) induces hematopoietic stem and progenitor cells to differentiate into megakaryocytes (MKs) in the bone marrow and other sites of megakaryopoiesis. The molecular mechanism that triggers MKs to undergo platelet production, or thrombopoiesis remains undefined. MKs in contact with blood vessels become polarized, releasing proplatelet fragments asymmetrically into the vascular lumen. The prevailing thinking is that activation signals and mechanical forces within sites of thrombopoiesis are the primary drivers of this process, however none of the factors or conditions tested thus far mediate physiological levels of thrombopoiesis. Inhibitory signals that maintain MKs in a refractory state in sites of thrombopoiesis have been conspicuously overlooked.</figcaption></figure>

proportion of primary MKs undergo proplatelet formation and release platelet-like particles in liquid culture under static conditions, albeit significantly fewer than in vivo.¹¹ Although viscosity, shear and turbulence contribute to thrombopoiesis of cultured primary MKs, the number of platelet-like particles produced under these nonphysiological conditions remains at best an order of magnitude below the 3000–5000 platelets estimated to be released per MK under physiological conditions.¹² Further, genetically ablating mechanosensing receptors in MKs only partially impacts platelet counts in mice,¹³ challenging mechanical forces as the driver of thrombopoiesis. A change is needed in how we conceptualize this fundamental process to circumvent the current impasse.

Equally important, but conspicuously overlooked are the inhibitory mechanisms that maintain MKs in a refractory state in sites of thrombopoiesis. We envisage the spatiotemporal release of such a brake system exclusively at the vessel wall being essential for MK polarization and asymmetric proplatelet formation to proceed towards the vessel lumen. Underpinning this model is the novel concept of an MK/

platelet checkpoint that safeguards against the haphazard release of platelets in sites of thrombopoiesis and the pathological consequences of this happening (Figure 3A). Parallels can be drawn with the conventional cell cycle checkpoint that acts as a quality control mechanism to ensure faithful cell replication,¹⁴ and the immune checkpoint that prevents immune cells from attacking themselves.¹⁵ Common to all cellular checkpoints is the notion of a gatekeeper, or inhibitory mechanism, controlled by intrinsic factors and extrinsic cues, and the central role of reversible phosphorylation in the assembly, compartmentalization and activity of regulatory protein complexes. We postulate that the MK/platelet checkpoint has hallmarks of the immune response checkpoint, but restrains MKs from making platelets until they are needed.

## The Co-Inhibitory Receptor G6b-B: Intrinsic Gatekeeper of Thrombopoiesis

Based on a growing body of evidence, we hypothesize the MK/platelet co-inhibitory receptor G6b-B (MPIG6B) is the main
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A

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B

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Figure 3. The megakaryocyte (MK)/platelet checkpoint. (A) Here we introduce the novel concept of an MK/platelet checkpoint that maintains MKs in a refractory state in sites of thrombopoiesis and is released when MKs come in contact with the vascular wall, allowing asymmetric thrombopoiesis to proceed. We postulate that the lineage-specific co-inhibitory receptor G6b-B together with non-transmembrane protein-tyrosine phosphatases (PTPs) Shp1 and Shp2 form an inhibitory complex that acts as a gatekeeper of this checkpoint, regulating activation signals from tyrosine kinase-linked receptors, including immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors and integrins. Contact of G6b-B with vascular heparan sulfates inhibits this inhibitory signaling, allowing MKs to make and release proplatelets into the circulation. Once in the circulation, G6b-B helps maintain platelets in a resting state through kiss-and-go interactions with heparan sulfates in the endothelial glycocalyx. Platelet-derived heparanase mediates release of heparan sulfate degradation products, that may also play a role in regulating G6b-B signaling. (B) G6b-B consists of a single extracellular immunoglobulin variable-like (IgV) domain, a transmembrane domain and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibitory and switch motifs (ITI/SM). Tyrosine phosphorylation of the ITI/SM by Src family kinases (SFKs) mediates binding and activation of the non-transmembrane Src homology 2 (SH2) domain-containing Shp1 or Shp2, which are essential for its inhibitory activity. Ligand-mediated heteroreceptor clustering brings G6b-B phospho-ITI/SM-associated Shp1 or Shp2 within the molecular reach of the proximal signaling complex of ITAM-containing receptors, such as the GPVI FcR γ-chain complex, including SFKs and phospho-ITAM-associated Syk tyrosine kinase that can be dephosphorylated and inactivated. Ligands of both G6b-B and GPVI are indicated. Immunoglobulin-like domains 1 and 2 (D1, D2).

gatekeeper of the MK/platelet checkpoint.¹⁶ The primary evidence is the profound effect of loss of G6b-B expression and function on MK/platelet homeostasis in mice and humans. No other immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor plays as important a role in regulating thrombopoiesis and platelet function as G6b-B, as evidenced by the phenotypes of knockout mouse models (Table 1). Although ablation of other genes, including cytokines, cytoskeletal proteins, transcription and translation

factors has equal or more severe consequences on megakaryopoieiss and thrombopoiesis, none of them only impact thrombopoiesis and possess the structural and functional criteria of a thrombopoiesis gatekeeper like G6b-B. Similar to the immune checkpoint regulator programmed cell death protein-1 (PD-1),¹⁷ G6b-B consists of a single extracellular immunoglobulin variable-like domain (IgV), a transmembrane domain, and a cytoplasmic tail containing an ITIM (I/V/LxYxxL/V) and immunoreceptor tyrosine-based switch motif
<figure><img src="image_24.png" /><figcaption>**Table 1.** Summary of ITIM-containing receptor null mouse phenotypes</figcaption></figure>

<table><thead><tr><th>Knockout mouse models</th><th>Platelet counts/ volume</th><th>MK/proplatelet formation</th><th>Platelet reactivity</th><th>Hemostasis</th><th>Thrombosis</th><th>Ref.</th></tr></thead><tbody><tr><td>PECAM-1 KO (CD31)</td><td>Platelet count: normal, delayed recovery after depletion<br>Platelet volume: normal</td><td>Normal MK maturation<br>↓ MK migration and polarization<br>Altered MK localization in the BM<br>↑ Extramedullary megakaryopoiesis</td><td>↑ Collagen, thrombin aggregation</td><td>↑ Tail bleeding</td><td>↑ Onset, size, stability in laser and ferric chloride injury models</td><td>69-71</td></tr><tr><td>G6b-B KO</td><td>Platelet count: ↓ 77%, increased clearance reduced production<br>Platelet volume: ↑ 38%</td><td>↑ Extramedullary megakaryopoiesis<br>Normal MK maturation<br>↓ MK spreading and PPF</td><td>↓ GPVI aggregation, ↑ CLEC-2 aggregation, thrombin aggregation reversible</td><td>↑↑ Tail bleeding</td><td>Unknown</td><td>19,23,24</td></tr><tr><td>TLT-1 KO</td><td>Platelet count: ↓ 20%<br>Platelet volume: unknown</td><td>Unknown</td><td>↓ ADP and U46619 aggregation, delayed collagen and thrombin aggregation</td><td>↑ Tail bleeding</td><td>Unknown</td><td>21,72,73</td></tr><tr><td>CEACAM1 KO (Bgp-1)</td><td>Platelet count: normal<br>Platelet volume: unknown</td><td>Unknown</td><td>↑ GPVI aggregation, granule release and flow adhesion</td><td>↑ Tail bleeding</td><td>Form larger, more stable thrombi</td><td>74</td></tr><tr><td>CEACAM2 KO (Bgp-2)</td><td>Platelet count: normal<br>Platelet volume: unknown</td><td>Unknown</td><td>↑ GPVI aggregation, granule release and flow adhesion, ↑ CLEC-2 aggregation</td><td>↑ Tail bleeding</td><td>Form larger, more stable thrombi</td><td>75</td></tr><tr><td>PIR-B KO (LILRB2)</td><td>Platelet count: ↑ 47%<br>Platelet volume: unknown</td><td>Unknown</td><td>↑ GPVI aggregation and granule release, ↑ ADP and thrombin aggregation</td><td>Unknown</td><td>Unknown</td><td>76</td></tr><tr><td>LAIR-1 KO</td><td>Platelet count: ↑ 25%<br>Increased half-life<br>Platelet volume: unchanged</td><td>Normal MK maturation<br>↑ MK spreading and PPF</td><td>↑ GPVI aggregation</td><td>Normal bleeding</td><td>Form larger, more stable thrombi on collagen<br>↑ Thrombus formation in chloride injury model</td><td>77</td></tr></tbody></table>

(ITSM, TxYxxV/I), abbreviated ITI/SM when merged (Figure 3B).¹⁸ Phosphorylation of the central tyrosine residues in these motifs by Src family kinases (SFKs) mediates binding and activation of either the non-transmembrane Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (PTP) 1 or 2 (Shp1, Shp2), which is essential for the inhibitory activities of both receptors. Binding of PD-1 to its counter-receptor PD-ligand 1 (PD-L1) on antigen presenting cells and platelets increases inhibitory PD-1 signaling, thereby preventing immune cells from attacking host cells.¹⁷ However, we postulate a unique mode of regulation of G6b-B, in which binding of G6b-B to heparan sulfate (HS) ligands in the vessel wall inhibits inhibitory G6b-B signaling in MKs, resulting in polarization and asymmetric thrombopoiesis. Conversely, kiss-and-go interactions of platelet G6b-B with HS ligands in the endothelial glycocalyx maintains tonic inhibitory G6b-B signaling and platelets inactive in the circulation. Theoretical mechanisms of these functions are described below. Other attributes of G6b-B lending it to this model are its lineage-specificity, constitutive expression and basal phosphorylation at the plasma membrane,¹⁹ suggesting it is essential for maintaining MKs/platelets in a refractory/resting

state. The only other lineage-specific member of this family is triggering receptors expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) stored in α-granules and upregulated on the MK/platelet plasma membrane following activation.²⁰,²¹ However, distinct structural features, compartmentalization and null mouse phenotypes demonstrate unique physiological functions compared with G6b-B.²²

Defining features of null and loss-of-function mutations in *Mpig6b* in mice include severe macrothrombocytopenia, expansion of the MK population, release of preplatelet fragments and platelet-like particles in sites of thrombopoiesis, and myelofibrosis (Figure 4A and B).²³⁻²⁵ G6b-B-deficient mice also exhibit mild-to-moderate bleeding following injury, due to low and aberrant platelet function, and sex-related osteosclerosis with age.²⁶ A similar phenotype has been reported in patients lacking G6b-B,²⁵ and replacing mouse *Mpig6b* with the human orthologue rescues the phenotype,²⁵ establishing its vital role in MK/platelet homeostasis in both species. The question is, how do these findings support the hypothetical gatekeeper function of G6b-B? We propose that all of these anomalies stem from G6b-B-deficient MKs/platelets being hyperactive, supported by elevated surface expression of P-
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<figure><img src="image_26.png" /><figcaption>**Figure 4.** Loss of G6b-B expression and function severely disrupts megakaryocyte (MK)/platelet homeostasis in mice and humans. (A) Summary of overlapping phenotypes exhibited by mouse models and human patients harboring null or loss-of-function mutations in G6b-B. (B) Sixteen patients with disease-associated mutations in *MPIG6B* have been reported since 2016 (2×, two patients). Clinical features and therapies are listed. Immunoglobulin variable-like (IgV) domain and immunoreceptor tyrosine-based inhibition and switch motifs (ITI/SM) are indicated.</figcaption></figure>

selectin, TLT-1 and the sheddase a distintegrin and metallo-
proteinase domain-containing protein 10 (ADAM10).²³,²⁴
Consequently, MKs become trapped and release platelet-like
particles in sites of thrombopoiesis, rather than at the vessel
wall, as well as profibrotic factors resulting in myelofibrosis.
Hyperactive platelets are rapidly cleared from the circulation
in the spleen and liver, and negative feedback pathways are
upregulated to mitigate their hyperactivity, including
degranulation, receptor downregulation (GPVI, α2β1 and
GPIbα) and altered expression and phosphorylation of signal-
ing proteins (SFKs and C-terminal Src kinase, Csk) resulting in
exhausted platelets, rather than developmentally defective.
An alternative explanation is that these defects are caused
by defective Mpl signaling and MK maturation, suggesting a
nonconventional function of G6b-B as a positive regulator of
cytokine receptor signalling.¹⁹ However, this is inconsistent
with findings that HSPC-derived MKs from G6b-B-deficient
mice proliferate and differentiate in response to Tpo
in vitro,²³,²⁴ and the Tpo mimetic romiplostim in vivo.²⁷
Nonetheless, it remains unclear whether the marked block in
proplatelet formation of G6b-B-deficient MKs is an indirect

consequence of MK hyperactivation, or a direct consequence
of an aberrant aspect of Mpl signaling. With recent landmark
discoveries on MK-biased HSPCs and context-dependent meg-
akaryopoiesis and thrombopoiesis,²⁸⁻³² it is tempting to
speculate that G6b-B plays a role in MK/platelet decision-
making, however this remains to be determined.

Vascular Heparan Sulfates: Extrinsic Regulators of G6b-B

Having established the vital role of G6b-B in MK/platelet
homeostasis, we turned our attention to identifying its ligand
and proximal signaling mechanism. Using an affinity chroma-
tography and proteomics-based approach, we discovered that
G6b-B binds the HS side-chains of perlecan, and the anti-
coagulant heparin.³³ HS/heparin are structurally-related anionic
glycosaminoglycans (GAGs), consisting of variably sulfated
repeated disaccharide units, diverse linkages, lengths and dis-
tributions, broadly described as the heparanome of a cell, tis-
sue, organ or organism.³⁴ Perlecan is a large multidomain
protein with three HS chains linked to its amino-terminus that
<figure><img src="image_27.png" /></figure>

is synthesized by endothelial and smooth muscle cells and secreted into the vessel wall.³⁵,³⁶ It plays a vital role in vascular development, providing structural support through its interactions with ECM proteins and docking site for cytokines and growth factors.³⁷ In contrast, heparin is synthesized exclusively by mast cells and distributed within intervascular spaces in the liver, lungs and gastrointestinal tract.³⁸

Binding of HS/heparin to G6b-B was subsequently validated through biophysical, cellular and genetic-based approaches.³³ Intriguingly, recombinant G6b-B ectodomain did not bind to other distinct HSPGs, namely syndecan-2 on the luminal surface of endothelial cells, or agrin in the vessel wall, demonstrating its specificity for perlecan-associated HS. Intermediate-high affinity (µM-nM) and intermediate/slow on/off rates (10⁵/10⁻⁴ M⁻¹s⁻¹) of HS/heparin and perlecan to recombinant G6b-B ectodomain by surface plasmon resonance suggest rapid/slow association/dissociation, which has important implications of how MK/platelet G6b-B may interact with its ligands in the vasculature.³³ The degree of sulfation of HS/heparin is critical for interacting with G6b-B, loss of which reduces or eliminates binding, as well as the degree of polymerization (dp). We showed that a dp12 heparin oligomer mediates dimerization of G6b-B ectodomain in-solution, and structural analysis revealed an intermolecular cleft within which heparin fits with pseudo 2-fold symmetry. Ligand binding is facilitated by charge interactions.

Importantly, different species of HS/heparin and how they are presented to MKs/platelets elicit distinct biological responses. For example, MK/platelets adhere and spread less well on fibrinogen and collagen surfaces co-coated with perlecan compared with either substrate alone.³³ This inhibitor effect was lost when perlecan was treated with heparanase that degrades the HS side-chains, suggesting that G6b-B/HS interaction increases inhibitory G6b-B signaling. In contrast, platelets pretreated with HS/heparin increased collagen-mediated aggregating in-suspension compared with collagen alone, suggesting that G6b-B-HS/heparin interactions decrease inhibitory G6b-B signalling.³³ However, platelets pretreated with a semisynthetic heparin-proteoglycan that mimics multichain heparin secreted by mast cells, referred to as dual antiplatelet, anticoagulant (APAC) heparin had the opposite effect of single-chained HS/heparin, inhibiting collagen-mediated platelet aggregation in suspension.³³,³⁹ Perlecan which is not normally found in-solution had no effect on platelet aggregation under these conditions.

## Knowns and Unknowns of Ligand-Mediated G6b-B Signaling

A primary objective now is elucidating how HS/heparin regulate G6b-B signaling and MK/platelet decisions. This is complicated by the high degree of heterogeneity and promiscuity of HS/heparin, and complexities of proximal PTP-linked receptor signaling. While ligand-mediated dimerization of protein-tyrosine kinase (PTK)-linked receptors induces trans-phosphorylation of opposing cytoplasmic tails and initiates signaling, the converse can be the case with PTP-linked receptors. For example, the cytoplasmic tail of one ligand-bound PTP-linked receptor may obstruct or dephosphorylate

the cytoplasmic tail of the neighboring receptor, thus preventing signaling.⁴⁰,⁴¹ In which case, ligands that maintain PTP-linked receptors monomeric may facilitate signaling.⁴²

The best characterized and conventional function of G6b-B to date is as an inhibitor of ITAM-containing receptor signaling. Our working model is that binding of HS/heparin to the G6b-B ectodomain increases SFK-mediated ITI/SM phosphorylation and SH2-mediated docking and activation of Shp1 or Shp2 (Figure 5A). HS/heparin complexity and context, whether the HS/heparin is cross-linked with other platelet agonists in the vessel wall, on a cell surface or in solution is critical for mediating homo- or hetero-receptor clustering (Figure 5B). Ligand-mediated positioning of the G6b-B-Shp1/2 inhibitory complex within the molecular reach of ITAM-containing receptors is necessary for Shp1/2 to access and dephosphorylate key tyrosine residues in the ITAM proximal signaling complex and inhibit signaling.⁴³,⁴⁴ However, many unknowns remain, including: (a) ligand-dependent and -independent accessibility of the G6b-B ITI/SM to SFKs; (b) dephosphorylation of G6b-B phospho-ITI/SM by Shp1/2; and (c) Shp1 versus Shp2 binding and downstream substrates of both PTPs. Presumably the same mechanisms also apply to the regulation of non-ITAM-containing receptors, including integrins, cytokine and G protein-coupled receptors by G6b-B. However, this remains speculation for now, with much of the evidence coming from genetically modified mouse models, which is complicated by developmental and compensatory feedback mechanisms.

Changes in the expression and degradation of vascular HS in different pathologies is likely to play a role in how G6b-B is differentially regulating in health and disease. Relatedly, unmasking and degradation of cryptic tethered and soluble HS ligands by MK/platelet-derived heparanase may control G6b-B activity at sites of thrombopoiesis and thrombosis (Figure 5C).⁴⁵ Circumstantial evidence for this model comes from patient studies linking changes in the endothelial glycocalyx to cardiovascular disease and sepsis,⁴⁶,⁴⁷ and mouse models showing that overexpression of heparanase promotes megakaryopoiesis, platelet adhesion and thrombogenicity.⁴⁸,⁴⁹ A further level of regulation may involve different isoforms of G6b-B that bind the same ligands and regulate downstream signaling, but evidence is lacking. Besides early transcriptomic and proteomics studies predicting a transmembrane (G6b-A) and soluble (G6b-C, -D, -E) isoforms of G6b-B,¹⁶,¹⁸ only G6b-A has been confirmed to be expressed in human platelets by immunoblotting using an isoform-specific antibody.²⁴ G6b-A shares the same ectodomain, transmembrane domain and juxtamembrane sequence as G6b-B, suggesting it may bind the same ligands and co-cluster with G6b-B. However, it has a unique cytoplasmic tail containing several serine/threonine residues rather than tyrosine residues that may be reversibly phosphorylated and act as docking sites for signaling proteins. Further research is needed to establish the function role of G6b-A and putative soluble isoforms.

## G6b-B Is a MK/Platelet-Specific Regulator of Shp1 and Shp2

Another way of thinking of G6b-B is as a MK/platelet-specific Shp1 and Shp2 regulator. Shp1 and Shp2 are the only
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<figure><img src="image_29.png" /><figcaption>**Figure 5.** Ligand-mediated regulation of proximal G6b-B signaling. (A) Hypothetical modes of regulation of G6b-B by heparan sulfate (HS), heparin and dual antiplatelet, anticoagulant (APAC) heparin. HS/heparin mediate dimerization of G6b-B and phosphorylation of the immunoreceptor tyrosine-based inhibition and switch motifs (ITI/SM) by Src family kinases (SFKs), Src homology 2 (SH2) domain-mediated association and activation of the non-transmembrane protein-tyrosine phosphatases (PTPs) Shp1 or Shp2, and inhibition of platelet activation through incompletely defined mechanisms. The order in which the SH2 domains of Shp1 and Shp2 dock to phospho-ITI/SM are indicated ([1], [2]) and block dephosphorylation of phospho-ITI/SM. (B) Several factors likely contribute to the specificity and biological effects of HS/heparin on G6b-B and megakaryocytes (MKs)/platelets, including variable sulfation of the disaccharide units, linkages, length, complexity, cell and tissue distribution. (C) MK/platelet-derived heparanase may degrade vascular HS G6b-B ligands, or conversely liberate cryptic soluble ligands in the form of HS degradation products, the cellular response being the net effect of a heterogeneous cocktail of HS species on G6b-B signaling and compartmentalization in the plasma membrane.</figcaption></figure>

SH2 domain-containing PTPs, and have been implicated as both negative and positive regulators of signaling from a variety of receptors and cellular responses. The biochemical

and biological functions of Shp1 and Shp2 are dictated by phosphotyrosine-binding proteins, such as ITIM-containing receptors like G6b-B that regulate when and where Shp1
<figure><img src="image_30.png" /></figure>

and Shp2 activated and the substrates they can access. Deletion of Shp1 and Shp2 in the MK/platelet lineage results in distinct and overlapping developmental and functional anomalies in mice lacking either PTP that are significantly exacerbated in mice lacking both PTPs.⁵⁰ Shp1/2 double-deficient mice exhibit a much more severe MK/platelet phenotype than mice lacking G6b-B, including < 5% platelet count, with severe morphological, functional and biochemical platelet anomalies. MKs from these mice do not develop, proliferate or survive in vitro. Since the phenotypes of these mice are the net effect of multiple signaling defects from multiple receptors, developmental and feedback mechanisms, it is difficult to attribute specific functions to either Shp1 or Shp2 based on these mouse models alone. However, by using genetic- and pharmacological-based approaches, we showed that Shp2 is a positive regulator of Mpl signaling, whereas Shp1 is not.⁵⁰ Both PTPs have been implicated in regulating signaling from other cytokine and growth factor receptors in other lineages, which is likely the case in MKs/platelets as well. Based on our findings and those of others, we hypothesize that Shp1 is a positive regulator of RhoA-mediated cytoskeletal remodeling, and that both PTPs inhibit ITAM-containing receptor signaling in MKs/platelets. There is also a growing body of evidence that both PTPs regulate MK/platelet integrin-mediated signaling and functions,⁵⁰ but the molecular mechanisms remain undefined. Importantly, G6b-B regulates only a fraction of all Shp1 and Shp2 functions in MKs/platelets, hence null and loss-of-function mutations in G6b-B result in milder phenotypes than lineage-specific ablation of Shp1 and Shp2.

## The Receptor-Type PTP CD148: Threshold Regulator of SFKs

We postulate that the receptor-type PTP J (PTPRJ, CD148) is another important component of the MK/platelet checkpoint, acting as a threshold regulator of SFKs upstream of G6b-B. Without CD148, SFKs are not properly activated and formation of the G6b-B-Shp1/2 inhibitory complex is abrogated. The way in which CD148 does so is by dephosphorylate key modulatory tyrosine residues in the C-terminal tail and activation loop of SFKs, which increases and decreases their catalytic activity, respectively.⁵¹ Unlike leukocytes that rely primarily on the structurally-distinct receptor-type PTP C (PTPRC, CD45) to carry out this function, MKs/platelets depend mainly on CD148.⁵² CD148 is part of a robust regulatory mechanism that rapidly switches SFKs through different states of activation (Figure 6). Other parts of this system include Csk and Csk homologous kinase (Chk) that phosphorylate the SFK C-terminal tyrosine residue and inhibit SFK activity, and SFKs that auto-phosphorylate activation loop and C-terminal tyrosine residues. Targeted deletion of CD148, Csk and Chk in mice has profound consequences on MK/platelet homeostasis, with knock-on effects on sites of thrombopoiesis.⁵³,⁵⁴ Phenotypes were especially severe in Csk/Chk double-deficient mice and partially rescued when CD148 is also ablated,⁵³,⁵⁴ demonstrating the interplay between these kinases and phosphatase in optimizing MK/platelet count

and function. Intriguingly, deletion of either G6b-B or Csk/Chk, all of which are inhibitors of MK/platelet activation results in severe thrombocytopenia, reduced thrombosis and bleeding due to MK/platelet hyperactivation and exhausted platelets. Seemingly counterintuitive, these phenotypes can be explained by negative feedback mechanisms that are activated in the absence of G6b-B or Csk/Chk to counteract MK/platelet hyperactivity.

A unique feature of CD148 is its large ectodomain, providing a direct link between the extracellular milieu and SFK activity. Comprised of nine consecutive fibronectin type III (FNIII) repeats, it reportedly binds the HSPG syndecan-2 on the luminal surface of vascular endothelial cells,⁵⁵ and the adhesion protein thrombospondin-1, secreted by endothelial cells, fibroblasts and platelets into the vessel wall.⁵⁶ However, it remains unclear how binding to these ligands regulates CD148 PTP catalytic activity and compartmentalization in the plasma membrane. Like the G6b-B-Shp1/2 inhibitory complex, the CD148 PTP domain must be within the molecular reach of substrates, including receptors, signaling and cytoskeletal proteins in order to dephosphorylate and regulate their functions.⁵⁷ A ligand-independent mode of regulation of CD148 referred to as the kinetic segregation model has also been described, whereby CD148 is excluded from tight cell-cell junctions, such as the immunological synapse by its large, rigid ectodomain.⁵⁸ However, this model has been questioned, primarily on biophysical grounds, and ligand-mediated regulation likely plays an important role.⁵⁹ What is unequivocal is that CD148 is the most abundant and essential receptor-type PTP in mouse and human platelets, its expression is tightly regulated, and it is constitutively active throughout the plasma membrane of resting and activated platelets.⁵²⁻⁵⁴,⁶⁰

## Harnessing G6b-B and CD148 to Reset MK/Platelet Homeostasis

Having established G6b-B and CD148 as critical regulators of MK/platelet homeostasis, we propose how collectively these PTP-linked receptors, downstream effectors and associated vascular HSPG ligands regulate the number and reactivity of platelets in the circulation (Figure 7A). Concurrent with fundamental research to elucidate the function and regulation of this mechanism, novel immunotherapeutic-based approaches are being developed targeting the ectodomains of these two receptors either individually or together to reset MK/platelet homeostasis in disease and transfusion medicine. The primary strategy involves the use of antibody fragment antigen-binding regions (Fabs), synthetic glycan (SG) mimetics of G6b-B and CD148 ligands, as described for coagulation factors,⁶¹ and synthetic heterobifunctional molecules, in the form of bispecific single-chain variable fragments (bi-scFvs), termed *cis*-acting platelet receptor inhibitors (CAPRIs), and SG-minibody conjugates (SG-Mibs) as a means of either mimicking or blocking ligand function. The premise being that such synthetic designer compounds will allow tuning of inhibitory G6b-B signaling to either increase or decrease thrombopoiesis and thrombosis (Figure 7B). Ren et al., provide proof-of-concept of *cis*-acting
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>**Figure 6.** CD148 threshold regulator of Src family kinase (SFK) activity. CD148 is a critical dual activator and attenuator of SFK activity in megakaryocytes/platelets. Dephosphorylation of the C-terminal inhibitory phospho-tyrosine residue (p-Y) by CD148 prevents the intramolecular interaction with the Src homology 2 (SH2) domain, thus activating SFKs. Disruption of the SH3 domain with the linker region also activates SFKs. Conversely, CD148-mediated dephosphorylation of the activation loop p-Y attenuates SFK activity. C-terminal Src kinase (Csk) and structurally related Csk homology kinase (Chk) inhibit SFK activity by phosphorylating the C-terminal tyrosine residue. SFKs also autoregulate their activities by *trans*-phosphorylating both residues. Collectively, these protein-tyrosine kinases and phosphatase form a robust regulatory mechanism that rapidly switches SFKs between different states of activity, depending on the stimulus and cellular response that is needed.</figcaption></figure>

synthetic bi-scFvs that mediate co-clustering, dephosphorylation and inhibition of inhibitory signaling from ITIM-containing receptors by receptor-type PTPs.⁶²,⁶³ They demonstrate that PD-1-CD45 bi-scFvs, termed receptor inhibition by phosphatase recruitment (RIPR) inhibited inhibitory PD-1 signaling in T cells, thus augmenting the immune response to cancer cells, compared with conventional PD-1 ligand blocking immunotherapeutics.⁶² They subsequently applied the same strategy to other immune receptor targets, including the ITIM-containing macrophage signal-regulatory protein α (SIRPα) "don't eat me" signal (SIRPα-CD45 RIPR),⁶² and natural killer cell receptors NKG2A and Ly49 (NKG2A-CD45 and Ly49-CD45 RIPRs).⁶³

We recently developed prototype CAPRIs that mediate hetero-clustering and inhibition of the ITAM-containing collagen receptor complex GPVI-FcR γ-chain and the immunoglobulin receptor FcγRIIA (CD32A) by G6b-B.⁶⁴ Potential advantages of CAPRIs compared with conventional monovalent Fab competitive antagonists include: (a) inhibition of basal and ligand-dependent platelet activation; (b) versatility and improved avidity; (c) inhibition of non-lineage-specific receptors in platelets; and (d) development of antidotes to reverse pharmacological effects of CAPRIs, such as incorporating protease cleavage sites within linker regions to uncouple receptors.⁶⁴ Collectively, CAPRIs offer a more nuanced way of modulating MK/platelet function and the possibility of improved efficacy, specificity and reduced bleeding outcomes compared with current antiplatelet therapeutics.

## Future G6b-B and CD148 Immunotherapeutics

Depending on pathological context, inhibiting inhibitory G6b-B signal may be clinically relevant, notably in instances

of thrombocytopenia and hemorrhaging. Further, an unlimited supply of biologically active transfusable platelets would address issues with donor-derived platelets, including short shelf-life, loss of reactivity and contamination. Industrial scale platelet production is currently not feasible, primarily because the molecular mechanism of thrombopoiesis has yet to be elucidated and properly exploited, but of invaluable benefit once it is.

Through fundamental research of ligand-mediated G6b-B and CD148 proximal signaling, we envisage antibody-based approaches to regulate the number and reactivity of platelets produced by MKs in vivo and in vitro. Proof-of-concept of antibody-based approaches to regulate G6b-B- and CD148-mediated platelet and endothelial cell functions respectively has already been established, albeit mechanistically ambiguous.⁶⁵⁻⁶⁷ Heterobifunctional compounds that mediate heteroreceptor clustering provides an alternative strategy of preventing inhibitory G6b-B signaling, to relieve the MK/platelet checkpoint and reduce the threshold of MK/platelet activation. The mode of action would involve harnessing CD148 PTP catalytic activity to dephosphorylate the G6b-B ITI/SM, thus preventing Shp1 or Shp2 docking to G6b-B (Figure 7B). In their simplest forms, such heterobifunctional compounds would be comprised of covalently linked antibody variable regions (VRs) to either receptor, or as more complex compounds consisting of SG ligands of G6b-B covalently linked to Fabs or Mibs targeting the CD148 ectodomains through click chemistry.⁶⁸ Other important considerations include linker lengths, guided by biophysical parameters of G6b-B and CD148, and stoichiometry of receptor clustering. The rationale being to block G6b-B ligand binding and downstream signaling by positioning multiple G6b-B receptors within the molecular reach of a single CD148 PTP domain. SG-Mibs may also provide a means of increasing the half-life and
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

**Figure 7.** Ligand-mediated regulation of CD148 and G6b-B. (A) CD148 and G6b-B bind and are regulated by vascular heparan sulfate (HS) proteoglycan ligands, namely syndecan-2 and thrombospondin-1 in the case of CD148, and the HS side-chains of perlecan in the case of G6b-B. The biochemical, biophysical and spatiotemporal distribution of HSs vary enormously in health and disease. CD148 regulates Src family kinase (SFK) activity upstream of G6b-B and assembly of the G6b-B-Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases (PTP) 1 and 2 (Shp1, Shp2) inhibitory complex. Fibronectin type III (FNIII) domain, immunoreceptor tyrosine-based inhibition and switch motifs (ITI/SM). (B) Designer synthetic regulators of G6b-B and CD148. Innovative immune-based approaches are being developed that target and regulate CD148 and G6b-B signaling transduction, including antigen-binding fragments (Fabs) and synthetic glycans (SGs) ligand mimetics. Heterobifunctional molecules are also under development, in the form of bispecific single-chain variable fragments (bi-scFvs), termed *cis*-acting platelet receptor inhibitors (CAPRIs), and minibody-SG conjugates (Mib-SG) as a means of harnessing CD148 to modulate inhibitory G6b-B signaling, thrombopoiesis and platelet activation. Immunoglobulin-like domains 1 and 2 (D1, D2).
<figure><img src="image_36.png" /></figure>

compartmentalization of SG G6b-B ligands to the MK/platelet surface, or sites of thrombopoiesis and thrombosis to better regulate these processes. Although findings from G6b-B null and loss-of-function mouse models suggest that inhibiting inhibitory G6b-B signaling severely impacts thrombopoiesis and platelet function, congenital defects and associated feedback mechanisms do not necessarily align with pharmacological effects of inhibitors, greatly depending on the degree and duration of inhibition of G6b-B signaling.

## Summary

The overarching hypothesis of this review is that an intrinsic MK/platelet checkpoint prevents MKs from randomly forming proplatelets and releasing hyperactive platelets into sites of thrombopoiesis. We envisage that this checkpoint is released when MKs come into contact with constituents of sinusoidal blood vessels, inducing MK polarity and asymmetric thrombopoiesis. We postulate that the co-inhibitory receptor G6b-B is the primary intrinsic gatekeeper of this checkpoint and that HSs embedded in the vessel wall are extrinsic regulators that engage and modulate G6b-B signaling in MKs. Once in the circulation, preplatelet fragments of MKs autoregulate, dissociating into mature platelets, with mechanical forces playing a minimal role. G6b-B is also vital role at this stage, preventing platelet activation in the circulation, but the regulation is distinct to MKs in sites of thrombopoiesis, due to the differential biochemical composition and presentation of HSs on the vascular wall versus luminal surface of endothelial cells, as well as the different sizes and signaling networks in MKs and platelets. Low affinity kiss-and-go interactions between platelet G6b-B and HSs in the endothelial glycocalyx help maintain platelets inactive. A further mode of regulation may involve MK/platelet-derived heparanase at sites of thrombopoiesis and thrombosis, unmasking or degrading cryptic G6b-B ligands that regulate its function. The biochemistry, size, complexity and tissue context of HSs all factor into whether G6b-B is inhibited or activated, homo- or heteroclustered with other receptors. CD148 is an integral component of the MK/platelet checkpoint as a threshold regulator of SFK activity upstream of G6b-B. By understanding how G6b-B and CD148 are regulated by physiological vascular HSPGs, synthetic designer heterobifunctional regulators can be engineered that either block or mimic G6b-B and CD148 ligands and utilized as a means of regulating the number and reactivity of platelets in vitro and in vivo for the treatment of MK/platelet-based disorders and potentially transfusion medicine. Whether these hypotheses turn out to be fact or fiction remains to be determined. Our ultimate objective is to stimulate discourse and a shift in how we conceptualize these fundamental processes, highlighting putative regulators and novel approaches of harnessing them. The rest is up to our collective efforts.

## Acknowledgments

We thank Oreste Acuto for insightful comments and discussion. Figures were prepared by Patrick Lane, ScEYEnce Studios, US.

## Author Contributions

A.M. wrote and revised the manuscript. Y.A.S. conceptualized, wrote and revised the manuscript.

## Disclosure Statement

No potential conflict of interest was reported by the authors.

## Funding

Authors are funded by INSERM, the European Research Council (YAS, ERC-MENTOR-101141783) and the Agence Nationale pour la Recherche (AM, ANR-22-REMYS-CE14-0061-01 and YAS, ANR-23-TARGITT-CE18-0020-01).

## ORCID

Alexandra Mazharian http://orcid.org/0000-0002-0204-3325

Yotis A. Senis http://orcid.org/0000-0002-0947-9957

## Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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