Of Mice and Man: The Unwinding of CLEC-2 as an Antithrombotic Target?

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

The COVID-19 (coronavirus disease 2019) pandemic has shone a bright light on the role of thromboinflammation in human disease.[1] [2] Venous thromboembolism, ischemia reperfusion injury (e.g., cardiac), sickle cell disease, and sepsis are other examples of diseases in which thromboinflammation is a central driver of pathology.[3] [4] Indeed, thrombus formation intersects with many aspects of the innate immune response, by inducing leukocyte recruitment, activation, neutrophil extracellular trap formation, and complement activation.[3] [5] The corollary is that these aspects of the innate immune system amplify the thrombotic response by activating platelets, upregulating tissue factor expression, and ultimately fibrin formation. Platelets sit at the crossroads of immunity, inflammation, and thrombosis, and as such, are now considered essential components of the innate immune system.[6] Accordingly, platelets express immune-like receptors such as glycoprotein (GP) VI, FcγRIIa, and C-type lectin-like receptor 2 (CLEC-2).[7] [8] More recently, CLEC-2 has been postulated to be an important regulator of the platelet response in thromboinflammation. Consequently, CLEC-2 has been proposed as a potentially novel therapeutic target, given the ongoing unmet clinical need for therapies to treat thromboinflammation.[7]

CLEC-2 is a platelet immune receptor initially described as playing an important role in regulating integrin αIIbβ3 (GPIIb/IIIa; CD41/CD61)-independent hemostasis and thromboinflammation[9] which is best exemplified by its role in upregulating VE cadherin expression in high endothelial venules, and providing hemostasis at lymphovascular valves, thus ensuring the vascular integrity of the lymphatic system and the prevention of blood–lymphatic mixing during development.[10] [11] Additionally, CLEC-2 has also been proposed to play an important role in regulating cerebrovascular development in mice by facilitating platelet adhesion, thereby preventing hemorrhage and nurturing the development of the nascent vasculature.[12] However, recent data have challenged these observations. Indeed, depending on the model of inflammatory hemostasis, platelet GPVI appears to be the predominant platelet receptor in mediating hemostasis in the face of vascular inflammation, with CLEC-2 playing a secondary role only in the absence of GPVI. [13] Moreover, with the recent development of more specific conditional mouse models generated by GPIbα-driven Cre expression to delete CLEC-2 in platelets, it appears that platelet CLEC-2 may not be important to separate the blood and lymphatic systems post-development in the absence of any inflammatory challenge.[14]

More recently, the role of platelet CLEC-2 in regulating thromboinflammation has come into sharp focus given that the upregulation of podoplanin expression, the major endogenous CLEC-2 ligand, on stromal cells and tissue-resident macrophages may drive CLEC-2-dependent platelet activation. In accordance with this concept, CLEC-2 has been demonstrated to regulate the development of hepatic intravascular thrombosis in the context of infection in a process linked to the upregulation of podoplanin expression in hepatic Kupffer cells and monocytes in mice.[15] Moreover, CLEC-2 deficiency protects mice from developing deep venous thrombosis, a prototypical thromboinflammatory disorder.[16] However, the role of CLEC-2 in thromboinflammation is complex, since CLEC-2 has also been demonstrated to have a protective role in other models of inflammation. Indeed, platelet CLEC-2 appears protective in mouse models of sepsis and experimental autoimmune encephalitis.[17] Thus, the role of CLEC-2 in mediating thromboinflammation is largely contingent upon the stimulus and disease model.

Intriguingly, genetic deletion or antibody depletion of CLEC-2 platelets has been demonstrated to afford mice protection from thrombus formation across a range of in vivo thrombosis models including the ferric chloride, laser injury, and mechanical perturbation of the endothelium.[18] These data have raised the prospect that CLEC-2 has ligands other than podoplanin to account for the apparent role of CLEC-2 in arterial thrombus formation, since podoplanin is not expressed by the normal vasculature. In this regard, several exogenous ligands of CLEC-2 have been proposed, including the snake venom toxin rhodocytin.[19] However, the endogenous ligand for CLEC-2 to account for its role in thrombus formation remains to be delineated. Moreover, the existence of potentially relevant nonprotein CLEC-2 ligands has been raised by the identification of katacine, a mixture of proanthocyanidin polymers, which can induce platelet aggregation and CLEC-2 phosphorylation.[20] Interestingly, previous data have demonstrated that mice expressing a CLEC-2 signaling-deficient mutant only display attenuated thrombus formation in the presence of a CLEC-2 blocking antibody, thus suggesting that CLEC-2 may support thrombus formation in a hemITAM-independent process.[21] Accordingly, recent data have demonstrated that CLEC-2 interacts with GPIbα thereby regulating the localization of GPIbα in lipid rafts as well as GPIb-mediated signaling.[22] Curiously, the expression of CLEC-2 on platelets differs markedly between mouse and human platelets. Indeed, while mouse platelets express approximately 40,000 copies of the CLEC-2 receptor, human platelets have been shown to express only 2,000 to 4,000 copies.[23] This is significant given the possibility that CLEC-2 may mediate GPIbα localization and signaling.

The manuscript by Bourne and colleagues in the current edition of Thrombosis and Haemostasis provides new insights into the differential role of CLEC-2 in mouse and human platelets.[24] Utilizing a genetic model of platelet-specific CLEC-2 deletion, in conjunction with in vivo and in vitro models of thrombosis, the authors could elegantly demonstrate that while CLEC-2 plays an important role in mediating thrombus growth in mice, in contrast, CLEC-2 appears to be dispensable for platelet thrombus formation under arterial shear in human platelets. Given previous data have highlighted the potential of CLEC-2 in regulating GPIbα-mediated integrin αIIbβ3 function, as opposed to triggering robust intracellular signaling, it perhaps stands to reason that this relatively low expression of CLEC-2 on human platelets underlies the reason why CLEC-2 does not appear to be important for platelet thrombus formation under arterial shear. The corollary is that this may be an important part of the CLEC-2 puzzle and it is tempting to speculate that this low level of CLEC-2 expression may provide reassurance that CLEC-2-targeting therapies may not cause bleeding and thus would make CLEC-2 a potentially attractive therapeutic target for the treatment of thromboinflammation. However, this needs to be tempered by the fact that clinically, there has been no description of CLEC-2 deficiency in humans to date to reassure that CLEC-2 is truly dispensable for hemostasis. Moreover, the preclinical bleeding models of hemostasis utilized in these studies have important limitations, which limit their translation to the clinic.[25] This is particularly relevant for platelet receptors that are differentially expressed on mouse and human platelets, as was demonstrated with the development of the PAR-1 antagonists, which ultimately translated to the clinic but whose use has been significantly limited by the associated increased bleeding risk.[26] [27]

Moreover, it is important to note the variable effects of CLEC-2 inhibition or deficiency in different models of thromboinflammation, coupled with the fact that CLEC-2 expression is not restricted to platelets makes the clinical translation of CLEC-2-targeted therapies a potentially unresolvable challenge. Additionally, Bourne et al report that CLEC-2 does not mediate human platelet or leukocyte adhesion on activated human endothelial cells, thus further dampening the enthusiasm for therapeutic CLEC-2 targeting in human venous thromboembolism. Lastly, there remains an ongoing need to identify the ligand(s) that regulate the role of CLEC-2 in thrombus formation in mice, which will shed further light on the apparent important species differences of platelet CLEC-2 function observed between mouse and human platelets. Therefore, although the potential for CLEC-2-targeted therapies in humans now appears to be elusive, this should not curb further investigation to understand the full scope and mechanisms of CLEC-2's role in thromboinflammation.

Overall, the manuscript by Bourne et al raises important questions regarding the biological role of CLEC-2 in mice and humans, especially when viewed through the prism of our ongoing quest for novel treatments for thromboinflammation.[24] Importantly, these studies emphasize the critical importance of trying to recapitulate phenotypes observed in mouse models of thrombosis with correlative human studies. Indeed, as demonstrated in the current study, given the ease of availability of human platelets for experimental purposes, and the expanding array of in vitro platelet function assays and microfluidic devices, thrombosis research has a particular advantage that allows the testing of potentially novel inhibitors identified in mouse studies[28] in the setting of human thrombus formation. Thus, studies such as those presented by Bourne et al[24] that define important species differences in mouse and human platelets are essential in the quest to identify potentially novel therapeutic targets and develop novel antithrombotic drugs that may (or may not) translate to clinical applications.

^* Equally contributing senior authors.

Publikationsverlauf

Eingereicht: 21. August 2022

Angenommen: 05. September 2022

Accepted Manuscript online:
07. September 2022

Artikel online veröffentlicht:
16. November 2022

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