CLEC-2 contributes to hemostasis independently of classical hemITAM signaling in mice

Platelets play an essential role in both development and hemostasis, and C-type lectin-like receptor 2 (CLEC-2) is the major platelet activatory surface receptor known to contribute to both of these platelet functions.^1-3  Furthermore, CLEC-2 is the only known platelet receptor to signal through a hemi-immunoreceptor tyrosine–based activation motif (hemITAM), and its only known physiological ligand, the transmembrane protein podoplanin, is not expressed in the vasculature in healthy conditions and is not known to participate in primary hemostasis.^4,5  The function of CLEC-2 in vivo has been primarily investigated through the generation of knockout (KO) mouse models and mice in which the receptor has been depleted from circulating platelets by injection of the anti–CLEC-2 antibody, INU1. Both of these approaches lead to a complete loss of the receptor and therefore its signaling in platelets.^1-3,6-11  These models have revealed the importance of platelet-expressed CLEC-2 in the separation of the blood and lymphatic systems and indicated a potential role for the receptor in thrombus stability in hemostasis and thrombosis. However, due to their nature, these studies have been unable to investigate any hemITAM-independent functions of CLEC-2 or dissociate its role in development from its function in hemostasis.

Animals were studied with approval from the district government of Lower Franconia. CLEC-2 Y7A knockin mice were generated by a homologous recombination approach onto a C57BL\6 background. Fetal liver chimeric mice and Clec1b^fl/fl PF4-cre mice were generated as previously described.^3,12 

Platelet isolation, subsequent flow cytometry, and western blotting experiments were performed as described previously.^1,6  Antibodies used in western blotting were as follows: anti–CLEC-2 (clone INU2, generated in house)⁶ ; anti-Syk and anti-Lyn (clone C13F9) (Cell Signaling Technology); and anti-Fyn (clone FYN3) and anti-PLCγ2 from Santa Cruz Biotechnology.

Platelet aggregate formation on collagen under flow (1000 s⁻¹) was previously described.¹³  Where used, INU1 Fab fragments¹  (5 µg/mL) were incubated with blood samples for 5 minutes at 37°C before beginning the flow experiments.

Chemical injury of the mesenteric arterioles and mechanical injury of the aorta were as previously described.¹⁴  Where used, INU1 Fab′ fragments at 2 µg/g mouse were injected retro-orbitally 5 minutes before vessel injury.

The data presented are means ± standard deviations and, unless otherwise stated, were analyzed by unpaired Student t test. P values <.05 were considered statistically significant.

Constitutive Y7A CLEC-2 knockin (Y7A KI) mice were generated by insertion of the point mutation into the first exon of the Clec1b gene (supplemental Figure 1, available on the Blood Web site). Homozygous Y7A KI embryos showed signs of hemorrhage in the brain at E12.5 and by E14.5 edema and blood-lymphatic mixing in the developing skin lymphatics (Figure 1A), closely resembling the developmental phenotype of CLEC-2 KO mouse models.^2,3,8,10,11  Because the constitutive Y7A KI mice died shortly after birth (supplemental Tables 1 and 2; supplemental Figure 2), we generated fetal liver chimeric mice. Again resembling (chimeric) CLEC-2 KO mice,^2,3,8,11  chimeric Y7A KI mice developed progressive blood-lymphatic mixing, with blood filling the mesenteric lymphatics (Figure 1B; supplemental Figure 3). These data confirmed the critical role of CLEC-2 hemITAM signaling to prevent blood filling of the lymphatics during development and postirradiation in adult mice.

Importantly, CLEC-2 expression was maintained on the platelet surface at levels comparable with wild-type, as were all other prominent platelet surface receptors (Figure 1C; supplemental Table 3) and major kinases and effector proteins of the hemITAM phosphotyrosine signaling cascade (Figure 1D). CLEC-2 Y7A KI platelets displayed normal responses to classic agonists, but a complete block in response to the CLEC-2 agonists rhodocytin and the activating anti–CLEC-2 antibody, INU1 (Figure 1E-H; supplemental Figure 4). Furthermore, megakaryocyte numbers in the bone marrow (Figure 1H; supplemental Figure 5) and platelet count and volume (Figure 1I and data not shown) were normal in the initial stages posttransplantation in Y7A KI mice, which supports the hypothesis that CLEC-2 hemITAM signaling is not critical for normal platelet production. Interestingly, platelet counts in Y7A KI mice significantly decreased over time as the blood-lymphatic mixing defect progressed to cause bloody ascites in the mice (supplemental Figure 6), suggesting that the reduced platelet counts seen in CLEC-2 KO mouse models^3,7,15  are most likely due to the blood-lymphatic mixing and vascular integrity defects.¹⁶ 

Remarkably, Y7A KI platelets formed aggregates on collagen under flow with similar kinetics and to a similar extent as wild-type platelets (Figure 2A), which was in clear contrast to the thrombus stability defect previously seen with CLEC-2–deficient platelets.^1,2,7  To assess the contribution of the signal-dead receptor to thrombus stabilization, the assay was repeated in the presence of Fab′ fragments of the monoclonal antibody, INU1, which blocks the interaction of CLEC-2 and rhodocytin.¹  Remarkably, the blockade of the CLEC-2 ectodomain resulted in a 50% decrease in platelet surface coverage with few 3-dimensional aggregates formed by Y7A KI platelets, which is reminiscent of the defect seen in CLEC-2–deficient platelets in this assay.^1,2  This reduction in aggregate formation was not due to an effect of the Fab′ fragment on Y7A KI platelet activation, because responses to GPVI and GPCR agonists were maintained in the presence of the antibody (supplemental Figure 7).

Importantly, chimeric Y7A KI mice displayed bleeding times comparable with chimeric wild-type mice in the filter paper tail bleeding assay, whereas treatment of the mice with INU1 Fab′ led to defective hemostasis (Figure 2B). A similar result was also seen in the 2 following in vivo thrombosis models: mechanical injury of the abdominal aorta (Figure 2C) and chemical injury of mesenteric arterioles by topical application of ferric chloride (Figure 2D-E). In both models, platelet-specific conditional CLEC-2 KO mice⁷  were analyzed in parallel with chimeric Y7A KI mice to allow direct comparison. Control experiments with chimeric Y7A KI and CLEC-2 KO mice injected with INU1 Fab′ confirmed that INU1 Fab′ does not exert any detectable nonspecific effects on platelet count, CLEC-2 surface expression, or thrombosis (supplemental Figures 8 and 9). The defect in thrombus formation in both CLEC-2 KO mice and INU1 Fab′–treated chimeric Y7A KI mice was not due to impaired thrombosis initiation (Figure 2D), but a destabilization of formed thrombi that resulted in frequent embolization, thus preventing vessel occlusion within the observation period (supplemental Videos 1-3).

In this study, we have generated a novel CLEC-2 KI mouse model that maintained the surface expression of a signaling-dead CLEC-2 receptor. The close developmental phenocopy of this mouse to other constitutive CLEC-2 knockout models is not unexpected given the clear contribution of CLEC-2 signaling to the prevention of lymphatic blood-filling, as indicated by the Syk, SLP-76, and PLCγ2 knockout mice.^3,11,17-19  However, this new model has identified an as yet undescribed signaling-independent function of CLEC-2. This is the first evidence that CLEC-2 does not contribute to thrombus stability through a hemITAM signaling cascade. The antibody Fab′ fragment used to block mutant CLEC-2 in this study has been previously shown to block the ability of rhodocytin to activate platelets,¹  indicating that the respective domain of CLEC-2 may be important for the observed thrombus-stabilizing role of the receptor. Further studies will be required to identify the blood-borne ligand/counterreceptor of CLEC-2^1,2,7  through which it mediates its signaling-independent adhesive role in thrombosis and hemostasis.

The authors would like to thank Sebastian Dütting and Julia Volz for contributing their technical expertise to some of the work in this manuscript.

This work was supported by Deutsche Forschungsgemeinschaft grants SFB688 (B.N.) and Eb177/13-1 (J.A.E.) and research fellowship CH 1734/1-1 (D.C.), and by British Heart Foundation grant RG/13/18/30563 (S.P.W.).

Contribution: E.J.H. performed the experiments, analyzed the data, and wrote the manuscript; K.W., I.C.B., S.B., and D.S. performed the experiments and analyzed the data; J.A.E. contributed vital reagents and proofread the manuscript; D.C. and S.P.W. wrote the manuscript; and B.N. supervised the research, analyzed the data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for E.J.H. is Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

The current affiliation for D.C. is Immune Disease Institute, Program in Cellular and Molecular Medicine, Children’s Hospital Boston and Department of Pediatrics, Harvard Medical School, Boston, MA.

Correspondence: Bernhard Nieswandt, Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Building D15, Josef-Schneider-Str 2, 97080 Würzburg, Germany; e-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de.