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

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1b fl/fl GPIbα-Cre + mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl 3 -induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1b fl/fl GPIbα-Cre + mice, revealed that the reduced thrombus size post-FeCl 3 -injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1b fl/fl GPIbα-Cre + mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1b fl/fl GPIbα-Cre + mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear.


Recombinant Protein
Human recombinant CLEC-2-Fc was generated in-house with a sequence:
All methodology for Supplementary Data is included in the main manuscript.
Thrombosis and Haemostasis © 2022. The Author(s). The statistical significance between two groups was analyzed using a paired t-test. **p < 0.01.
Supplementary Fig. S4 Transfused washed platelets contribute to thrombus formation in murine whole blood when perfused over collagen at arterial shear. Washed mouse platelets were donated to whole mouse blood, before perfusion through a Horm collagencoated (100 µg/mL) µ-Slide VI 0.1 flow chamber at 1,000 s −1 . The washed platelets (magenta) were incubated with a GPIbβ-AF647 conjugated antibody, before addition to whole blood containing DiOC6 (green). Arrow indicates direction of flow. Image is representative of three mice.  (C) Whole blood from healthy volunteer donors was perfused over Horm collagen-coated (100 µg/mL) µ-Slide VI 0.1 flow chamber at 1,000 s −1 , in the presence or absence of F (ab) (10 µg/mL) and F(ab) 2 (10 µg/mL) for minutes prior to perfusion. (Ci) Thrombus surface coverage over 4 minutes, measured using DiOC6 fluorescence (2 µM), (Cii) subsequent area under the curve (AUC) was calculated (a.u. = arbitrary units), and (Ciii) thrombus formation after 4 minutes. (Civ) Representative images shown after 4 minutes (n = 8 donors; arrow indicates direction of flow). The statistical significance between two groups was analyzed using an unpaired t-test and the statistical significance between multiple groups was analyzed using one-way ANOVA with Tukey's multiple comparisons test. ****p < 0.0001.
Supplementary Fig. S6 The effect of inhibition of CLEC-2 inhibition by AYP1 fragments on platelet activation on collagen under flow conditions. Whole blood from healthy volunteer donors was perfused through a Maastricht flow chamber at 1,000 s −1 in the presence or absence of F(ab) (10 µg/mL) 10 minutes prior to perfusion. The flow chamber was coated with Horm collagen (100 µg/mL). Representative images of three donors shown after 3.5 minutes of (A) phosphatidylserine (PS) exposure, (B) P-selectin expression (CD62P), and (C) fibrinogen coverage. Quantification was assessed with a semi-automated ImageJ script from these images, shown in Fig. 3Div-vi.
Supplementary Fig. S7 Recombinant human CLEC-2-Fc does not alter thrombus formation in human blood perfused over Horm collagen. Whole blood from healthy volunteer donors was perfused through a Maastricht flow chamber at 1,000 s −1 , in the presence or absence of hFc-CLEC-2 (10 µg/mL) for 10 minutes prior to perfusion. (Ai) Thrombus surface coverage and (Aii) representative images shown after 4 minutes (n = 3 donors; arrow indicates direction of flow). The statistical significance between two groups was analyzed using an unpaired t-test.