Combined in vivo depletion of glycoprotein VI and C-type lectin-like receptor 2 severely compromises hemostasis and abrogates arterial thrombosis in mice

Abstract

Objective: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice.

Approach and results: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2.

Conclusions: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.

Figure 1

Analysis of mice deficient in Glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) on antibody injection. A, Mice were intravenously injected with 100 µg JAQ1 and 200 µg INU1 in sterile PBS, and platelet counts were determined on a FACSCalibur at the indicated time points post injection. Results are mean±SD in % of control animals (n=5 mice per group, representative for 2 individual experiments). B and D, Flow cytometric analysis of surface protein expression 5 days post injection with the indicated antibodies. Platelets were stained for 15 minutes at room temperature with the indicated fluorophore-labeled antibodies and directly analyzed. Platelet count in number of platelets/µL. Platelet size is given as mean forward scatter (FSC) and was determined by FSC characteristics. Results are mean fluorescence intensities (MFI)±SD (n=5, representative of at least 3 independent measurements). *P<0.05; **P<0.01; ***P<0.001. C and E, Flow cytometric analysis of integrin αIIbβ3 activation (JON/A-PE) and degranulation-dependent P-selectin exposure on platelets on day 5 post injection. Washed blood was incubated with the indicated agonists for 15 minutes and analyzed on a FACSCalibur. Results are mean±SD (n=5 mice per group, representative of 3 independent experiments). ***P<0.001. ADP: 10 µmol/L; U46619: 3 µmol/L; thrombin: 0.01 U/mL; rhodocytin (RC): 1 µg/mL; collagen-related peptide (CRP): 10 µg/mL; convulxin (CVX): 1 µg/mL. All experiments were performed on day 5 to 6 after antibody injection. FITC indicates fluorescein isothiocyanate.

Figure 2

Determination of hemostatic function and pathological thrombus formation in Glycoprotein VI (GPVI)/C-type lectin-like receptor 2 (CLEC-2)–depleted mice. A, A 1-mm segment of the tail tip was cut, and bleeding was determined to have ceased when no blood drop was observed on the filter paper. Each symbol represents 1 individual. B, A 1-mm segment of the tail tip was cut, and the tail tip was immersed in saline. Each symbol represents 1 individual. Differences of bleeding times between wild-type (WT), single GPVI-depleted, and single CLEC-2–depleted mice are nonsignificant. C, Mesenteric arterioles were treated with 20% FeCl₃, and adhesion and thrombus formation of fluorescently labeled platelets were monitored by in vivo fluorescence microscopy. Statistical evaluation of the time to appearance of a first thrombus (left) and percentage of maximal vessel stenosis (right) are depicted, n≥10. At most 2 arterioles of each mouse were analyzed. **P<0.01; ***P<0.001. Vessel stenosis was determined by measuring maximal thrombus size divided by vessel diameter using the Metamorph software (Visitron). Representative images are shown. White asterisk indicates occluded vessel. All experiments were performed on day 5 to 6 after antibody injection.

Figure 3

Analysis of Glycoprotein VI (Gp6^−/−)/C-type lectin-like receptor 2 (CLEC-2)–depleted mice. A, Flow cytometric analysis of surface protein expression of Gp6^−/− platelets 5 days post injection with the anti–CLEC-2 antibody INU1. Platelets were stained for 15 minutes at room temperature with the indicated fluorophore-labeled antibodies and directly analyzed. Platelet count in number of platelets/µL. Platelet size is given as mean forward scatter (FSC) and was determined by FSC characteristics. Results are mean fluorescence intensities (MFI)±SD (n=5, representative of at least 3 independent measurements). **P<0.01; ***P<0.001. B, Flow cytometric analysis of degranulation-dependent P-selectin exposure and integrin αIIbβ3 activation on platelets. Washed blood was incubated with the indicated agonists for 15 minutes at room temperature and analyzed on a FACSCalibur. Results are mean±SD (n=5 mice per group, representative of 3 individual experiments). ***P<0.001. ADP: 10 µmol/L; U46619: 3 µmol/L; thrombin (thr): 0.01 U/mL; rhodocytin (RC): 1 µg/mL; collagen-related peptide (CRP): 10 µg/mL; convulxin (CVX): 1 µg/mL. C, A 1-mm segment of the tail tip was cut, and bleeding was determined to have ceased when no blood drop was observed on the filter paper. Each symbol represents 1 individual. Differences of bleeding times among control, Gp6^−/− mice, and CLEC-2–-depleted mice are nonsignificant. D, An 1-mm segment of the tail tip was cut, and the tail tip was immersed in saline. Each symbol represents 1 individual. All experiments were performed on day 5 to 6 after antibody injection. Bleeding time of Gp6^−/−/CLEC-2–depleted mice is significantly prolonged compared with control and Gp6^−/− mice ***P<0.001, and to CLEC-2–depleted mice **P<0.01. Bleeding time of CLEC-2–depleted mice is prolonged compared with control **P<0.01. WT indicates wild-type mice.

Figure 4

Analysis of megakaryocyte/platelet-specific C-type lectin-like receptor 2 (CLEC-2)–deficient mice. A, Representative images of the intestine are shown. L indicates lymphatic vessel; A, arteriole; and V, vein. B, Platelet count was determined by flow cytometric analysis. ***P<0.001. C, Flow cytometric analysis of surface protein expression. Platelets were stained for 15 minutes at room temperature with the indicated fluorophore-labeled antibodies and directly analyzed. Platelet size is given as mean forward scatter (FSC) and was determined by FSC characteristics. Results are mean fluorescence intensities (MFI)±SD (n=4, representative of at least 3 independent measurements). ***P<0.001. D, Flow cytometric analysis of degranulation-dependent P-selectin exposure and integrin αIIbβ3 activation on platelets. Washed blood was incubated with the indicated agonists for 15 minutes at room temperature and analyzed on a FACSCalibur. Results are mean±SD (n=5 mice per group, representative of 3 individual experiments). *P<0.05; ***P<0.001. ADP [µmol/L]; U46619: [µmol/L); thrombin (thr): [U/mL]; rhodocytin (RC): [µg/mL]; collagen-related peptide (CRP): [µg/mL]; convulxin (CVX): [µg/mL]. E, Mesenteric arterioles were treated with 20% FeCl₃ and adhesion, and thrombus formation of fluorescently labeled platelets were monitored by in vivo fluorescence microscopy. Evaluation of the time to appearance of a first thrombus (left) and time to vessel occlusion (right) are depicted. n≥10. Time of first appearance of thrombi is nonsignificant. Time to occlusion is *P<0.05. GPVI indicates Glycoprotein VI.

Figure 5

Analysis of Glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) double-mutant mice. A, Flow cytometric analysis of surface protein expression of Gp6^−/−/Clec-2^{fl/fl, Pf4-Cre} platelets. Platelets were stained for 15 minutes at room temperature with the indicated fluorophore-labeled antibodies and directly analyzed. Platelet count in number of platelets/µL. Platelet size is given as mean forward scatter (FSC) and was determined by FSC characteristics. Results are mean fluorescence intensities (MFI)±SD (n=5, representative of at least 3 independent measurements). *P<0.05; **P<0.01; ***P<0.001. B, Flow cytometric analysis of degranulation-dependent P-selectin exposure and integrin αIIbβ3 activation on platelets. Washed blood was incubated with the indicated agonists for 15 minutes at room temperature and analyzed on a FACSCalibur. Results are mean±SD (n=4 mice per group, representative of 3 individual experiments). **P<0.01; ***P<0.001. ADP [µmol/L]; U46619: [µmol/L]; thrombin (thr): [U/mL]; rhodocytin (RC): [µg/mL]; collagen-related peptide (CRP): [µg/mL]; convulxin (CVX): [µg/mL]. C, A 1-mm segment of the tail tip was cut, and bleeding was determined to have ceased when no blood drop was observed on the filter paper. Each symbol represents 1 individual. Fisher test: Gp6^−/−/Clec-2^{fl/fl, Pf4-Cre} vs control: 0.0351; Gp6^−/−/Clec-2^{fl/fl, Pf4-Cre} vs Clec-2^{fl/fl, Pf4-Cre}: 0.0087. Other conditions nonsignificant. D, An 1-mm segment of the tail tip was cut, and the tail tip was immersed in saline. Each symbol represents 1 individual. Bleeding time of Gp6^−/−/Clec-2^{fl/fl, Pf4-Cre} mice is prolonged compared with control and Clec-2^{fl/fl, Pf4-Cre*}, P<0.05.