Murine cadherin-6 mediates thrombosis in vivo in a platelet-independent manner

Abstract

Background: Platelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin-6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation.

Objectives: We aimed to further characterize the importance of cadherin-6 in thrombosis in vivo.

Methods: Cadherin-6 platelet expression was evaluated by western blotting, flow cytometry, and immunoprecipitation. Thrombosis was evaluated using the FeCl₃ and Rose Bengal carotid artery models in C57Bl6 mice treated with anti-cadherin-6 or IgG and wild-type or Cdh6^-/- mice. Platelet function was compared in wild-type and Cdh6^-/- mice using tail-clip assays, aggregometry, and flow cytometry.

Results: Human platelet expression of cadherin-6 was confirmed at ~3000 copies per platelet. Cdh6^-/- mice or those treated with anti-cadherin-6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin-6 was not expressed on mouse platelets, and there were no differences in tail bleeding times, platelet aggregation, or platelet activation in wild-type versus Cdh6^-/- mice.

Conclusions: Cadherin-6 plays an essential role in thrombosis in vivo. However, cadherin-6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin-6/catenin complex. The essential, platelet-independent role for cadherin-6 in hemostasis may allow it to be an effective and safe therapeutic target.

Keywords: arterial thrombosis; cadherin‐6; mouse model; platelet adhesion.

FIGURE 1

Human platelets express cadherin‐6. (A) Human platelet lysate was probed with mouse monoclonal anti–cadherin‐6; CHO cells transfected with human cadherin‐6 (Cdh6) was used as a control. A standard curve was generated using recombinant human cadherin‐6 Fc chimera protein. The blot shown is representative of n = 2. (B) Human platelet lysate was immunoprecipitated with anti‐cadherin‐6 antibody. Cadherin 6, α‐catenin, and β‐catenin were detected by western blot. (C) Quantitative flow cytometry was performed using an APC‐conjugated anti‐cadherin‐6 antibody on nonstimulated platelets or those activated with PAR1 (SFLLRN, 25 μmol/L) or PAR4 (AYPGKF, 250 μmol/L) stimulating peptides, ADP (5 μmol/L), or convulxin (5 nmol/L). CHO, Chinese hamster ovary; PAR, protease‐activated receptor; PLTs, platelets

FIGURE 2

Blocking cadherin‐6 disrupts thrombosis. C57BL/6J mice were treated with sheep polyclonal anti–cadherin‐6 (Cdh6, 0.8 mg/kg) 15 minutes before initiating thrombosis with FeCl₃ or Rose Bengal. (A) Intravital microscope images obtained following FeCl₃ treatment. In IgG‐treated mice at 8 minutes, the proximal end of the thrombus was monitored to accurately detect full occlusion. Time to stable vessel occlusion was observed and represented as Kaplan‐Meier curves for (B) FeCl₃ (n = 3 per group) and (C) Rose Bengal models (n = 5 for no treatment, n = 4 for IgG or Cdh6). The curves were compared using the Mantel‐Cox log‐rank test

FIGURE 3

Thrombosis is delayed in CDH6 knockout mice. Thrombosis was initiated in Cdh6 ^−/− and wild‐type mice using the (A) FeCl₃ (n = 9 per group) or (B) Rose Bengal (n = 5 for wild‐type and n = 6 for Cdh6^−/−) models and time to vessel occlusion was observed. The vessels were monitored until a full occlusion was formed or the experiment ended and represented as Kaplan‐Meier curves. The curves were compared using the Mantel‐Cox log‐rank test. (C) Platelet count was measured in whole blood from wild‐type and Cdh6 ^−/− mice and compared using Student's t test. (D) Cdh6 ^−/− and wild‐type mice were anesthetized before clipping 3 mm of tail to measure time to bleeding cessation; significance was evaluated with Student's t test, and bars represent mean ± SEM

FIGURE 4

Cadherin 6 is absent on murine platelets. (A) Platelet lysates from Cdh6−/− and wild‐type mice were probed with mouse monoclonal anti–cadherin‐6. CHO cells transfected with mouse cadherin‐6 (Cdh6) was used as a control. A standard curve was generated using recombinant mouse cadherin‐6 protein. (B) Surface expression of cadherin‐6 was measured on platelets from wild‐type or Cdh6−/− mice via flow cytometry with an APC‐conjugated cadherin‐6 antibody. (C) Representative (n = 3) aggregometry tracings of Cdh6−/− and wild‐type platelets stimulated with AYPGKF (150 μmol/L) and convulxin (0.5 nmol/L). CHO, Chinese hamster ovary; PAR, protease‐activated receptor