Cadherin 6 has a functional role in platelet aggregation and thrombus formation

Abstract

Objective: Thrombosis occurs at sites of vascular injury when platelets adhere to subendothelial matrix proteins and to each other. Platelets express many surface receptor proteins, the function of several of these remains poorly characterized. Cadherin 6 is expressed on the platelet surface and contains an arginine-glycine-aspartic acid motif, suggesting that it might have a supportive role in thrombus formation. The aim of this study was to characterize the role of cadherin 6 in platelet function.

Methods and results: Platelet aggregation was inhibited by both antibodies and exogenous soluble cadherin 6. Platelet adhesion to immobilized cadherin 6 was inhibited by arginine-glycine-aspartic acid-serine tetrapeptides. Antibodies to α(IIb)β(3) inhibited platelet adhesion to cadherin 6. Because platelet aggregation occurs in fibrinogen and von Willebrand factor double-deficient mice, we investigated whether cadherin 6 is an alternative ligand for the integrin α(IIb)β(3). Platelet aggregation in fibrinogen and von Willebrand factor double-deficient mice was significantly inhibited by an antibody to cadherin 6. In flow-based assays, inhibition of cadherin 6 caused a marked reduction in thrombus formation in both human and mouse blood.

Conclusions: This study demonstrates the role of cadherin 6 as a novel ligand for α(IIb)β(3) and highlights its function in thrombus formation.

Figure 1

Cadherin 6 is present on platelets. A, Total platelet lysate from 3 different donors was analyzed by Western blot for the presence of cadherin 6 using a sheep polyclonal antibody against cadherin 6. Lane 1, Cdh6_IgG; lane 2, sheep IgG; lane 3, human IgG; lanes 4–6, total platelet lysate. Cdh6_IgG is composed of the extracellular portion of cadherin 6 fused to the Fc domain of human IgG and therefore has a higher molecular weight than the native protein. B, Platelets were incubated with a polyclonal antibody and analyzed by flow cytometry to confirm the presence of cadherin 6 on the platelet surface. Cdh6_IgG indicates cadherin 6_IgG fusion protein.

Figure 2

Platelet aggregation is inhibited by an antibody against cadherin 6 and an excess of cadherin 6 protein. A, A polyclonal antibody against cadherin 6 significantly inhibits thrombin receptor activating peptide (TRAP)-induced platelet aggregation in gel-filtered platelets (P<0.0001) (n=15). B, 10 µg/mL Cdh6_IgG fusion protein inhibits TRAP-induced aggregation in gel-filtered platelets (P<0.0001). The P-Cdh_IgG control fusion protein had no effect (n=5). C, 3×10⁸ platelets per mL from Fg/VWF−/− platelet-rich protein were stimulated by either 100 or 250 mmol/L TRAP4. Coincubation of anti-cadherin 6 resulted in decreased platelet aggregation in response to 100 mmol/L TRAP4. Fg/VWF−/− indicates fibrinogen and von Willebrand factor double-deficient mice; Cdh6_IgG, cadherin 6_IgG fusion protein; and P-Cdh_IgG, placental cadherin_IgG recombinant fusion protein.

Figure 3

Platelet adhesion to immobilized cadherin 6 is αII_bβ₃ dependent. A, Platelets adhere to 1 µg/mL cadherin 6, 20 µg/mL fibrinogen, and 1 µg/ml VE-Cdh. Adhesion to 1 µg/mL P-Cdh or 10 µg/mL BSA is greatly reduced in comparison with Cdh6_IgG. B, RGDS peptides inhibit platelet adhesion to immobilized cadherin 6 (P<0.005), n=3. C, Antibodies to integrin αII_bβ₃ inhibit platelet adhesion to cadherin 6 (P<0.005), n=7. D, Cdh6_IgG inhibits PAC1 binding to αII_bβ₃. VE-Cdh indicates vascular endothelial cadherin; P-Cdh, placental cadherin; Cdh6_IgG, cadherin 6_IgG fusion protein.

Figure 4

Platelets adhere to the RGD motif presented as part of an extended cadherin 6 (Cdh6) sequence. Platelets from healthy human donors (A) and wild-type mice (B) adhered to glass slides coated with a peptide corresponding to Cdh6 amino acids 73–95, containing the Cdh6 RGD motif. Mutating the RGD to RGE abrogated platelet adhesion. RGD indicates arginine-glycine-aspartic acid.

Figure 5

Cadherin 6 contributes to platelet-rich thrombus formation at moderate shear. Whole blood from healthy human donors or wild-type mice treated with an anti-cadherin 6 antibody or Cdh6_IgG and perfused at 600 per inverse second over collagen. Cadherin 6 treatment caused a significant reduction in thrombus size, thrombus volume, and surface area covered by thrombi as assessed by confocal microscopy (P<0.0001). Cdh6_IgG indicates cadherin 6_IgG fusion protein.