GPIbα-selective activation of platelets induces platelet signaling events comparable to GPVI activation events

Abstract

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbα (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbα-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbα- and α_IIbβ₃-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbα and anti-α_IIbβ₃ antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbα-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbα engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbα can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

• Glycoprotein Ibα
• glycoprotein VI
• platelets
• platelet activation
• GPIb-IX-V
• VWF

Acknowledgments

We thank Cheryl Berndt and Jing Jing for excellent assistance. This work was supported by the National Health and Medical Research Council of Australia and Monash University.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.