Interaction between Platelet Glycoprotein Ibα and Filamin-1 Is Essential for Glycoprotein Ib/IX Receptor Anchorage at High Shear

Abstract

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIb␣ and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIb␣ intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569 -579 of the GPIb␣ cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant ⌬557-568 and ⌬569 -579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIb␣ for interaction with filamin-1.

Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

“…However, there are two major differences between Williamson's and our studies (and the results of Crammer et al using human VWF (44)). Williamson et al (45) examined the interaction of GPIb-IX-expressing cells with bovine VWF, which is similar to the result of Cranmer et al using bovine VWF (44). In contrast, we (38) and Cranmer et al (44) showed that wild type GPIb-IX-expressing cells adhere poorly to immobilized human VWF under high shear rate (and static) conditions.…”

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…FlnA binding facilitates GPIb surface expression in Chinese hamster ovary (CHO) cells (Feng et al, 2005), and the interaction between FlnA and GPIb has been reported to influence VWF receptor function, although conflicting effects are found in the literature. CHO cells transfected with GPIb mutants that lack the FlnA binding site have decreased VWF binding (Dong et al, 1997;Schade et al, 2003), VWF-induced cell aggregation (Mistry et al, 2000), and/or adhesion to a VWF matrix under high shear (Cranmer et al, 1999;Williamson et al, 2002;Cranmer et al, 2005). In contrast, another study has shown that FlnA binding to GPIb negatively regulates VWF binding to CHO cells and CHO cell adhesion under both static and flow conditions (Englund et al, 2001).…”

“…Whether FlnA influences the ligand binding properties of GPIb has been controversial in the literature. Some groups have reported that it enhances (Dong et al, 1997;Cranmer et al, 1999Cranmer et al, , 2005Mistry et al, 2000;Williamson et al, 2002;Schade et al, 2003) and others that it diminishes (Englund et al, 2001). Our studies directly confirm the former, pointing out the protein tyrosine kinase Syk.…”

The structure of the GPIb–filamin A complex

“…11 The GPIb␣-FLNa interaction is essential for the platelet adhesion to VWF that binds extracellular domain of the GPIb-V-IX receptor, for maintaining normal platelet integrity and shape, and for normal signal transduction reactions involved in platelet activation. 12,13 The absence of, or mutations in, VWF or in the GPIb-V-IX receptor are responsible for von Willebrand disease 14 or Bernard-Soulier syndrome, 15 respectively, disorders characterized by either bleeding or increased thrombosis. Additional roles of the GPIb-V-IX receptor in vascular biology have been recognized due to the identification of novel adhesive ligands for the receptor such as P-selectin, ␣M␤2 integrin, the coagulation factors thrombin and factors XI and XII, and the major extracellular matrix protein, collagen.…”