Platelets Having a W127X Mutation in GPIX Express Sufficient Residual Amounts of the GPIbα Chain to Support Ristocetin-Mediated Agglutination and Adhesion to VWF and Collagen Under Conditions of Flow.

A W127X mutation in platelet glycoprotein (GP)IX is the most common genetic defect in Japanese Bernard-Soulier Syndrome (BSS). Patients having this mutation are often misdiagnosed as having immune thrombocytopenia (ITP) because of residual expression of GPIbα on the platelet surface. The purpose of the present investigation was to determine whether the residual amount of GPIbα present on the surface of GPIX^W127X BSS platelets might be sufficient to support adhesive functions of the GPIb complex. Flow cytometric analysis of platelets from our GPIX^W127X BSS patient revealed 7–11% of the normal level of GPIbα (3,700 receptors/platelet), although the expression of GPIbα estimated by western blot analysis was only 1% of normal. Expression levels of other receptors such as GPIIb, GPVI, and α2 integrin were 3–4 times higher per platelet than that of normal controls–probably due to the increased size of the BSS platelets. To characterize the ability of GPIX^W127X BSS platelets to bind VWF, we evaluated their agglutination in the presence of various concentrations of ristocetin, and found that ristocetin-induced agglutination was absent at 1.2 and 1.5 mg of ristocetin/ml, but present at 2.0 mg/ml. We then evaluated the ability of GPIX^W127X BSS platelets to adhere under conditions of flow to immobilized VWF, type 1 collagen, and type III collagen. Whereas platelets derived from a Type I BSS patient having a TGTG deletion within the GPIbα gene in which GPIbα is completely absent (Thrombosis and Haemost 77:1055,1997) were unable to adhere to immobilized VWF under conditions of high shear, GPIX^W127X BSS platelets bound well, demonstrating that the residual amount of GPIbα present GPIX^W127X BSS platelets is sufficient to mediate adhesion to VWF. Finally, recent studies (J Thromb Haemost 5:797,2007) have shown that platelet adhesion to Type III collagen can be mediated by either interaction of GPIbα with VWF, or by interaction of the integrin α2β1 with collagen under high shear flow. In support of this notion, adhesion of GPIbα^{TGTG del} BSS platelets to immobilized Type III collagen could be completely inhibited by addition of the anti-α2β1 blocking mAb, Gi9, while adhesion of GPIX^W127X BSS platelets to Type III collagen was only marginally affected. Taken together, we conclude that

1. platelets having a W127X mutation in GPIX express residual amounts of functional GPIbα, and

2. both GPIb-VWF and integrin α2β1-collagen interactions are physiologically important under conditions of high shear stress.

Residual expression of GPIbα in GPIXW127X platelets, therefore, likely accounts for the relatively mild bleeding phenotype in this variant form of BSS.