Abstract
von Willebrand factor (VWF) binding to platelet glycoprotein (Gp) Ib-IX-V triggers platelet activation. Under conditions of pathologically elevated arterial wall shear stress, VWF-dependent platelet adhesion is coupled to aggregation and thrombus formation principally through GpIbα-induced signaling to α IIbβ 3. To elucidate the mechanism of GpIbα signaling to α IIbβ 3, we have examined molecular interactions involving structural proteins that bind to the cytoplasmic domains of GpIbα and β 3. In CHO cells co-expressing human GpIb-IX and α IIbβ 3, the activation of α IIbβ 3 as reported by monoclonal antibody PAC-1 binding is stimulated by ristocetin (1 mg/ml) + purified human VWF(5 μg/ml). When filamin binding to the cytoplasmic domain of GpIbα is eliminated by deleting GpIbα residues 560-570, PAC-1 binding is eliminated. When human platelets in reconstituted whole blood are treated with a peptide that interferes with filamin binding to GpIbα (as reported in Blood2003;102:2122–2129), shear-dependent (1500 sec−1 shear rate or 60 dynes/cm2 shear stress) platelet deposition onto bovine type I collagen is inhibited. In washed resting platelets and platelets activated by 120 dynes/cm2 shear stress, filamin co-immunoprecipitates with both GpIbα and α IIbβ 3; only its association with β 3 is eliminated by DNaseI (1 mg/ml), demonstrating that filamin binds indirectly to α IIbβ 3 through other cytoskeletal elements. One such element is observed to be talin, which co-immunoprecipitates with filamin, α IIbβ 3 and small amounts of GpIbα in resting platelets. When platelets are sheared for two minutes at 120 dynes/cm2, talin’s DNaseI-resistant association with filamin is decreased and its DNaseI-sensitive association with α IIbβ 3 is increased. These changes are prevented when shear-dependent VWF binding to GpIb-IX-V is blocked by monoclonal antibody AK2. Shear-dependent VWF binding to GpIb-IX-V also results in the proteolysis of talin, which is considered to be one mechanism by which the N-terminal head domain of talin regulates α IIbβ 3 activation. Shear-dependent talin proteolysis is not affected by blocking VWF binding to α IIbβ 3 with a RGD peptide. These results demonstrate that the cytoplasmic domain of GpIbα transduces signals to activate α IIbβ 3 through its interactions with filamin. These signals depend only on VWF binding to GpIb-IX-V. Under pathologically elevated wall shear stress in vitro, the mechanism of signaling may be the release of talin by filamin, thus permitting the proteolysis of talin and enhancing talin’s interaction with α IIbβ 3.
A Monoclonal Antibody Against the Oncogenic Mucin 1 Cytoplasmic Domain
