Targeting Platelet GPIbβ Reduces Platelet Adhesion, GPIb Signaling and Thrombin Generation and Prevents Arterial Thrombosis

Abstract

Objective-The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets.We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIbβ, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIbβ by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation. Approach and Results-We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca 2+ signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)′2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)′2 did not prolong the tailbleeding time or increase the volume of blood lost. Conclusions-These

Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets

“…However, it cannot adequately account for the lack of activation by certain anti-LBD (ie, AK2), most anti-GPIbb, anti-GPIX, and anti-MSD antibodies, which should also be capable of lateral dimerization ( Figure 1A). 15,[24][25][26] In comparison, the platelet-crosslinking model we propose here is the first model to fully explain the aforementioned 4 observations about anti-GPIb-IX antibodies, in addition to the shear requirement of anti-LBD antibody-induced signaling ( Figure 6). First, the dimeric structure of an antibody, but not the Fc region, is used to crosslink platelets, which explains the requirement of bivalency and the feature of Fc independence.…”

“…Conversely, mAbs FMC25, RAM.1, and 5G6, which target GPIX, GPIbb, and the MSD of GPIba, respectively, 29,31,36 did not induce P-selectin exposure in human platelets under either static or sheared conditions ( Figure 1D), confirming the difference between them and most anti-LBD mAbs in their abilities to activate GPIb-IX. 24,31 Anti-LBD mAb AK2 neither activates GPIb-IX nor induces IVIg-resistant clearance of platelets…”

“…Third, most mAbs targeting the LBD, but not other regions in GPIb-IX, including the juxtamembrane portion of GPIba, induce Fc-independent clearance ( Figure 1A). 15,[24][25][26] This indicates that there is something particular about the LBD that allows antibodies targeting this region to activate the receptor and/or clear platelets in an Fc-independent manner. Finally, although almost all anti-LBD antibodies induce platelet clearance, 1 anti-LBD mAb, VM16D, does not ( Figure 1A).…”

Identification of a juxtamembrane mechanosensitive domain in the platelet mechanosensor glycoprotein Ib-IX complex

“…This mechanotransducing model, instead of the receptor clustering model, can explain that anti-GPIbb monoclonal antibody RAM.1 blocks VWF-initiated GPIb-IXmediated signaling into the platelet without affecting VWF binding to GPIb-IX. 49,56 It may also help to explain the critical role of GPIbb in mediating the procoagulant activity of platelets. 57 The identification of MSD in GPIb-IX also provides new insights into the pathogenesis of BSS.…”

Immunomodulatory Nanomedicine

“…The platelet expressed collagen receptor GP VI was shown by Boilard et al to be a key trigger for platelet microparticle generation in arthritis pathophysiology . Their GP Ib‐V‐IX receptor affects their adhesion, activation, and procoagulant activity and has been explored as novel preventive treatment of thrombus formation using therapeutic antibodies . Furthermore, platelets are targeted in chronic HIV infection where different anti‐inflammatory drugs are currently compared at a stage II clinical trial (NCT02578706) (Table ).…”