Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Abstract

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.