Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibß function.

UNLABELLED: Essentials A signaling role of glycoprotein (GP)Ibß is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbß function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbß subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbß in hemostasis and thrombosis. Methods GPIbß(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbß were transplanted into lethally irradiated GPIbß(-/-) recipient mice. Results hGPIbß transplanted into the bone marrow of GPIbß(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbß(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbß intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbß in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

ß-arrestin-1 participates in thrombosis and regulates integrin aIIbß3 signalling without affecting P2Y receptors desensitisation and function.

ß-arrestin-1 (ß-arr1) and ß-arrestin-2 (ß-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of ß-arr1 or ß-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in ß-arr1-/- and ß-arr2-/- platelets. In addition, deficiency in ß-arr1 or ß-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between ß-arr1 and ß-arr2 may explain these unchanged platelet responses. Interestingly, ß-arr1-/- but not ß-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in ß-arr1-/- and ß-arr2-/- mice, suggesting no defect in haemostasis. ß-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbß3. ß-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbß3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for ß-arr1 in promoting thrombus formation, in part through its participation in αIIbß3 signalling, and no role of ß-arr1 and ß-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

BACKGROUND: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. OBJECTIVE: The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. RESULTS: In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. CONCLUSIONS: Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.