A Model to Study Platelet Integrin Variants Using Transfected Human Embryonic Kidney-293 Cells Expressing Fluorescent αIIbβ3-Fusion Proteins
Authors: Stoldt Volker Rainer , Khattouti Abdelouahid El , Scharf Rudiger E. 
Published: April 2020

The HPA-1 polymorphism of platelet integrin αIIbβ3 (GPIIb-IIIa) arises from a thymidine to cytosine transition in position 1565 of the ITGB3 gene. This transition leads to an amino acid exchange at residue 33 of the mature β3 subunit. The resulting isoforms are HPA-1a (Leu33) or HPA-1b (Pro33). We have shown that the HPA-1b variant of αIIbβ3 is associated with premature manifestation of myocardial infarction in patients suffering from coronary artery disease (Zotz et al. J Thromb Haemost 2005). This observation has lead to the hypothesis that the HPA-1b variant of αIIbβ3 may increase platelet thrombogenicity. Recently, we have also demonstrated that HPA-1b/1b platelets adhering onto fibrinogen are more resistant than HPA-1a/1a platelets when exposed to arterial shear rates of 1000 to 1500 sec-1 (Loncar et al. Thromb J 2007). To explore the molecular nature of the postulated prothrombotic phenotype of HPA-1b in further detail, we have now overexpressed the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP) fused to the cytoplasmic tails of the αIIb or β3 subunit of both αIIbβ3 variants in human embryonic kidney-293 (HEK293) cells. Clones were screened for their cyan and yellow fluorescence. Ten positive clones of each αIIbβ3 variant were detected and characterized by Western blotting identifying the 140 kD αIIb-CFP fusion protein and the 113 kD β3-YFP fusion protein with appropriate antibodies directed against the αIIb subunit, the β3 subunit or the fluorescent proteins. Stable HEK293 clones expressing the HPA-1 receptor isoforms on the cell surface were functionally analyzed by flow cytometry (Becton Dickinson), confocal laser scanning microscopy (Zeiss), and a fluorescence imager (Thermo). We used fluorophore (PE)-conjugated complex-specific (PM6/248) and activation-dependent (PAC-1) antibodies and fluorescently tagged fibrinogen (Alexa647- Fg) in combination with phorbol-12-myristate-13-acetate (PMA) or organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol, SAG). Corresponding controls were performed with the chimeric antibody abciximab (ReoPro) to block fibrinogen binding to αIIbβ3 or with pertussis toxin (PTX) to inhibit G-protein-coupled inside-out signal transduction. Functional integrity of the integrin variants (HPA-1a and HPA-1b) was demonstrated by intact activation through G-protein-coupled receptors with SAG and by specific binding of Alexa647 fibrinogen to αIIbβ3 indicating proper insertion of the receptor complex into the plasma membrane of transfected cells. In the presence of PTX or abciximab, activation and ligand binding function of αIIbβ3 were completely (&gt;95%) inhibited in both isoforms of HPA-1. Cytoplasmic conformational changes upon integrin activation using either PMA or SAG were followed by fluorescence resonance energy transfer (FRET) and quantified by FRET signal disappearance due to allosteric changes of the cytoplasmic domains. Stimulation with PMA and SAG caused FRET signal disappearance to same extent in each HPA-1 variant. However, the dynamics of signal disappearance appeared to be faster in the HPA-1b than in the HPA-1a variant of the cell clones studied so far. This observation corresponds to the prothrombotic phenotype of HPA-1b. In conclusion, our results demonstrate that we have generated a cellular model which can be useful to study molecular properties of αIIbβ3 variants and to explore the nature of the prothrombotic HPA-1b phenotype in further detail.