Utilizing CRISPR-CAS9 Gene Editing Technology in Human Pluripotent Stem Cells to Study Platelet Integrin αIIbβ3 Function

Integrins are a family of cell surface receptors that transmit signals bidirectionally across plasma membranes. Cellular agonists induce 'inside-out' signals that cause ligand binding to integrins; in turn, this causes integrin-mediated 'outside-in' signals that control cellular functions such as adhesion and migration. The platelet integrin αIIbβ3 has served as a paradigm for deciphering these bidirectional signaling mechanisms. Moreover, site-directed mutagenesis has been essential for dissecting integrin structure-function relationships. For platelet integrins such as αIIbβ3, the structure-function consequences of the site-directed mutations have previously been studied in epithelial cell lines, such as Chinese hamster ovary (CHO) cells. Only recently has technology advanced sufficiently to enable study of platelet integrins in their normal cellular environment, the platelet-producing megakaryocyte. The advent of human induced pluripotent stem cells (iPSC), coupled with the ability to gene edit these cells using the CRISPR-CAS9 technology provides a way to study αIIbβ3 mutants in megakaryocytes. Following platelet stimulation, αIIbβ3 rapidly undergoes a global rearrangement in which a clasp composed of its extracellular stalk, transmembrane, and membrane-proximal cytoplasmic domains is disrupted causing the αIIbβ3 headpiece to open exposing a ligand binding site. Using computational methods, we predicted mutations that would destabilize the stalk domain, thereby causing αIIbβ3 activation. These predictions were confirmed by showing that the mutations were indeed able to cause constitutive αIIbβ3 activation in CHO cells (Donald et al, J. Biol. Chem. 2010; Tan et al, Biochemistry 2019). To translate these findings to human cells and compare the functional consequences of the mutations when endogenously expressed in iPSC-derived megakaryocytes, we chose a highly activating V760A missense mutation located in the stalk region of the αIIb subunit. Using an established control iPSC line designated CHOPWT14, both heterozygous and homozygous V760A missense mutations were created in the ITGA2B gene encoding αIIb using our CRISPR-CAS9 protocol (Maguire et al. Current Protocols in Stem Cell 2019). The generation of these mutant cell lines from a genetically identical control cell line eliminates the heterogeneity and variability in downstream applications that was observed when using lines of different genetic backgrounds. To ensure genetic identity of the parent and mutant lines, copy number variation or CNV analyses were performed and shown to be identical in all lines. To study ligand binding of αIIbβ3 expressing the V760A mutation in iPSC-derived megakaryocytes, the isogenic control and mutant lines were differentiated to blood using our serum- and stromal-free directed differentiation protocol (Mills et al. Methods in Mol. Biol. 2014). Briefly, the differentiation of human iPSCs to blood follows primitive hematopoiesis which recapitulates the developmental stages observed in the embryo of primitive streak formation, mesoderm specification, and hematopoietic development, including megakaryocytes. To measure conformational change in αIIbβ3, the activation-dependent monoclonal antibody PAC-1 was used as a surrogate for fibrinogen. By analyzing CD41⁺CD42b⁺ megakaryocytes by flow cytometry, PAC-1 was shown to bind constitutively to 23.4% and 26.0% of the megakaryocytes expressing heterozygous and homozygous V760A mutations, respectively, compared to 9.04% of control megakaryocytes. The specificity of PAC-1 binding was confirmed by a reduction in binding to <1% following pretreatment with EDTA. In addition, thrombin stimulation increased PAC-1 binding to >65% in all lines, indicating normal overall αIIbβ3 function. Essentially identical results were obtained when FITC-fibrinogen was used instead of PAC-1. These data show that 1) structure-function studies of computationally identified mutations confirmed in CHO cells can be analyzed using human iPSC-derived megakaryocytes, 2) mutations shown to be highly active in CHO cells appear to be constrained or less constitutively active in human megakaryocytes, and 3) more in-depth analyses of platelet integrin structure-function relationships will be possible using human megakaryocytes.