Background: For platelet integrins such as αIIbβ3, the functional consequences of 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 CRISPR-CAS9 technology, has provided a way to study αIIbβ3 mutants in megakaryocytes. Following platelet stimulation, αIIbβ3 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 previously predicted mutations that would destabilize the αIIbβ3 stalk, causing αIIbβ3 activation.

Aims: To translate these findings to iPSC-derived megakaryocytes, we studied a V760A missense mutation located in the αIIb stalk that is highly activating in CHO cells.

Methods: Using an established iPSC line designated CHOPWT14, we created heterozygous and homozygous V760A missense mutations using a CRISPR-CAS9 protocol.

Results: Cell lines were differentiated into megakaryocytes and binding of the activation-dependent monoclonal antibody PAC-1 was used to measure constitutive and agonist-induced αIIbβ3 ligand binding activity. PAC-1 bound constitutively and specifically to 23.4% and 26.0% of megakaryocytes expressing heterozygous and homozygous V760A mutations, respectively, compared to 9.04% of control megakaryocytes. In addition, thrombin stimulation increased PAC-1 binding to >65% in all lines, indicating normal overall αIIbβ3 function.

Conclusions: 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.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/utilizing-crispr-cas9-gene-editing-technology-in-human-pluripotent-stem-cells-to-study-platelet-integrin-%ce%b1iib%ce%b23-function/