Abstract Number: VPB0394
Meeting: ISTH 2022 Congress
Background: Integrin IIb3, is the major fibrin(ogen) receptor on activated human platelets, supporting platelet aggregation and clot contraction during the hemostatic response to injury. IIb3 is expressed on megakaryocytes (MKs) as well as platelets. Historically, methods to study IIb3 structure/function relationships have been limited to humans with naturally occurring sequence variants, transgenic mouse models, and expression studies in cultured cell lines.
Aims: To determine whether the IIb3 receptor expressed on human induced pluripotent stem cell (iPSC)-derived CD41+CD42+ iMKs is functional, and whether CRISPR/Cas9 gene editing can be used to generate isogenic lines to support integrin structure/function studies.
Methods: Hematopoietic progenitor cells (HPC) were generated from iPSCs using a 2D directed differentiation protocol. iMKs were generated from HPCs cultured in previously established cytokine mixtures. IIb3 activation was analyzed by flow cytometry using the activation-dependent monoclonal antibody, PAC-1. Clot contraction was measured using a Thromboimager Analyzer with iMKs resuspended in platelet-poor plasma in the presence of 2U/ml thrombin and 3mM calcium.
Results: The mean fluorescence intensity (MFI) for PAC-1 binding was 6128±203 (n=4) for thrombin-stimulated, 241±17 (n=4) for unstimulated and 185±70 (n=4) for EDTA-treated cells. Previous work in CHO cells showed the IIb calf domain variants V760A and H787A to be constitutively and non-constitutively active, respectively. The V760A constitutively active variant created in an established control human iPSC line showed a two-fold increase in PAC-1 binding to iMKs (432±144, n=3) compared to the non-constitutively active H787A variant (223±91, n=4, p=0.06) and control iMKs (241±17, n=3, p=0.07). All of the control and variant iMKs resuspended in platelet poor plasma were able to support clot contraction.
Conclusion(s): The ability to generate and study specific variants in the platelet integrin IIb3 receptor using iPSCs and the CRISPR/Cas9 technology demonstrates the use of iMKs in studying platelet integrin-structure function relationships.
To cite this abstract in AMA style:Fong K, Kim O, Gagne A, Jobaliya C, Maguire J, Poncz M, French D, Litvinov R, Weisel J, Degrado W, Brass L. iPSC-Derived Megakaryocytes as a Model to Study Human Platelet Integrin IIb3 Function [abstract]. https://abstracts.isth.org/abstract/ipsc-derived-megakaryocytes-as-a-model-to-study-human-platelet-integrin-%ef%81%a1iib%ef%81%a23-function/. Accessed September 21, 2023.
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