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iPSC-Derived Megakaryocytes as a Model to Study Human Platelet Integrin IIb3 Function

1Hematology-Oncology Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States, 2Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 3Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States, 4Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States, 5University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania, United States, 6Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 7Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States

Abstract Number: VPB0394

Meeting: ISTH 2022 Congress

Theme: Platelets and Megakaryocytes » Platelet Receptors

Background: Integrin IIb3, is the major fibrin(ogen) receptor on activated human platelets, supporting platelet aggregation and clot contraction during the hemostatic response to injury. IIb3 is expressed on megakaryocytes (MKs) as well as platelets. Historically, methods to study IIb3 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 IIb3 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. IIb3 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 IIb3 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 IIb3 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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