VPB0394 - iPSC-Derived Megakaryocytes as a Model to Study Human Platelet Integrin IIb3 Function

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: Virtual

Presenting Author(s)

KF

Karen Pei Yi Fong, PhD

Research Scientist
Hematology-Oncology Division, University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania, United States

Co-Author(s)

OK

Oleg V. Kim

Research Scientist
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania
Philadelphia, Pennsylvania, United States
AG

Alyssa L. Gagne

Research Assistant
Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
CJ

Chintan D. Jobaliya

Research Scientist
Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
JM

Jean Ann Maguire

Research Scientist
Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
MP

Mortimer Poncz

Professor
Department of Pediatrics, Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
DF

Deborah L. French

Research Professor
Department of Pathology, Perelman School of Medicine at the University of Pennsylvania
Philadelphia, Pennsylvania, United States
Rustem I. Litvinov, MD, PhD

Senior researcher
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania
Philadelphia, Pennsylvania, United States
John W. Weisel, PhD

Professor
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania
Philadelphia, Pennsylvania, United States
WD

William F. Degrado

Professor
Department of Pharmaceutical Chemistry, University of California, San Francisco
San Francisco, California, United States
LB

Lawrence F. F. Brass

Professor
University of Pennsylvania
Philadelphia, Pennsylvania, United States

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.