OC 59.3 - Megakaryocyte Engraftment and altered Platelet Function in a Murine Model of Hematopoietic Stem Cell Transplantation

Tuesday, July 12, 2022

11:15 AM – 11:30 AM

Location: ExCel Center, ICC Capital Suite Room 1

Presentation Format: In-Person

Presenting Author(s)

KM

Kristina Mott, MSc

PhD Student
University Hospital Wuerzburg
Würzburg, Bayern, Germany

Co-Author(s)

CK

Cornelia Keß

Student
Institute of Experimental Biomedicine, Chair I, University Hospital Wuerzburg, Wuerzburg, Germany
Würzburg, Bayern, Germany
AF

Anna Frank

Student
Institute of Experimental Biomedicine, Chair I, University Hospital Wuerzburg, Wuerzburg, Germany
Würzburg, Bayern, Germany
SH

Sarah Hölle

Medical Student
Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Children's Hospital, University Medical Center, University of Wuerzburg, Wuerzburg, Germany
Würzburg, Bayern, Germany
ZN

Zoltan Nagy

Project Leader
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany
NS

Nicolas Schlegel

Professor
Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Wuerzburg, Wuerzburg, Germany
Würzburg, Bayern, Germany
ME

Matthias Eyrich

Professor
Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Children's Hospital, University Medical Center, University of Wuerzburg, Wuerzburg, Germany
Würzburg, Bayern, Germany
HS

Harald Schulze, PhD

Professor
Institute of Experimental Biomedicine I, University Hospital Würzburg, Germany
Würzburg, Bayern, Germany

Background: Total body irradiation (TBI) preceding hematopoietic stem cell transplantation (HSCT) remains a treatment option for hemato-oncological diseases. Complications include bleeding due to poor megakaryocyte (MK) engraftment and prolonged thrombocytopenia. Underlying mechanisms for delayed platelet production are yet ill-defined.

Aims: To analyze how TBI clinically impacts platelet engraftment and how TBI-induced bone marrow (BM) remodeling influences MK engraftment and platelet function using a reporter-based murine HSCT model.

Methods: C57B6/J wildtype mice were subjected to sublethal or lethal TBI followed by HSCT with BM from dsRedβ-actin mice. Platelet production and function was assessed by flow cytometry and immunoblotting. MK number and location were determined in respect to BM sinusoids and matrix proteins in femur sections by confocal microscopy. MMP activity was assessed by immunoblot and zymography using MMP9-/- mice as controls. Plasma chemokine levels were measured by ProcartaPlex luminex. Platelet engraftment kinetics was assessed in a retrospective single-center cohort of 350 pediatric HSCT recipients.

Results: Our human HSCT analysis revealed that TBI-conditioning is associated with prolonged thrombocytopenia and increased platelet transfusion demand compared to chemo-based conditioning regimens. In TBI-conditioned mice after HSCT, platelets were hyporeactive towards thrombin and CRP-XL (Fig.1A-C,E). While dsRed- (recipient-derived) platelets were hyporeactive, dsRed+ (donor-derived) platelets displayed over-activation towards CRP-XL (Fig.1D), implying that two functionally distinct platelet populations are present in circulation. Syk phosphorylation was markedly reduced in response to CRP-XL (Fig.1F). Donor cell engraftment occurred in large clusters, containing Ly6G+ cells and MKs, while Ly6C+, B220+ and Ter119+ cells were homogeneously distributed (Fig.2A), implying a specific MK/granulocytes engraftment pattern. Massive vasodilation occurred already six hours after irradiation (Fig.2B,C), including sinusoid-specific MMP9-mediated collagen IV degradation (Fig.2D-F). CXCL1, CXCL9 and CCL7 were selectively upregulated (Fig.2G-I), suggesting a role for HSC engraftment.

Conclusion(s): A specific MK engraftment pattern in response to TBI-induced BM remodeling is associated with a mixed platelet chimerism comprising markedly different reactivity.