Heme and Cell-Free Hemoglobin Drive RBC-Endothelial Adhesion in Critical Illness

Tuesday, March 24, 2026

8:45 AM - 9:45 AM Central Time

Location: Connections Central - RST 04

DL

Dhriti Lathker, B.S. Candidate, Biology (Expected June 2026) (she/her/hers)

California

Disclosure(s): No relevant financial relationship(s) to disclose.
MS

Mikhail Stiffler, M.D. Student, Year 2

Medical Student
Case Western Reserve University

Disclosure information not submitted.
ES

Emma Seibert, M.D. Student, Year 1

Medical Student
Cornell Medical School

Disclosure information not submitted.
AS

Andrew Saluan, B.S

Researcher
Case Western Reserve University

Disclosure information not submitted.
DC

Daniel Chen, N/A

Lab Manager
Case Western Reserve University

Disclosure information not submitted.
SP

Soumya Panigrahi, , PhD

Researcher
Case Western Reserve University

Disclosure information not submitted.
MM

Monty Mazer, MD

Assistant Professor of Pediatrics
University Hospitals of Cleveland/Case Western

Disclosure information not submitted.
PB

Priya Borker, MD

Physician
UPMC Presbyterian

Disclosure information not submitted.
Kenneth Remy, MD, MSc, MSCI, FCCM

Case Western Reserve University/University Hospitals Cleveland Medical Center, Ohio

Disclosure(s): No relevant financial relationship(s) to disclose.
JR

James Ross, MD, MS

Assistant Professor of Surgery
University Hospitals Cleveland Medical Center, Case Western Reserve University School of Medicine, Cleveland OH

Disclosure information not submitted.

Introduction: Endothelial dysfunction and microvascular injury are major drivers of organ failure in critical
illness. Hemolytic conditions—seen in sepsis, trauma, sickle cell disease, and transfusion of
stored red blood cells—release red cell–derived damage-associated molecular patterns
(DAMPs), including cell-free hemoglobin (CFH) and free heme. These DAMPs promote
oxidative injury, inflammation, and endothelial activation. While their effects on leukocyte and
platelet adhesion are well described, their impact on red blood cell (RBC) adhesion to the
endothelium is less understood. Enhanced RBC-endothelial interactions may contribute to
microvascular occlusion, tissue ischemia, and organ dysfunction. We hypothesized that CFH
and free heme increase RBC adhesion to human endothelial cells in a dose-dependent
manner.

Methods: Human aortic endothelial cells (HAECs) were exposed for 6 hours to CFH (10 or 20 µM), free
heme (1–80 µM), lipopolysaccharide (LPS; 100 ng/mL, positive control), or vehicle (PBS).
After washing, HAECs were incubated with isolated RBCs or fresh whole blood for 5 minutes.
Adherent RBCs were imaged using EVOS M500 microscopy and quantified as RBCs per
endothelial nucleus via MATLAB. Experiments were performed in triplicate; data were
analyzed by ANOVA with Dunnett’s post-hoc test.

Results: CFH and free heme significantly increased RBC adhesion compared to vehicle in a dose-
dependent fashion. In the whole blood assay, vehicle-treated cells showed 1.1 ± 0.2
RBCs/nucleus. CFH at 10 µM and 20 µM increased adhesion to 4.5 ± 0.4 and 5.7 ± 0.3,
respectively (p < 0.01). Free heme raised adhesion from 3.6 ± 0.2 (1 µM) to 12.5 ± 0.6
RBCs/nucleus (80 µM, p < 0.001), an >10-fold increase. LPS increased adhesion (3.8 ± 0.3)
but was less potent than CFH or high-dose heme. Similar trends were seen with isolated
RBCs, suggesting direct RBC-endothelial interaction.

Conclusions: Red cell–derived DAMPs, particularly CFH and free heme, markedly enhance RBC adhesion
to human endothelial cells. These findings suggest a mechanistic link between hemolysis and
microvascular dysfunction in critical illness. Therapeutic targeting of DAMP-induced
endothelial activation may mitigate tissue hypoxia and organ injury in hemolytic disease
states.