Hemin regulates platelet clearance in hemolytic disease by binding to GPIbα

Abstract

Plain Language Summary
What is the context? Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of several diseases.
Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more potent agonist than hemoglobin for directly activating platelets.
Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet hemostasis.
Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological platelet clearance through liver macrophages and hepatocytes.
What is new? In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the influence of hemin at 0-50 μM on platelets in vitro before exploring the mechanism underlying hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.
Our analyses suggest that: Hemin bound to GPIbα on the platelet surface with high affinity.
Platelet clearance occurred slowly in the liver and spleen after hemin treatment.
Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.
Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation after hemin treatment.
What is the impact? This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated platelets activation and clearance in diseases associated with hemolysis.

Full text

RESEARCH ARTICLE
Hemin regulates platelet clearance in hemolytic disease by binding to
GPIbα
Man Zhao, Dongxin Peng, Yuxuan Li, Minwei He, Yulong Zhang, Qianqian Zhou, Sujing Sun, Ping Ma, Liping Lv,
Xiaohui Wang, & Linsheng Zhan
Field Blood Transfusion, Institute of Health Service and Transfusion Medicine, Beijing, China
Abstract
Hemolysis is associated with thrombosis and vascular dysfunction, which are the pathological
components of many diseases. Hemolytic products, including hemoglobin and hemin, activate
platelets (PLT). Despite its activation, the effect of hemolysis on platelet clearance remains unclear,
It is critical to maintain a normal platelet count and ensure that circulating platelets are functionally
viable. In this study, we used hemin, a degradation product of hemoglobin, as a potent agonist to
treat platelets and simulate changes in vivo in mice. Hemin treatment induced activation and
morphological changes in platelets, including an increase in intracellular Ca
2+
levels, phosphatidyl-
serine (PS) exposure, and cytoskeletal rearrangement. Fewer hemin-treated platelets were cleared by
macrophages in the liver after transfusion than untreated platelets. Hemin bound to glycoprotein Ibα
(GPIbα), the surface receptor in hemin-induced platelet activation and aggregation. Furthermore,
hemin decreased GPIbα desialylation, as evidenced by reduced Ricinus communis agglutinin I (RCA- I)
binding, which likely extended the lifetime of such platelets in vivo. These data provided new insight
into the mechanisms of GPIbα-mediated platelet activation and clearance in hemolytic disease.
Keywords
GPIbα, hemin, platelet; , platelet clearance
History
Received 4 April 2024
Revised 15 July 2024
Accepted 18 July 2024
Plain Language Summary
What is the context?
●Hemolysis is a primary hematological disease. Hemolysis is a pathological complication of
several diseases.
●Hemin, a degradation product of cell-free hemoglobin, has been proven to be a more
potent agonist than hemoglobin for directly activating platelets.
●Platelet membrane glycoproteins (GP), including GPIb-IX and GPIIb/IIIa complexes, play
crucial roles in platelet hemostasis.
●Desialylation (loss of sialic acid residues) of GPIbα, is believed to regulate physiological
platelet clearance through liver macrophages and hepatocytes.
What is new?
●In this study, we evaluated the effects of hemolysis on platelet clearance. We first analyzed the
influence of hemin at 0-50 μM on platelets in vitro before exploring the mechanism underlying
hemin-induced platelet activation and its role in platelet clearance in vitro and in vivo.
Our analyses suggest that:
●Hemin bound to GPIbα on the platelet surface with high affinity.
●Platelet clearance occurred slowly in the liver and spleen after hemin treatment.
●Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation
after hemin treatment.
●Platelets exhibited significant significantly reduced GPIbα surface expression and desialylation
after hemin treatment.
Correspondence: Xiaohui Wang, Field Blood Transfusion, Institute of
Health Service and Transfusion Medicine, 27 Taiping Road, Beijing
100850, China. E-mail: lovechina1980@163.com
Linsheng Zhan, Field Blood Transfusion, Institute of Health Service and
Transfusion Medicine, 27 Taiping Road, Beijing 100850, China.
E-mail: amms91@126.com
This is an Open Access article distributed under the terms of the Creative
Commons Attribution-NonCommercial License (http://creativecommons.
org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use,
distribution, and reproduction in any medium, provided the original work
is properly cited. The terms on which this article has been published
allow the posting of the Accepted Manuscript in a repository by the
author(s) or with their consent.
http://www.tandfonline.com/iplt
ISSN: 0953-7104 (print), 1369-1635 (electronic)
Platelets, 2024; 35(1): 2383642
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
DOI: https://doi.org/10.1080/09537104.2024.2383642

What is the impact?
●This study provides new insights into the role of hemin in the mechanisms of GPIbα-mediated
platelets activation and clearance in diseases associated with hemolysis.
Introduction
Hemolytic disease is a condition in which the lifespan of red
blood cells is reduced. Several hematologic diseases, includ-
ing paroxysmal nocturnal hemoglobinuria (PNH), thalassemia,
and glucose-6-phosphate dehydrogenase deficiency, are pri-
marily characterized by hemolysis. Hemolysis is also a patho-
logical complication of diseases such as malaria,
1
sepsis
2
and
sickle cell disease,
3
and is accompanied by platelet activation,
hypercoagulability, thrombocytopenia, platelet-leukocyte
aggregates and inflammation. Hemolysis is associated with
thrombosis and vascular dysfunction. Hemolytic products,
including hemoglobin and hemin, could activate platelets
(PLT) directly. Meanwhile, a broad range of plasma Hb con-
centrations (0.37–6 μM) has been demonstrated to be directly
correlated with the occurrence and severity of intravascular
thrombosis and thromboembolism in PNH patients.
4
Hemin, a
degradation product of cell-free hemoglobin, has been shown
to be a more potent agonist than hemoglobin for directly
activating platelets.
5
It is a signaling molecule that mediates
various biochemical processes, such as inflammation, tran-
scription, and signal transduction, via transient binding to
various proteins.
6,7
Annarapu GK. et al. showed that hemin-
stimulated human platelet mitochondrial oxidant production
induces targeted granule secretion.
8
Moreover, recently,
hemin was shown to activate human and mice platelets via
CLEC-2 and GPVI/FcRγ.
8
Platelets adhere to and are activated via ligand-receptor
recognition and intracellular signaling pathways. Platelet adhe-
sion, activation, and aggregation are crucial for hemostasis and
thrombosis. Platelet membrane glycoproteins (GP), including
GPIb-IX and GPIIb/IIIa complexes, play crucial roles in platelet
hemostasis. The former is the second-most abundant platelet
membrane glycoprotein.
9
GPIb is composed of subunits of
GPIbα and GPIbβ that noncovalently combine with glycoprotein
GPIX to form GPIb-IX.
10,11
The extracellular domain binding of
the von Willebrand factor is critical for initial platelet adhesion
at the site of injury,
12
and triggers downstream signaling in
platelets, including activation, aggregation and phosphatidylser-
ine (PS) exposure.
13
The most heavily glycosylated platelet
membrane surface protein is GPIbα.
14
The terminal glycan resi-
due of GPIbα is sialic acid, linked to a penultimate β-galactose
(β-gal) and antepenultimate GlcNAc. Desialylation (loss of sia-
lic acid residues) of major platelet surface glycoproteins, pri-
marily GPIbα, is believed to regulate physiological platelet
clearance and lead to the clearance of human and mouse plate-
lets by liver macrophages and hepatocytes.
15–19
Another
mechanism that contributes to the clearance of old platelets
from mice is apoptosis.
20
Despite its known effects on platelet activation, the role of
hemin in platelet clearance remains unclear. We investigated the
effect of hemin at increased labile (free and weakly bound) heme/
hemin in plasma concentrations between 2 μM and 50 μM
21,22
in
patients with hemolytic diseases. Subsequently, we assessed the
mechanism underlying hemin-induced platelet activation and its
role in platelet clearance via in vitro and in vivo experiments. To
the best of our knowledge, this is the first study to systematically
investigate the interactions between hemin and platelets and is
expected to help us understand the mechanism by which hemin
regulates platelet clearance in hemolytic diseases.
Materials and methods
Animals
Male wild-type C57BL/6J mice (6–8 weeks) were purchased from
Vital River (Beijing, China). C57BL/6-Tg (GFP) transgenic GFP
and tdTomato mice were purchased from the Model Animal
Research Center of Nanjing University (Nanjing, China). All
animal studies were performed in accordance with the guidelines
of the National Beijing Center for Drug Safety Evaluation and
Research (IACUC-DWZX-2023-528).
Isolation of apheresis platelets (APs) from humans
APs were isolated from healthy human volunteers at the Chinese
People’s Liberation Army (PLA) General Hospital (Beijing, China).
This study was designed and conducted in accordance with the
Declaration of Helsinki and approved by the Ethics Committee of
the National Beijing Center for Drug Safety Evaluation and Research.
All the donors met the criteria for AP donation in China. No patient-
identifying information was involved in this study; therefore, informed
consent was not required. The APs were washed twice with calcium-
free Tyrode’s buffer [1 L Tyrode’s buffer contains 8.0 g NaCl, 0.2 g
KCl, 2.383 g HEPES, 0.056 g NaH
2
PO
4
*2 H
2
O, 0.203 g MgCl
2
*6
H
2
O and 1.0 g glucose] (Yuanye Biotech, China), centrifuged at
800 × g for 15 min at 37°C, and then suspended in calcium-free
Tyrode’s buffer.
Preparation of platelets from mice
Mice were anesthetized, and whole blood was collected via cardiac
puncture using a 1-mL syringe containing 14% anticoagulant (CPDA-
1). Leukoreduced blood samples were collected and pooled into sterile
microcentrifuge tubes. Platelet-rich plasma (PRP) was prepared by
gentle centrifugation (two rounds of 15 min each at 150 × g).
Preparation of hemin
A 50 mM hemin (from Porcine, ≥ 96.0% (HPLC), Sigma-
Aldrich) stock solution was dissolved with 1.4 M sodium hydro-
xide and diluted to 50 μM working stock in modified calcium-free
Tyrode’s buffer for all experiments.
Co-culture experiments
For platelet-hemin co-culture experiments, 5–10 × 10
6
platelet sus-
pension was treated with six concentrations of hemin (0, 5, 10, 25, 40
and 50 μM) for 15 min at 37°C or pre-treated with a GPIbα blocking
anti-GPIbα mAb (5 μg/mL; Biolegend, clone H1P1) in 1 mL Tyrode’s
buffer for 30 min at 37°C before the cells were washed with Tyrode’s
buffer. The platelets were then used for flow cytometry and morpho-
logical observation by microscopy.
Flow cytometry
Platelet activation was measured by staining with FITC-labeled anti-
human CD62P (P-selectin) antibody (5 μg/mL) (Biolegend, Clone
AK4) or PE-labeled anti-human CD42b (GPIbα) antibody (5 μg/mL,
Biolegend, Clone H1P1) for human platelets. PS exposure was
assessed using 5 μL annexin V-PE (BD Biosciences). Desialylation
was detected using fluorescein-labeled RCA-1 (Vector Laboratories,
USA) on human platelets. An ROS assay kit (Beyotime, China) was
used to measure the total intracellular reactive oxygen species (ROS)
2M. Zhao et al. Platelets, 2024; 35(1): 1–11

production according to the manufacturer’s instructions.
Mitochondrial membrane potential was determined using the JC-1
Assay Kit (Beyotime, China) according to the manufacturer’s instruc-
tions and quantified by flow cytometry. The fold increase was repre-
sented as a percentage increase from the gated control platelets set at
1% positivity. All isotype control antibodies were purchased from the
same manufacturer as the detection antibodies.
Intracellular free-calcium measurements
For imaging intracellular Ca
2+
([Ca
2+
]i) transients induced by
hemin treatment, human platelets were incubated with 2 μM
Fluo-4 AM (Molecular Probes, DojinDo) for 30 min at 37°C.
Cells were washed with HBSS, incubated for 20 min at 37°C in
HBSS medium, and then stimulated with hemin. Images were
captured immediately every 10 s using a confocal microscope
(Zeiss, Germany). The recorded images were analyzed and quan-
tified using Image J software (NIH).
For image analysis, background correction was performed by
subtracting the intensity of untreated control platelets from that of
hemin-treated platelets. F0 was calculated by subtracting the
initial background from the fluorescence intensity. F indicates
fluorescence intensity at each time point. ΔF/F0 = (F – F0)/F0,
indicated the Ca
2+
concentration change after hemin stimulation.
Confocal microscopy for studying cytoskeletal protein
dynamics in hemin-treated platelets
To evaluate the cytoskeletal protein dynamics in hemin-treated plate-
lets, human platelets were allowed to settle on collagen-coated cover-
slips for 1 h, followed by incubation, washing with PBS, fixing with
4% paraformaldehyde (Merck), and permeabilization with 0.1% Triton
X-100 (Sigma-Aldrich). Finally, platelets were stained with FITC-
conjugated phalloidin (Sigma-Aldrich) to analyze F-actin and subse-
quent incubation with anti-β-tubulin antibodies (Abcam, Clone
EPR1330) followed by Cy3-labeled secondary antibodies. Finally,
all the coverslips were washed and viewed under a confocal laser
scanning microscope (Perkin Elmer, USA).
Light transmission aggregometry
Human PRP (1 × 10
9
/mL; 250 μL) was incubated with concentration-
dependent hemin (5–50 μM) for 15 min at 37°C or pre-treated with
anti-GPIbα blocking antibody (H1P1) (20 ug/mL) for 30 min at
37°C before aggregation. Platelet aggregation was assessed by light
transmission aggregometry using Agg RAM
TM
(Roche
Diagnostics, UK) in response to TRAP-6 (1.5 μM). Platelet-poor
plasma (PPP) represented 100% aggregation as the control,
whereas hemin-treated PRP showed 0% aggregation before stimu-
lation. The results are presented as % aggregation, representing
the maximum amplitude after stimulation.
Platelet aggregometry by confocal microscopy
For the assessment of platelet aggregation by confocal microscopy, 10
µL human PRP (1 × 10
9
/mL) was allowed to settle in a collagen-
coated chamber for 30 min at 37°C, followed by incubation with
different concentrations of hemin (5–50 μM) for 1 h at 37°C and
washed by calcium-free Tyrode’s buffer. All samples were photo-
graphed using a confocal microscope (Zeiss) after the platelets adhered
to the bottom of the chamber. The platelets that adhered to the col-
lagen-coated pore walls were activated and aggregated.
Western blotting
Platelets were washed twice and lysed. Total proteins in the cell
lysates were quantified using a BCA assay kit. The same amounts
of protein aliquots were fractionated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and the resulting protein bans
were transferred to PVDF membranes. The membranes were
blocked with 4% bovine serum albumin in TBST and incubated
with anti-human CD42b antibody (Abcam, Clone EPR6995) and
anti-human GAPDH antibody (Abcam) at 4°C overnight. The
membranes were washed and incubated with horseradish perox-
idase-conjugated secondary antibodies and then developed using
an ECL Kit (Sigma-Aldrich).
Scanning electron microscopy (SEM)
For SEM imaging, the platelets were washed and fixed in 2.5%
glutaraldehyde for 24 h at 4°C and treated for gradual desiccation.
The specimens were sputter-coated with 20 nm gold and used for
SEM observation (Hitachi, Japan).
Transmission electron microscopy (TEM)
For TEM imaging, the platelets were washed and fixed in 2.5%
glutaraldehyde overnight at 4°C and washed with PBS, followed by
post-fixation with 1% osmium tetroxide on ice for 1 h. Samples were
dehydrated using a graded ethanol series and embedded in Epon. The
specimens were used for TEM observations (Hitachi, Japan).
Generation of mouse bone marrow-derived macrophages
(BMDMs)
As BMDM were usually employed to represent Kupffer cell in the
liver in the researches that focus on the clearance of blood cells
that transfused, the phagocytosis experiment of platelets in vitro
was conducted by BMDM.
Bone marrow monocytes prepared from the femurs and tibias
of tdTomato mice were cultured at a density of 2 × 10
6
cells/mL
in 6-well plates with 25 ng/mL murine macrophage colony-stimu-
lating factor (Peprotech, Israel) supplemented with complete
Dulbecco’s modified Eagle medium (DMEM) (Life
Technologies Corporation, USA). The medium was replaced
with 2 mL of fresh medium containing M-CSF on days 3 and 5.
Adherent cells were used as BMDMs on day 6.
Platelet clearance in vivo
A novel model was established to monitor the lifespan of platelets
in circulation after transfusion. Platelets from GFP+ transgenic
mice were GFP-positive (GFP+) and distinguishable from wild-
type (WT) recipient platelets by FACS. GFP+ murine platelets
were incubated with hemin (0–50 μM) for 15 min at 37°C and
washed. Subsequently, mice were intravenously injected with 2 × 10
8
platelets in 300 μL PBS. The control mice received the same
volume of untreated GFP+ platelets under identical conditions.
After transfusion of GFP+ platelets, whole-blood samples were
obtained from mice anesthetized by retro orbital bleeding at 4, 24,
and 48 h. Platelet circulation was measured by the enumeration of
fluorescently labeled platelets in the blood. The 20-min recovery of
GFP+ murine platelets in each mouse was set at 100%, that is 0%
clearance.
Platelet clearance in vitro
Platelet clearance in vitro was quantified by measuring the levels of
GFP+ murine platelets in tdTomato BMDMs using flow cytometry.
Briefly, 1–5 × 10
7
GFP+ platelets treated with hemin (0–50 μM)
alone were added to 2 × 10
6
tdTomato BMDMs in 2 mL of complete
DMEM. In another setup, platelets were pre-treated with a specific
blocking antibody to GPIbα (20 µg/mL) for 30 min before being
DOI: https://doi.org/10.1080/09537104.2024.2383642 Hemin Regulates Platelet Clearance 3

added. After 4 h of phagocytosis, BMDMs were washed for removal
of noningested platelets and detected by FACS.
Immunohistochemistry
After the in vivo platelet clearance studies, the mice were eutha-
nized, and their livers and spleens were harvested at 48 h and
snap-frozen in liquid nitrogen. The frozen tissues were sectioned
(5-mm-thick) and fixed onto slides. Slides were washed in 5%
BSA and then incubated with primary rat anti-mouse F4/80
(Clone BM8; eBioscience) overnight at 4°C and then stained by
the corresponding secondary antibody for 2 h. Cell nuclei were
stained with 5 μg/mL 4,6-diamidino-2-phenylindole (DAPI).
Images were acquired using an Olympus fluorescence
microscope.
Hemin-platelets binding kinetic characterization
The binding kinetics of hemin to human platelets were measured
using the bio-layer interferometry (BLI) biosensor (Gator Bio,
USA). Briefly, hemin (100 nM) was loaded onto the sensors
(SA, Gator Bio) and exposed to 62.5–1000 nM Recombinant
Human CD42b/GP1BA Protein (His Tag) (HPLC-verified, Sino
Biological, Cat: 11765-H08H) for association and dissociation
with PBST buffer. The binding affinity was measured by the
wavelength shift (nm) and used as the binding ratio to compare
the binding affinities between the different groups. The binding
ability was normalized to that of the relative control under the
same conditions.
Statistical analysis
Values were represented as the mean ± standard deviation (SD).
Statistical comparisons between different treatments were shown
in the respective figure legends. Differences with p values less
than 0.05 were considered significant.
Results
Extracellular hemin activates platelets
To investigate the platelet activity under hemolytic diseases, the
effects of extracellular hemin on human platelets were studied in
vitro. Our findings showed that hemin raised the expression of P-
selectin upon platelet activation in a dose-dependent manner
(Figure 1A and S1). PS exposure was evaluated by measuring
annexin V binding. Hemin significantly increased annexin V
binding, however, by less than 5%, even at the highest concentra-
tion (Figure 1B and S2), demonstrating PS exposure or generation
of a procoagulant surface. Simultaneously, increased platelet
aggregation at high concentrations was observed using light trans-
mission aggregometry (Figure 1C). Moreover, platelet aggrego-
metry was directly observed using confocal microscopy (Figure
1D). Larger platelet aggregates were observed in high-concentra-
tion hemin-treated platelets.
Hemin can induce apoptosis and necroptosis in RBCs by
increasing intracellular Ca
2+
levels and eliminating cellular
glutathione.
23
High intracellular Ca
2+
enhances dynein activity
and suppresses kinesin activity, producing a dynamic and rapid
change in the cytoskeleton. In our study, we observed that a rapid
Ca
2+
burst occurred upon the addition of 25 μM hemin (Figure
1E,F). Changes in the platelet ultrastructure were also observed
using SEM (Figure 1G) and TEM (Figure 1H). As shown in
Figure 1G. platelet morphology changed and pseudopodia
extended after hemin treatment, reflecting the increased degree
of activation, which was consistent with the increased expression
of CD62P in Figure 1A. First, typical and distinguishable platelets
with a discoid shape were predominant in the hemin-free group.
Subsequently, 5 μM hemin stimulation facilitated cell adhesion.
Thereafter, filopodia in 25 μM hemin-treated platelets formed a
spindle-like morphology. However, in the case of 50 μM, lamelli-
podia filled the area between individual filopodia before a so-
called “fried-egg” morphology was formed.
TEM analysis revealed that untreated platelets had a discoid
shape, and granules were heterogeneously distributed in the cyto-
plasm (Figure 1H). Furthermore, 5 μM hemin-treated platelets
displayed minor shape changes, retaining the basic discoid form
to some extent. Most of the cells formed short pseudopods with
evident centralization of α-granules, and the integrity of granules
was moderately maintained and visible throughout the cytoplasm.
Upon 25 μM hemin stimulation, the classic activation signs
emerged and dramatic alterations in shape and noticeable degra-
nulation, such as loss of the discoid shape, longer pseudopodia
formation, enlargement of vacuoles, internal contraction of the
platelet marginal band and aggregate formation with reduced
cytoplasmic granules, were observed. In the 50 μM group, large
vacuoles were seen in nearly all the platelets.
We analyzed the cytoskeleton dynamics in hemin-treated pla-
telets using confocal microscopy. As shown in Figure 1I, resting
platelets maintained their discoid shape with a well-demarcated
and highly specialized cytoskeleton, while hemin treatment
caused cytoskeleton reorganization, inducing depolymerization
of the microtubule coil and loss of the discoid shape of platelets
compared to controls. Further analysis suggested that hemin-trea-
ted platelets contracted and elongated, forming stress fibers, filo-
podia, and lamellipodia. Altogether, hemin-activated platelets
increased intracellular Ca
2+
and induced platelet PS exposure or
the generation of a procoagulant surface apoptosis and morpho-
logical changes (Figure 1). However, from a macro perspective,
the morphological changes of platelets were hemin concentration-
dependent and gradual.
Hemin-inhibited platelet clearance in vivo
In general, the clearance of apoptotic cells by macrophages plays
a central role in tissue homeostasis. BMDMs are typically used to
investigate the phagocytosis of apoptotic cells. Because macro-
phages are critical for clearing aged platelets,
16
we used BMDMs
for phagocytosis experiments to simulate the platelet clearance
process in vivo. FACS analysis revealed that after 4 h of co-
incubation, hemin inhibited GFP+ mouse platelet phagocytosis
by tdTomato BMDMs compared to the hemin-free group (Figure
2A,B). This observation was also confirmed by analyzing GFP+
platelets treated with hemin (0–50 μM) localization after 4 h of
co-culture of tdTomato BMDMs by confocal microscopy. Figure
2C shows colocalization images of GFP+ platelets (green) and
tdTomato BMDMs (red). The effect of hemin on the lifespan of
platelets in vivo was investigated, and the post-transfusion
recovery of hemin-treated platelets was evaluated by transfusing
GFP+ mouse platelets into wild-type C57BL/6J mice. The per-
centage of GFP+ platelets from among the total platelets 20 min
after transfusion was considered as 100% recovery in circula-
tion. Notably, after 25 or 50 μM hemin-treatment, the platelets
were cleared slowly at 24 h and 48 h, which was consistent with
the results in vitro (Figure 2D). Furthermore, routine blood tests
of blood samples from mice transfused with hemin-treated pla-
telets showed no significant differences in platelet count com-
pared to the control group at different timepoints (Figure 2E),
4M. Zhao et al. Platelets, 2024; 35(1): 1–11

Figure 1. Hemin induced human platelet activation. (A, B) P-selectin and annexin V binding expression on the surface of hemin-treated platelets analyzed by
FACS. The positive gate and specificity of these antibodies were controlled by corresponding isotypes. (C) Percentage aggregation measured using light
transmission aggregometry in response to TRAP-6 (1.5 μM). Data are presented as the mean ± SD (panels A, B, and C); n = 3. *Compared with the hemin-free
group (0 μM) (p<.05). Statistical analysis by one-way ANOVA with Tukey’s multiple comparison in panels A, B, and C. (D) Fluorescent images showing the
formation and size of aggregates after incubation with hemin for 1 h. (E) Ten representative traces of intracellular Ca
2+
dynamics in platelets treated with hemin.
(F) Intracellular [Ca
2+
] transient amplitudes quantified by measuring the maximal fluorescence intensity change of fluo-4 AM in control and hemin-treated
platelets. Data are presented as the mean ± SD (panels F); n = 10. *Compared with the hemin-free group (0 μM) (p<.05). Statistical analysis by independent-
sample t-tests in panel F. (G) SEM observation of hemin-treated platelets in vitro. Scale bars = 10 μm. (H) TEM observation for studying platelet structure. Hemin
(0 μM) represented resting human platelets; hemin (5–50 μM) represented hemin-activated human platelets. Scale bars = 2 μm. The pictures shown in G and H are
DOI: https://doi.org/10.1080/09537104.2024.2383642 Hemin Regulates Platelet Clearance 5

suggesting that treatment with hemin affected the fate of plate-
lets in vivo.
Next, tissues from recipient mice were examined after intrave-
nous injection of GFP+ platelets by immunofluorescence stain-
ing. As expected, GFP+ platelets appeared in the liver and spleen
(Figure 2F,G). Labeling macrophages with monoclonal antibody
F4/80 revealed that most platelets were colocalized with macro-
phages in the liver, while a few were colocalized with macro-
phages in the spleen. These results suggest that macrophages play
a key role in the clearance of hemin-treated platelets.
Hemin binds to GPIbα
The molecular mechanisms underlying hemin-induced platelet
activation were examined to elucidate the role of hemin in platelet
clearance. Hemoglobin induced platelet activation and apoptosis
through interactions with surface GPIbα,
4
suggesting GPIbα as
the main receptor of hemin. The BLI biosensor was used to
determine the binding affinity between hemin and GPIbα.
Hemin bound to GPIbα with high affinities; the affinity constant
(K
D
) of GPIbα to hemin was 15.7 nM (Figure 3A). We then
investigated the role of GPIbα signaling in hemin-treated human
platelet activation. The pretreatment of platelets with HIP1, an
anti-GPIbα antibody, to block GPIbα prevented platelet
aggregation,
24
inhibiting hemin-induced activation, apoptosis,
and aggregation of platelets to some degree (Figure 3B–D).
Collectively, these results allowed us to speculated that hemin
could probably bind to GPIbα on the platelet surface and it might
partly activate platelets this way. Simultaneously, it is worth
noting that the downstream signalings such as P-selectin expres-
sion and PS exposure, were not inhibited completely. Thus, we
suspect that there was other pathways involved in the binding
events in hemin treating.
Hemin-mediated reduction in human platelet desialylation
attenuated platelet clearance
Previous studies have demonstrated that GPIbα plays a pivotal
role in the rapid clearance of transfused cold-stored platelets.
18
Therefore, we evaluated the expression of GPIbα in hemin-treated
platelets. Hemin treatment could down-regulate the surface
expression of GPIbα (CD42b). Notably, FACS analysis revealed
that as hemin concentrations increased, GPIbα surface expression
decreased (Figure 4A,B). However, Western blot data showed no
significant difference in platelet GPIbα levels after hemin treat-
ment (Figure 4C), indicating that total GPIbα remained
unchanged.
Previous studies have shown that mitochondrial ROS play a
critical role in DOX-induced platelet apoptosis and GPIbα
shedding,
25
and that ROS play a crucial role in thrombin and
collagen-induced GPIbα ectodomain shedding.
26
Therefore, we
analyzed mitochondrial membrane activity in the presence of
different concentrations of hemin (5–50 μM). As shown in
Figure 4D,E, human platelets treated with hemin showed reduced
mitochondrial membrane potential and increased ROS generation
in a dose-dependent manner.
Because hemin bound to GPIbα with high affinity, we tested
the effect of hemin treatment on platelet GPIbα desialylation by
quantifying the binding of fluorescein-conjugated RCA-1 lectins,
which specifically target exposed galactose residues following
desialylation.
17,18
RCA-1 binding to human platelets was reduced
and desialylation significantly reduced after hemin treatment
(Figure 4F).
To clarify the role of platelet GPIbα expression in hemin-
treated platelet clearance by macrophages, we then examined the
mouse platelet clearance by BMDMs after anti-GPIbα antibody
pretreatment of platelets in vitro. Phagocytosis of platelets by
BMDMs was reduced in low concentration hemin groups (0, 5
and 10 μM) to a certain extent after GPIbα blocking. However, it
was worth noting that the role of GPIb blocking disappeared in
the case of 50 μM hemin group (Figure 4G), indicating that
GPIbα is involved in platelet clearance. It is reasonable to
deduce that the dose-dependent activation and aggregation of
platelets toward hemin exposure probably accelerate the clear-
ance of platelets by BMDM, neutralizing the effect of GPIba
blocking, especially in the high levels hemin groups. We spec-
ulate that besides the GPIbα desialylation pathway, other clear-
ance mechanisms might also be at work in hemin-mediated
platelet clearance.
Hemin may inhibit the desialylation of GPIbα through binding
or occupation, which might affect its simultaneous recognition by
macrophages and hepatocytes and in turn slow down the platelet
clearance in vivo (Figure 4).
Discussion
Previous studies have shown that hemin is a ligand of various
platelet receptors such as CLEC-2, GPVI,
8
and TLR4, and
activates human and mouse platelets. In this study, we analyzed
the effect of hemin on platelets platelet activation and clearance
through in vitro and in vivo experiments. BLI analysis and K
D
value of 15.7 nM after platelet treatment suggested high affinity
binding between hemin and GPIbα on platelets. Moreover, anti-
GPIbα antibody pretreatment inhibited hemin-induced platelet
activation, indicating that hemin binds GPIbα, reinforcing that
GPIbα mainly could mediate the effect of hemin on platelets.
Previous studies have shown that VWF-GPIb-IX interaction
induces platelet activation, leading to platelet adhesion and
aggregation.
27,28
Similarly, in this study, we found that hemin
(5–50 μM) induced platelet activation in a concentration-depen-
dent manner in vitro. Hemin can induce apoptosis and necrop-
tosis in RBCs by increasing intracellular Ca
2+
levels and
eliminating cellular glutathione.
23
High intracellular Ca
2+
enhances dynein activity while reducing kinesin activity, produ-
cing a dynamic and rapid change in the cytoskeleton. Another
study showed that myeloperoxidase binding to platelets induced
actin cytoskeleton reorganization that elevated cytosolic Ca
2+
concentrations.
29
Therefore, hemin-GPIbα interaction was
speculated to initiate cytoskeleton reorganization to increase
[Ca
2+
]
i
. As expected, in the current study high [Ca
2+
]
i
enhanced
microtubule coil expansion and produced dynamic and rapid
changes in the cytoskeleton. Our data also showed that hemin-
activating platelets induced changes in platelet shape, including
the formation of filopodia and lamellipodia from actin filaments
and the dispersal of microtubule ring fragments, indicating that a
hemin-dependent increase in [Ca
2+
]
i
likely regulates redistribu-
tion of the cytoskeleton network in platelets, leading to the
formation of larger aggregates, which marks a procoagulant
state and thrombosis.
Billions of platelets are cleared every day from circulation
through efficient regulatory mechanisms, which can be regulated
representative of all experiments conducted. (I) Cytoskeleton staining in platelets. Washed platelets were treated with hemin and analyzed by confocal microscopy.
Representative actin staining with phalloidin (red) and tubulin stained with anti-β-tubulin antibodies (green) in platelets preincubated with 0, 5, 25, and 50 μM
hemin, respectively (scale bars = 20 μm).
6M. Zhao et al. Platelets, 2024; 35(1): 1–11

Figure 2. Treatment with hemin inhibited the clearance of mouse platelets in vivo. (A, B, C) phagocytosis of GFP+ platelets (green) by BMDMs (red)
quantified by FACS (A, B) and confocal microscopy (C) in vitro. (D) Percentage clearance of GFP+ platelets after intravenous injection of GFP+
hemin-opsonized platelets. (E) Routine blood test results of platelets (PLT) number in different groups. (F, G) tissue sections of the liver (F) and spleen
(G) harvested from mice stained with anti-F4/80 (liver and spleen macrophages; red). Nuclei were counterstained with 4,6-diamidino-2-phenylindole
(DAPI) (blue). F: white scale bars, 50 μm. G: white scale bars, 40 μm. Data are presented as the mean ± SD; n = 3–5. *Compared with the hemin-free
group (0 μM) (p<.05). Statistical analysis by one-way ANOVA with Tukey’s multiple comparison in panel A. Statistical analysis by two-way ANOVA
with Tukey’s multiple comparison in panels D and E.
DOI: https://doi.org/10.1080/09537104.2024.2383642 Hemin Regulates Platelet Clearance 7

by a variety of factors, such as exogenous reagents or environ-
mental changes. Two case reports have shown that the platelet
count decreases after hemin injection.
30,31
In contrast, a study in
rats showed that platelet count increased after an intraperitoneal
injection of hemin.
32
In our study, we found that platelet count
decreased after hemin treatment in vitro (Fig. S3), probably due to
increased platelet aggregation. However, when residual hemin-
treated platelets were injected intravenously into mice, the post-
transfusion recovery of these platelets rose in vivo at 24 h and
48 h after transfusion, indicating that the hemin-treated platelets
cleared slowly. The in vitro data also showed that the presence of
hemin inhibited platelet phagocytosis by BMDMs. However, this
is inconsistent with the findings of previous studies that show
platelet clearance from circulation is accelerated under (pro)
thrombotic conditions once they have executed their functions.
33
We speculated that this bias might be because no significant
aggregates or apoptosis were formed despite the apparent platelet
activation observed in vitro.
Intersecting molecular mechanisms regulate platelet clearance.
The binding of VWF or antibodies to the GPIbα on platelets can
activate GPIb-IX, triggering downstream signaling in the platelet
such as activation and PS exposure. It has been shown that the
loss of terminal sialic acid (a derivative of neuraminic acid) from
the platelet surface is directly correlated with senescent platelet
clearance from circulation.
34
Deglycosylation of GPIbα, a subunit
of GPIb, also causes hepatic clearance of cold-stored mice plate-
lets after transfusion.
35,36
We observed a significantly reduced
surface GPIbα (CD42b) in platelets upon the addition of hemin,
however, no significant changes in GPIbα expression were
observed from western blot results. It is can be deduced that the
reduced exposure of galactose residues may be due to the
decreased sialic acid loss at the end of GPIba instead of GPIba
extracellular domain loss, for there was no glycocalicin loss
detected in the medium after hemin treatment (data not shown).
Simultaneously, we speculated that hemin might have probably
occupy the position of GPIbα instead of inducing the structural
changes of it (such as cluster formation). It is known that glycan
modification is involved in platelet clearance in human and mice
after the stage of desialylation.
35,37
In our study, a significantly
reduced exposure of galactose residues (RCA-1) was detected,
which indicated reduced GPIbα-mediated desialylation of hemin-
treated platelets, however, we also can’t exclude the possibility
that hemin remaining in the system after washing interfered with
the binding of RCA-I to GPIbα. Notably, hemin treatment
induced PS exposure, which was another mechanism that con-
tributed to platelet clearance. However, despite the significant
increase in platelet PS exposure after hemin treatment, in vitro
and in vivo data showed that platelet clearance was slowed.
Therefore, it was likely a balanced process and inhibition of
desialylation might be dominating and responsible for the slowing
of hemin-mediated platelet clearance.
Finally, previous studies have demonstrated that GPIbα clus-
tering induces liver macrophage-mediated platelet clearance in
guinea pigs and macaques.
24
And platelet clearance in immune
thrombocytopenia (ITP) is mediated by FcγR via macrophages of
Figure 3. Hemin induced human platelet activation by binding to GPIbα. (A) The binding kinetic characteristics of the hemin-platelet complex were
detected by the BLI biosensor. (B-D) blocking effect of anti-GPIbα antibody (H1P1) (20 μg/mL) on hemin (0, 25, 50 μM)-induced platelet activation,
PS exposure and aggregation. Washed platelets were pretreated with anti-GPIbα antibody and incubated with hemin. The expressions of P-selectin (B),
and annexin V binding (C) on the platelet surface were measured by FACS. The positive gate and specificity of these antibodies were controlled by
corresponding isotypes. (D) Quantification of maximum light transmission of the platelets. Data are presented as the mean ± SD; n = 3. *Compared
with the anti-GPIbα antibody-free group (p<.05). #Compared with anti-GPIbα antibody (5 μg/mL)/hemin-free group (p<.05). Statistical analysis by
two-way ANOVA with Tukey’s multiple comparison in panel B-E.
8M. Zhao et al. Platelets, 2024; 35(1): 1–11

the reticuloendothelial system through Fc - FcγR interactions
primarily in the spleen.
38
We found that platelets were mainly
cleared by macrophages in the liver, which is consistent with
previous studies in rats and mice.
39,40
In summary, hemin binding
to GPIbα induced platelet activation, but inhibited platelet desia-
lylation and in turn extended the circulation time of platelets,
suggesting that the hemin-treated platelets by allogeneic transfu-
sion obtained more recovery in our mouse model (Figure 5).
However, the connection between these processes remains
unclear, which necessitate further experimental investigation.
Figure 4. Hemin inhibited human platelet clearance via decreased desialylation of GPIbα (A, B) the expression level of GPIbα in hemin-treated human
platelets assessed by FACS using FITC-conjugated CD42b. (C) Western blot analysis of GPIbα. (D) FACS analysis of mitochondrial membrane
potential (ΔΨm) in human platelets. (E) FACS analysis of ROS generation. (F) Hemin inhibited GPIbα desialylation of human platelets. The positive
gate and specificity of these antibodies were controlled by corresponding isotypes. Data are presented as the mean ± SD; n = 3. *Compared with the
hemin-free group (0 μM) (p<.05). Statistical analysis by one-way ANOVA with Tukey’s multiple comparison in panels A, D, E, and F. (G) Blocking
GPIbα inhibited platelet clearance. GFP+ mouse platelets pre-incubated with anti-GPIbα blocking antibody (20 μg/mL) for 30 min prior to hemin
treatment, then phagocytosis of GFP+ platelets by BMDMs was quantified by FACS. Data are presented as the mean ± SD; n = 3. *Compared with the
anti-GPIbα antibody-free group (p<.05), #Compared with the anti-GPIbα antibody (5 μg/mL)/hemin-free group (p<.05). Statistical analysis by two-
way ANOVA with Tukey’s multiple comparison in panel G.
DOI: https://doi.org/10.1080/09537104.2024.2383642 Hemin Regulates Platelet Clearance 9

Conclusion
In summary, our study shows that hemin plays an important role in
regulating normal platelet clearance during severe hemolysis. Herein,
we demonstrated that hemin activates platelets, as evidenced by down-
stream signaling pathways, such as: Increased intracellular Ca
2+
levels, morphological changes, and platelet aggregation response in a
dose-dependent manner. The binding of hemin to GPIbα on platelets
could be involved in inducing platelet activation, which might slow
platelet clearance by decreasing GPIbα desialylation in vivo. Further
research is needed to elucidate the precise mechanism linking hemo-
lysis to platelet activation and clearance regulation. Our findings high-
light the need to focus on the status and function of platelets in the
diagnosis and therapy of hemolytic disorders, which may, in turn,
provide insights into novel clinical strategies.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The author(s) reported there is no funding associated with the work
featured in this article.
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