Keywords
carbamylation - von Willebrand factor - coagulation - platelet - chronic kidney disease
Introduction
Chronic kidney disease (CKD), a complex condition characterized by high morbidity
and mortality with no definitive cure, affects approximately one-tenth of the global
population.[1] The interplay between the kidney and circulating metabolites is intricate. These
metabolites play pivotal roles in the kidney, acting as ligands for specific receptors,
substrates for posttranslational modifications (PTMs), or as inflammatory chemoattractants.[2] Thus, circulating metabolite alterations in CKD are a complex tally of impaired
filtration, absorption, and secretion, acting as functional mediators of disease.
The reduction of glomerular filtration rate during CKD results in the accumulation
of many protein-bound and water-soluble metabolites in plasma, referred to as uremic
solutes that can exert devastating effects on almost all organ systems. The improvement
of uremic thrombocytopathies by modern hemodialysis techniques indicates that uremic
toxins are, at least partially, responsible for the observed platelet dysfunctions,[3] e.g., phenolic acids and guanidinosuccinic acid inhibited platelet aggregation,
while urea or creatinine did not.[4]
[5] Urea, quantitatively the most abundant retention solute in the body, is in equilibrium
with the reactive product of its decay, cyanate. Cyanate ions react with amino groups
of protein N-terminus and lysine residues, thus converting them to noncharged carbamyl
residues in a process known as carbamylation.[6] This irreversible nonenzymatic PTM of proteins and peptides can lead to their structural
and functional changes. In fact, several carbamylated proteins have been reported
to exhibit reduced functional capacity and higher immunogenicity, such as fibrinopeptide
B, serum albumin, erythropoietin, matrix metalloprotease-2, and others.[7]
[8]
[9]
In CKD, cyanate levels can rise severalfold to those found in healthy individuals.
This elevation leads to an increased production of carbamylated compounds, which not
only play a role in the progression of kidney failure but also in the onset of CKD-related
complications. Due to their strong association with cardiovascular diseases and overall
mortality, these carbamylated compounds have garnered clinical interest as potential
biomarkers. Carbamylated proteins can accumulate across various tissues, including
the skin, heart, liver, and kidneys.[10]
[11]
[12] A significant consequence of raised serum cyanate is its detrimental effect on cell-surface
proteins. For instance, carbamylation of erythrocyte membrane proteins in patients
with uremia has been linked to decreased membrane stability and the premature breakdown
of erythrocytes.[13]
[14]
Coagulopathies represent a significant complication associated with renal insufficiency,
affecting nearly half of all patients with end-stage kidney disease (ESKD).[15]
[16] Our previous research demonstrated that the exposure of platelets to cyanate led
to structural changes in integrin αIIbβ3, a critical fibrinogen-binding protein, resulting
in a fibrinogen-binding defect that impairs the aggregation of uremic platelets.[17] Furthermore, the carbamylation of fibrinogen itself influences the kinetics of fibrin
clot formation, affecting the clot's structure and degradation. These changes are
thought to contribute significantly to the disrupted hemostasis observed in ESKD patients.[18]
von Willebrand factor (vWF), an important ligand of αIIbβ3, is one of the largest
glycoproteins (GPs) circulating in plasma and plays a pivotal role in hemostasis.
In ESKD patients, the reported plasma levels of vWF were either higher than in healthy
volunteers or within a normal range, suggesting that the vWF levels do not contribute
to uremic diathesis.[19]
[20] Interestingly, vWF binding to αIIbβ3 was decreased, while binding to GPlb receptor
was not altered in ESKD patients.[21] A reduced protein tethering and platelet adhesion to subendothelial surfaces has
been reported, while binding to collagen was normal[22]
[23] and the concentration of the vWF:FVIII complex was higher in the ESKD group than
in healthy volunteers.[24]
The impact of carbamylation on vWF functionality and its implications for platelet
adhesion remain largely unexplored. This study aims to elucidate whether carbamylation
of vWF establishes a mechanistic connection between uremia and coagulation dysfunction
in patients with ESKD. Our investigation centers on the susceptibility of vWF to carbamylation,
its influence on protein–protein interactions, and subsequent effects on platelet
adhesion. Additionally, using cell models we have assessed the direct impact of uremia
and carbamylation on platelet activation and their adherence to the endothelium.
Materials and Methods
Study Subjects
Blood from anonymous healthy volunteers was collected at the local blood bank (Department
of Immunology and Transfusion Medicine, Haukeland University Hospital, Bergen, Norway)
using acid citrate dextrose collection tubes (Greiner Bio-One, Monroe, North Carolina,
United States).
Platelet Isolation and Quantification
The collected blood was centrifuged for 20 minutes at 200 g, and platelet-rich plasma
was collected, diluted with an equal volume of HEP buffer (140 mM NaCl, 2.7 mM KCl,
3.8 mM HEPES, 5 mM EDTA, pH 7.4), and centrifuged for 20 minutes at 100 g to remove
red and white blood cells. The supernatant was then centrifuged for additional 20 minutes
at 300 g. The platelet-containing pellet was resuspended in 2 mL of platelet buffer
(145 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM Na2HPO4, 6 mM glucose, pH 7.4). All centrifugations were performed at room temperature (RT).
For platelet quantification, the density was measured at OD800 and platelet number was calculated as described by Tamang et al.[25] Platelets were then diluted to a concentration of 2 × 108/mL, allowed to rest for an hour at RT, and then used in subsequent experiments.
Platelet Carbamylation
Platelets were carbamylated in 1, 5, or 10 mM KCNO (Merck) for 30 minutes at 37°C.
Samples were then centrifuged for 5 minutes at 300 g, resuspended in binding buffer
(140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES), and allowed to rest for 30 minutes at RT.
Platelet Activation
Samples were incubated with either 0.4 U/mL human thrombin (Merck), 50 ng/mL convulxin
(Cayman Chemical, Ann Arbor, Michigan, United States), 25 mM TRAP-6 (Abcam, Cambridge,
United Kingdom), or 25 mM of AY-NH2 (Tocris Bioscience, Bristol, United Kingdom) in binding buffer for 20 minutes at
37°C.
Tandem Mass Spectrometry
vWF (Haematologic Technologies Inc., Essex Junction, Vermont, United States) was carbamylated
in 1 or 10 mM KCNO in phosphate-buffered saline (PBS) for 2, 6, and 16 hours at 37°C.
Samples were then deglycosylated with PGNase F (Promega, Madison, Wisconsin, United
States) and separated via SDS-PAGE. Protein bands were visualized using Coomassie
Blue, bands corresponding to vWF were excised, submerged in HPLC-grade water, and
further processed at the Proteomics and Mass Spectrometry Core Facility of Małopolska
Centre of Biotechnology, Krakow, Poland.
Samples were destained, reduced with 50 mM dithiothreitol for 45 minutes at 37°C,
and alkylated with iodoacetamide before digesting with 6.15 ng/µL chymotrypsin solution
overnight (16 hours) at 25°C. Peptides were extracted with 100% acetonitrile (can)
by sonication and subsequently lyophilized. Samples were resuspended in 2% ACN and
analyzed with a Q-Exactive mass spectrometer coupled with a nanoHPLC UltiMate 3000
RSLCnano System (ThermoFisher Scientific, Waltham, Massachusetts, United States) using
an Acclaim PepMap 100 C18 (75 μm × 20 mm, 3 μm particle, 100 Å pore size) trap column
(ThermoFisher Scientific) in 2% ACN with 0.05% trifluoroacetic acid at a flow rate
of 5 μL/min. Samples were separated on Acclaim PepMap RSLC C18 (75 μm × 500 mm, 2
μm particle, 100 Å pore size) analytical column (ThermoFisher Scientific) at 50°C
in a 60 minute 2–40% ACN gradient in 0.05% formic acid at a flow rate of 250 nL/min.
The eluted peptides were ionized using a Digital PicoView 550 nanospray source (New
Objective, Littleton, Massachusetts, United States). The Q-Exactive was operated in
a data-dependent mode using top eight method with 35 seconds of dynamic exclusion.
Full-scan mass spectrometry (MS) spectra were acquired with a resolution of 70,000
at m/z 200 with an automatic gain control (AGC) target of 1e6. The MS/MS spectra were acquired
with a resolution of 35,000 at m/z 200 with an AGC target of 3e6. The maximum ion accumulation times for the full MS
and the MS/MS scans were 120 and 110 milliseconds, respectively. Raw files acquired
by the MS system were processed using the Proteome Discoverer platform (v.1.4, ThermoFisher
Scientific). The obtained peak lists were searched using an inhouse MASCOT server
(v.2.5.1, Matrix Science, London, United Kingdom) against the cRAP database (https://www.thegpm.org/crap/, released August 2019) with manually added sequences of the protein of interest.
During search, up to five missed chymotrypsin cleavages were allowed. Peptide mass
tolerance and fragment mass tolerance were 10 ppm and 20 mmu, respectively. Carbamidomethyl
(C) was set as a fixed modification and oxidation (M), deamidated (NQ), carbamyl (K)
as variable modifications.
von Willebrand Factor Adhesion to Collagen, Factor VIII, and Fibrinogen
A 96-well microplate was coated with collagen (30 µg/mL in PBS; MP Biomedicals, Santa
Ana, California, United States), factor VIII (FVIII; 10 µg/mL in PBS) for 24 hours
at RT, or fibrinogen (50 µg/mL in PBS, Merck) for 2 hours at 4°C, washed twice with
PBS and carbamylated with 1 mM KCNO (Merck) for 24 hours at 37°C. Wells were washed
with PBS (2×), blocked with 200 µL 5% BSA in 1× PBS for 2 hours at 37°C, and then
washed with 200 µL PBS 0.05% Tween-20 (3×). Next, 50 µL of 5 µg/mL of human vWF (Merck)
in PBS 0.01% Tween-20 was added and plates were incubated for 2 hours at RT. After
washing with 200 µL of PBS 0.05% Tween-20 (3×), wells were incubated with 100 µL of
HRP-conjugated rabbit polyclonal anti-human vWF (1:20,000 in 2.5% BSA, 0.01% Tween-20;
P0226, Dako) for 1 hour at RT and then washed with 200 µL of PBS 0.05% Tween-20 (3×).
The reaction was then started by adding 100 µL of TMB substrate (BD Biosciences, Franklin
Lakes, New Jersey, United States), and stopped within 30 minutes by adding 50 µL 2N
H2SO4. Absorbance was measured at 450 nm (reference wavelength 570 nm) via SpectraMax iD5
microplate reader (Molecular Devices, San Jose, California, United States).
Thromboxane A2 Release
After activation, the 5 × 107 platelets were centrifuged for 10 minutes, 10,000 g at 4°C to obtain the supernatant.
The concentration of thromboxane B2 (TxB2), stable product of nonenzymatic hydration of Thromboxane A2 (TxA2), was measured using ELISA (R&D Systems, Minneapolis, Minnesota, United States) following
the manufacturer's instructions. Results were normalized to TxB2 released by noncarbamylated controls.
Platelet Exposure of Phosphatidylserine
After carbamylation and activation, 5 × 107 platelets were then stained with FITC-Annexin V (ThermoFisher Scientific) for 10 minutes
at RT, diluted with 200 µL binding buffer, and analyzed by flow cytometry using BD
LSRFortessa (BD Biosciences). The percentage of positively stained platelets was calculated
using FlowJo software (v10.9.0, BD Biosciences).
Platelet Adhesion to von Willebrand Factor
Wells of 96-well microplate were coated with 100 µL of human vWF (10 µg/mL) in coating
buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) overnight at 4°C. Bound vWF was subsequently carbamylated in 1, 5, or 10 mM
KCNO overnight at 37°C. Then, wells were washed and blocked with 2.5% BSA in PBS for
2 hours at RT. Wells were washed (3 × ) and incubated with native or carbamylated
thrombin-activated platelets (1 × 106). After 30 minutes at 37°C, adherent platelets were washed twice, fixed with 1% glutaraldehyde
in PBS, and then stained with 0.5% crystal violet in H2O for 10 minutes at RT. Formed violet crystals were dissolved in 1% SDS. Absorbance
was measured at 570 nm using Synergy H1 Hybrid Multi-Mode Reader (BioTek, Bad Friedrichshall,
Germany). The results were calculated as percentage of noncarbamylated control.
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were maintained on collagen-coated
(Advanced BioMatrix, Carlsbad, California, United States) 75 cm2 bottles in DMEM/F12, 10% FBS, Large Vessel Endothelial Supplement (ThermoFisher Scientific),
100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C/5% CO2. Cells were passaged at 90 to 95% confluence. For all experiments, cell passages
between five to eight were used.
Platelet Adhesion to Endothelium
HUVECs were seeded onto clear bottom black 96-well plates (Corning, New York, United
States) and, upon reaching full confluence, washed once with PBS, and activated with
50 ng/mL tumor necrosis factor (TNFα; ThermoFisher Scientific) and carbamylated with
1 mM KCNO (Merck) at 37°C for 24 hours.
Native and carbamylated platelets were stained with CellTrace Far Red (1 µM; ThermoFisher
Scientific) for 45 minutes at 37°C. The platelets were then activated with thrombin.
Platelets were diluted with DMEM/F12 (1.5 × 108/mL) and allowed to adhere to endothelium for 30 minutes at 37°C. Unbound platelets
were washed out with PBS (2 × ). Cells were fixed with 4% paraformaldehyde for 10 minutes
at RT. Cells were visualized using Cytation 5 Cell Imaging Multimode Reader (BioTek)
and one image per well was acquired (Texas Red filter cube, 10× magnification). Analysis
of fluorescence intensity was performed using Fiji software.[26] To identify platelets in the images, “Adaptive Thresholding” plugin [Tseng Q] was
used, with threshold calculated as a weighted mean, and C value was empirically determined
for each experiment. The thresholded images were used as an input for “analyze particles”
command and the result allowed for conversion of identified objects into regions of
interests. These were overlaid onto corresponding raw images and the “integrated density”
was measured.
Statistical Analysis
ANOVA with Tukey's post-hoc test was used to analyze the results. The results are
presented as mean ± standard error of the mean. Statistical significance was evaluated
with GraphPad Prism 9.5.1 (La Jolla, California, United States). The differences were
considered significant: * if p < 0.05, ** if p < 0.01, or *** if p < 0.001.
Results
von Willebrand Factor Is Efficiently Carbamylated at Multiple Sites
To assess vWF's vulnerability to carbamylation, recombinant vWF was incubated with
1 mM and 10 mM KCNO, and the carbamyl-lysines were detected through MS/MS. Modified
lysines were observed in most vWF domains, with lysines within A1 exhibiting increased
susceptibility to carbamylation ([Fig. 1A], [Supplementary Table S1] [available in the online version). In total, at 1 mM KCNO, 22 carbamylated lysine
residues were identified, increasing to 47 at 10 mM KCNO. This signifies clear concentration-
and time-dependence of carbamylation. In the A3 domain, pivotal for vWF interactions
with collagen I, type III, and ADAMTS13, we detected three carbamylated lysines, including
K1720 and K1850, which were readily modified at 1 mM KCNO ([Fig. 1B], [Supplementary Table S1] [available in the online version]). The D' and D3 domains, known for their role
in FVIII binding, harbored one carbamylated lysine each (K773 and K912, respectively)
at lower KCNO concentrations (1 mM) and shorter incubation times (2 and 6 hours).
Extended incubation at 10 mM KCNO for 16 hours produced additional modifications (K834,
K843 in D' and K1026, K1063 in D3; [Fig. 1C]). Normally, vWF has an average circulation half-life of 16 hours[27]
[28]; however, the plasma half-life of vWF in ESKD might be increased due to its impaired
clearance, as suggested by increased plasma vWF in some studies.[29] Herein, even incubation times significantly shorter than the circulation half-life
(2–6 hours) produced multiple carbamyl-lysines. Moreover, we have used KCNO concentration
of 1 mM, which resembles the uremic conditions in vivo,[30] thus confirming the high likelihood of carbamylation of circulating vWF.
Fig. 1 Carbamylation sites on vWF identified in vitro and modeled in silico. (A) Three-dimensional in silico model of the vWF A1 domain showcasing five carbamylated
lysine residues (K1348, K1371, K1408, K1432, K1430; depicted in red) as determined
by orbitrap mass spectrometry following 6 hours of carbamylation with 1 mM KCNO (left
panel). An increase in KCNO concentration to 10 mM for 6 hours revealed three additional
carbamylated lysines (K1312, K1362, K1436; indicated in red with black arrows) (right
panel). (B) In silico three-dimensional model of the vWF A3 domain illustrating two carbamylated
lysines (K1720, K1850; in red) identified post 6-hour carbamylation with 1 mM KCNO
(left panel). Carbamylation with 10 mM KCNO identified an additional lysine (K1794;
in red with a black arrow) (right panel). (C) Three-dimensional in silico representation of the D'D3 dimer, highlighting two carbamylated
lysines following 6 hours of carbamylation with 10 mM KCNO (vWF domain D': depicted
in gray with K773; D3: in light green with K912; both residues marked in red) (left
panel). Extended carbamylation for 16 hours at 10 mM KCNO revealed two more carbamylated
lysines in each domain (D': K834, K843; D3: K1026, K1036; indicated in red with black
arrows) (right panel). vWF, von Willebrand factor.
Carbamylation Affects Binding of von Willebrand Factor to Its Partners
To further investigate impact of the modifications observed via MS/MS analyses, we
conducted binding assays with collagen (types I and III), FVIII, and fibrinogen (Fib).
Our findings reveal that carbamylation significantly impairs the binding affinity
between vWF and collagen, with a notable decrease observed after carbamylation of
both proteins ([Fig. 2A]). Interestingly, the reduction in binding affinity was more pronounced when collagen
itself was carbamylated, indicating that the collagen carbamylation status plays a
critical role in this interaction. The binding assay results for FVIII presented a
contrasting outcome. When FVIII was carbamylated in 10 mM KCNO, its binding to vWF
was enhanced compared with the native form of FVIII ([Fig. 2B]). This suggests that carbamylation may facilitate a stronger association between
FVIII and vWF, potentially influencing coagulation dynamics. Contrary to the effects
seen with collagen and FVIII, carbamylation of vWF did not alter its binding capacity
to fibrinogen, even in the presence of thrombin ([Fig. 2C]). This indicates that the carbamylation process does not significantly impact the
vWF–fibrinogen interaction, underscoring the specificity of carbamylation effects
on protein interactions within the coagulation cascade.
Fig. 2 The effect of carbamylation on protein–protein interactions in vitro. (A) Plates were coated with collagen and carbamylated with 1 mM KCNO. Native (N) or
carbamylated (Carb; 1 mM KCNO) vWF was added and incubated for 2 hours at 37°C. Bound
vWF was determined by HRP-conjugated anti-vWF antibody. (B) Plates were coated with factor VIII and carbamylated by 1 or 10 mM KCNO. Native
vWF was added, incubated, and detected as above. (C) Plates were coated with fibrinogen, and native (N) or carbamylated (C; 10 mM) vWF
was added. The reaction was performed with or without the presence of thrombin. Data
are presented as mean ± SEM, *p < 0.05, **p < 0.01, n = 6 (A, C) or 7 (B) per group. HRP, horseradish peroxidase; SEM, standard error of
the mean; vWF, von Willebrand factor.
Carbamylation Hampers Adhesion of Platelets to vWF but Increases Rate of Binding to
Endothelium
To analyze whether carbamylation influences the platelet–vWF adhesion, we carbamylated
platelets, activated them using thrombin, and examined their binding to immobilized
vWF. Our results indicate a KCNO concentration-dependent reduction in the adhesion
of platelets to vWF ([Fig. 3A]). Subsequently, we examined the binding behavior of noncarbamylated and carbamylated
platelets to carbamylated vWF. While noncarbamylated platelets exhibited a trend toward
increased adhesion to carbamylated vWF, this difference did not reach statistical
significance ([Fig. 3B]). In contrast, simultaneous carbamylation of both vWF (10 mM KCNO) and platelets
led to a significant decline in platelet adhesion, also in a KCNO-concentration–dependent
manner ([Fig. 3C]).
Fig. 3 The effect of platelet carbamylation on their binding capacity. (A) Platelets were carbamylated by either 1 or 5 mM KCNO and incubated with vWF-coated
plates for 30 minutes at 37°C. The number of adhered platelets was determined spectrophotometrically
after staining the platelets with crystal violet. (B) vWF was carbamylated by 1, 5, or 10 mM KCNO at 37°C for 16 hours. Noncarbamylated,
thrombin-activated platelets were incubated for 30 minutes at 37°C and analyzed as
before. (C) vWF was carbamylated by 1, 5, or 10 mM KCNO at mM KCNO at 37°C for 16 hours. Carbamylated,
thrombin-activated platelets were incubated for 30 minutes at 37°C and analyzed as
before. (D) Platelets were carbamylated, stained using CellTrace Far Red dye, activated by thrombin,
and incubated with cultured HUVECs for 30 minutes. Bound platelets were washed, fixed,
and visualized under a fluorescence microscope. (E) Platelets were carbamylated by 1, 5, or 10 mM KCNO and incubated with carbamylated
HUVECs (1 mM). Data are presented as mean ± SEM, dotted line represents noncarbamylated
platelets set as 100% (A–C), or nonactivated HUVECs (D, E); *p < 0.05, **p < 0.01, ***p < 0.001 vs. 100%, #
p < 0.05 vs. 1 mM, n = 3 (A), 6 (B), 5 (C, D), or 4 (E) per group. vWF, von Willebrand factor.
We extended our investigation to examine how carbamylation influences the binding
interaction between platelets and endothelial cells. In this series of experiments,
platelets were carbamylated, stained, and activated with thrombin before being incubated
with TNFα-prestimulated HUVECs. Our findings demonstrate a significant enhancement
in the adhesion of carbamylated platelets to the endothelium, with the extent of adhesion
increasing in a KCNO concentration–dependent manner ([Fig. 3D]). In a subsequent analysis, both the endothelium and platelets were subjected to
carbamylation. This condition suggested a trend toward increased platelet adhesion
compared with native platelets. However, the observed increase in adhesion was markedly
lower than that observed when employing native endothelium, suggesting that while
carbamylation of platelets promotes adhesion, simultaneous carbamylation of endothelium
hampers that interaction ([Fig. 3E])
Carbamylation Limits Release of Thromboxane A2 upon Platelet Activation
TxA2, a metabolite of arachidonic acid produced by activated platelets, serves as a critical
mediator in platelet activation and aggregation. Therefore, we next measured the concentration
of its stable derivative, TxB2, to assess the impact of carbamylation on TxA2 release. Platelets underwent carbamylation prior to their activation with either
thrombin or convulxin, and TxB2 concentrations in the supernatants were measured. Our results clearly indicate that
carbamylation significantly diminishes the release of TxA2 following activation by both thrombin and convulxin, as evidenced by the reduced
levels of TxB2 ([Fig. 4A] and [B], respectively).
Fig. 4 Carbamylation of platelets reduces the release of TXA2. (A) Platelets were carbamylated by 1 or 5 mM KCNO for 30 minutes and activated by thrombin
for 10 minutes. The platelets were then centrifuged, the supernatant was collected,
and Thromboxane B2 was measured using ELISA. (B) Platelets were carbamylated as before and activated with convulxin for 10 minutes.
Data are presented as mean ± SEM, dotted line represents noncarbamylated platelets
set as 100%. *p < 0.05, ***p < 0.001, n = 3 per group.
Thrombin-Induced Phosphatidylserine Exposure Is Increased after Platelet Carbamylation
Platelet activation is a critical step in the coagulation cascade, marked by the exposure
of phosphatidylserine (PS) on the platelet surface, which plays a pivotal role in
creating a procoagulant environment. This study sought to examine the influence of
carbamylation on PS exposure following platelet activation. Carbamylated platelets
were activated with either thrombin or convulxin and subsequently stained with annexin
V. Flow cytometry analysis revealed a significant increase in PS exposure on carbamylated
platelets following thrombin activation, with a 190% increase compared with 100% in
native platelets ([Fig. 5A]). Conversely, the response of carbamylated platelets to convulxin was notably diminished
in comparison to the nonmodified counterparts ([Fig. 5B]).
Fig. 5 Carbamylation of platelets alters the surface exposure of phosphatidylserine depending
on the activating agent. (A) Platelets were carbamylated by 1 or 5 mM KCNO for 30 minutes, activated by thrombin
for 10 minutes, stained with Annexin V and immediately analyzed using flow cytometry.
(B) Platelets were carbamylated as before and activated with convulxin. Data are presented
as mean ± SEM, **p < 0.01, n = 4–5 per group.
Given the specific pathways through which thrombin mediates platelet activation—namely,
the protease-activated receptors 1 and 4 (PAR-1 and PAR-4)—we further investigated
the effect of carbamylation on platelet activation through these receptors. Utilizing
the specific agonists TRAP-6 (PAR-1 agonist) and AY-NH2 (PAR-4 agonist), our investigations
revealed no discernible impact of carbamylation on platelet activation via either
PAR receptor (data not shown).
Discussion
Carbamylation is an unavoidable outcome of elevated levels of plasma cyanate in patients
with ESKD. This PTM impacts a broad spectrum of proteins and peptides in circulation
as well as the cell-bound proteins. Among others, the carbamylation targets components
of the coagulation cascade, serving as a direct catalyst for the development of coagulopathies
frequently seen in individuals with ESKD. Our previous research has demonstrated that
carbamylation significantly alters the structure and function of fibrinogen and αIIbβ3,
two critical components involved in blood clotting and platelet function. These alterations
contribute to the impaired function of platelets, a key factor in the abnormal clotting
observed in ESKD patients.[17]
[18] In this study, we further explore the mechanisms by which carbamylation influences
the interaction between vWF and its partner proteins, examining its impact on platelet
aggregation and function. Our findings illuminate the complex ways carbamylation disrupts
these essential processes, highlighting the multifaceted complications associated
with ESKD.
The association between vWF and coagulopathies in patients with ESKD has been predominantly
linked to the compromised functionality of vWF, rather than a decrease in its levels.
Interestingly, vWF concentrations in the plasma of these patients are often normal
or slightly elevated.[20] vWF is a large multimeric GP essential for hemostasis and thrombosis regulation.
It circulates in plasma, where it binds and stabilizes FVIII and is also found intracellularly
within endothelial cells and platelets. Upon vascular injury, vWF plays a pivotal
role by recruiting platelets to the site of damage, facilitating their adhesion and
aggregation. Consequently, deficiencies or abnormalities in vWF can lead to bleeding
disorders. In our research, we identified multiple lysine residues within vWF domains
that are susceptible to carbamylation. This modification could impair vWF's function,
shedding light on the molecular mechanisms underlying coagulation disorders in ESKD.
Our in vitro analyses revealed that exposure to cyanate leads to differential carbamylation
across vWF domains, with domain A1 exhibiting the most substantial level of modification,
marked by the carbamylation of nine lysine residues. These modifications occurred
at pivotal sites within the A1, A3, D3, C4, and CK domains, where lysins have been
implicated to take part in the binding or are in the immediate vicinity of crucial
vWF-interactions ([Table 1]). The A1 domain is involved in vWF binding to sulfatides on the surface of platelets,
and its readily undergoing carbamylation lysine residues - K1408, K1423, and K1430
- are involved in of vWF-sulfatide binding.[31] K1408 plays a crucial role in vWF's interaction with the scavenger receptor LRP1,
instrumental in vWF clearance from circulation.[32] Whether carbamylation of K1408 in ESKD patients might contribute to decreased vWF
elimination requires further investigation. Whereas within the A3 domain, lysine residues
K1720 and K1850, crucial for binding to the GPIb platelet surface receptor, were carbamylated.
This modification may disrupt the vWF–GPIb interaction, pivotal for platelet adhesion
under high shear conditions. Moreover, lysine K1312, essential for vWF's binding to
GPIb, was susceptible to carbamylation under various conditions, potentially impairing
native platelet binding to carbamylated vWF, as evidenced in our findings ([Fig. 3B]).[33] Domain A3 plays a pivotal role in the immobilization of vWF onto the subendothelial
matrix, primarily through interactions with collagen type I and type III.[23] This interaction is facilitated by several key amino acids located at the domain's
upper epitope, critical for mediating collagen binding.[33]
[34] Under shear stress, vWF's domain A2 unfolds, revealing a cleavage site between residues
Y1605 and M1606 for the specific metalloproteinase ADAMTS13 (a disintegrin and metalloproteinase
with thrombospondin motif 13).[35] We identified two carbamylated lysine residues, K1518 and K1617, within this domain,
with K1617 playing a role in the ADAMTS13 recognition sequence essential for vWF binding.
It remains to be determined whether the carbamylation patterns observed in vitro mirror
those in ESRD patients. Nonetheless, previous studies on albumin[10] and integrin αIIbβ3[17] strongly suggest that this is highly probable. Overall, carbamylation of vWF at
specific lysine residues, observed across various domains, could significantly influence
its multifaceted roles, particularly in protein–protein interactions critical for
hemostatic processes.
Table 1
List of vWF domains with identified carbamylated lysines and their respective interacting
domains (lysines were identified at two different carbamylation conditions [1 mM and
10 mM KCNO])
|
Domain
|
Carbamylated lysine
|
Interactions
|
|
A1
|
K1312, K1348, K1408
|
GP1bα, β2-integrins, β2-glycoprotein I, PSGL-1, Col VI, ADAMST13
|
|
A3
|
K1720, K1850
|
Col I and III, ADAMST13, thrombospondin
|
|
D3
|
K1935
|
ADAMST13, IGFBP7 (insulin growth factor-binding protein-7)
|
|
C4
|
K2537
|
αIIbβ3, αVβ3, ADAMST13, fibrin, IGFBP7
|
|
CK
|
K2757
|
ADAMST13, IGFBP7, CTGF/CCN2 (connective tissue growth factor)
|
Abbreviation: vWF, von Willebrand factor.
Although the direct role of the K1720 and K1794 residues in collagen interaction has
not been previously described, their proximity to crucial amino acids suggests a potential
impact on the binding efficiency. In vitro analyses demonstrated a diminished interaction
between carbamylated vWF and a collagen mixture (types I and III), hinting at the
significant role these modified residues may play in the collagen–vWF binding process,
potentially obstructing access to R1726 and H1786 directly interacting with collagens.
Intriguingly, this reduction in binding affinity was more pronounced when both vWF
and collagen were carbamylated, presenting a contrast to previous research that generally
reports unaffected vWF–collagen binding in ESKD patients.[36] Furthermore, vWF domains Dˊ and D3 are known to engage with FVIII, serving as a
crucial cofactor in the coagulation cascade, with the vWF:FVIII complex circulating
in an inactive form in plasma.[24] In the context of ESKD, there is an upsurge in circulating levels of the vWF:FVIII
complex. Notably, our study revealed that carbamylation of FVIII in vitro enhances
its binding to vWF, potentially contributing to the elevated plasma levels of the
complex as observed in ESKD. Additionally, the identification of carbamylated lysine
K773 within the Dˊ domain of vWF, a residue known to be integral to FVIII binding,[31] provides further potential mechanisms through which carbamylation could augment
the vWF:FVIII interaction.
Contrary to expectations, our study revealed no significant effect of vWF carbamylation
on its binding to fibrinogen. This suggests that the carbamylation-induced alterations
in vWF do not hinder its competition with fibrinogen for the αIIbβ3 integrin, a key
mediator of platelet aggregation. Contrary to the effects observed with immobilized
vWF, we demonstrated that carbamylation significantly enhances platelet adhesion to
endothelial cells in vitro. The adhesion to HUVECs is primarily mediated through ICAM-1
and Integrin αIIbβ3, offering a potential explanation for the observed discrepancies
with vWF.[37] While platelets typically adhere to the subendothelial extracellular matrix, our
findings indicate that carbamylation facilitates a notable increase in the direct
platelet–endothelium binding. This effect is evident in both isolated and carbamylated
platelets, as well as under conditions simulating ESKD and uremia.[38]
Interestingly, when both HUVECs and platelets undergo carbamylation, the rate of platelet
adhesion, although still elevated in carbamylated platelets, is marginally reduced
compared with their adhesion to noncarbamylated HUVECs. This nuanced result might
shed light on the complex and ambivalent nature of hemostasis in ESKD patients—fluctuating
between diathesis and prothrombotic states. However, our observations of predominantly
reduced platelet adhesion in this context suggest a more intricate interplay at the
endothelial interface than previously understood. Our investigations into the effects
of carbamylation on platelet–vWF interactions showed that carbamylated platelets exhibit
reduced binding to native vWF. Interestingly, native platelets displayed a trend toward
increased binding to carbamylated vWF. This suggests that platelet surface carbamylation
influences their interaction with vWF potentially more than the carbamylation of vWF
itself. However, in vivo, both carbamylated platelets and vWF likely contribute to
impaired hemostasis in ESKD.
We also explored the effects of carbamylation on the release of TxA2 and the exposure of PS upon platelet activation. A reduction in TxA2 secretion following carbamylation and activation of platelets aligned with previous
observations of diminished TxA2 generation in ESKD.[39] Additionally, carbamylation led to an increased exposure of PS on activated platelets,
supporting in vivo findings of enhanced procoagulant activity in ESKD. The exposure
is crucial for creating a procoagulant surface by providing binding sites for factor
X leading to assembly of prothrombinase complex and releasing thrombin. In ESKD, the
exposure of PS by platelets was increased.[40]
In conclusion, our findings elucidate the multifaceted impact of carbamylation on
vWF and platelets, highlighting its significant role in modifying their interactions
and functions. These alterations could contribute to the complex hemostatic imbalance
in ESKD, underscoring the need for further research to fully understand these mechanisms
and their clinical implications.
What is known about this topic?
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Coagulopathies affect almost half of the patients suffering from the ESKD.
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Carbamylation of integrin αIIbβ3 impairs aggregation of uremic platelets.
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Structure and formation of carbamylated fibrinogen is significantly affected.
What does this paper add?
