Keywords
thrombospondin-1 - neutrophil elastase - cathepsin G - proteolysis - platelet adhesion
- platelet string formation
Introduction
Thrombospondin-1 (TSP-1) is a multi-domain glycoprotein formed by three identical
185 kDa sub-units that are connected via disulphide bonds.[1] The protein comprises a heparin-binding domain (HBD) of globular structure which
is N-terminal of the connecting region, followed by a pro-collagen-homology domain,
three properidin-like TSP type 1 modules, three epidermal growth factor (GF)-like
TSP type 2 elements that provide structural stability, seven calcium-binding TSP type
3 repeats and a unique lectin-like C-terminal globular domain.[1] Each domain enables TSP-1 to fulfil distinct functions in various biological processes
and to interact with a variety of binding partners such as fibrinogen, von Willebrand
factor (vWF), the scavenger receptor CD36 or the cell surface receptor CD47.[2]
[3] The main producers of TSP-1 are platelets and endothelial cells (ECs) where it is
stored in α-granules or Weibel–Palade bodies.[4]
[5] TSP-1 is constitutively expressed and protein levels between 20 and 40 ng/mL are
found in human plasma under physiological conditions.[6]
[7] However, a rapid increase of TSP-1 release can be observed after activation of platelets
and ECs.[4]
[5]
Among its various biological functions, the role of TSP-1 in angiogenesis has been
extensively studied over the past years.[8] Both pro- and anti-angiogenic properties are attributed to TSP-1.[9]
[10]
[11] While the N-terminus mediates adhesion and motility of ECs, the remaining molecular
core inhibits angiogenesis by antagonizing survival pathways while also activating
apoptotic pathways.[8]
[12] In particular, TSP-1 binding to CD47 or CD36 surface receptors was found to inhibit
nitric oxide (NO) signalling.[13]
[14] Lee et al reported in 2006 that the matrix metalloprotease a disintegrin and metalloproteinase
with thrombospondin motif (ADAMTS)-1 is able to cut TSP-1 leading to a matrix-bound
trimer of the 36-kDa N-terminal domain and a soluble, monomeric 110 to 125 kDa C-terminal
fragment acting as a potent angiogenesis inhibitor.[15]
[16] Thus, by removal of the N-terminal HBD the anti-angiogenic effect of TSP-1, mainly
attributed to the type 1 repeats and C-terminal globular domain, is promoted.
In addition to its prominent functions in angiogenesis, TSP-1 is also known to play
a role in haemostasis. It enhances platelet aggregation by forming a bridge between
fibrinogen molecules bound to platelet integrin αIIbβ3.[17] Furthermore, TSP-1 promotes platelet aggregation through binding to CD36 and CD47
receptors which results in the activation of platelets and intra-cellular signalling.[18] Another essential function of TSP-1 is the ability to stabilize platelet aggregates
under shear stress. While the plasma protease ADAMTS-13 cuts multimeric vWF to resolve
platelet strings, TSP-1 is able to bind to and stabilize vWF thereby protecting it
from ADAMTS-13-mediated degradation.[19]
In the context of haemostasis, it is still unclear whether proteolytic processing
of TSP-1 and removal of the N-terminus promotes a functional change of the protein
as observed in angiogenesis. We previously found that two different TSP-1 isoforms
of 185 and 160 kDa are constitutively present in human plasma.[6] A substantial increase of the smaller TSP-1 isoform was detected after surgical
intervention when blood coagulation and post-operative wound healing were triggered.
However, the source and mechanism generating the 160-kDa TSP-1 variant have not been
revealed to date and the functional consequences on haemostasis have not been addressed.
During the past years, several plasma or leukocyte-derived proteases were found to
have the ability to cut TSP-1 resulting in fragments of variable size ranging from
25 to 160 kDa, but with unknown functional or in vivo relevance. In addition to plasmin,
TSP-1 is reportedly susceptible to cleavage by the neutrophil serine proteases cathepsin
G and elastase which may remove the N-terminal domain.[20]
[21] Since it is known that neutrophils are crucially involved in platelet aggregation
and in the formation and degradation of fibrin during thrombus deposition,[22] we hypothesized that their activation and protease release might also affect TSP-1
function in haemostasis or thrombosis where the so-called neutrophil extracellular
traps (NETs) are implicated.
NETs are a network of extracellular deoxyribonucleic acid (DNA) associated with histones
and neutrophil-derived proteins such as elastase and have recently been discovered
to contribute to thrombus formation.[23]
[24] During the process of NETosis DNA is decondensed by modification of histones, for
example, via citrullination by peptidylarginine deiminase 4 (PAD4) and subsequently
released to the extracellular space.[25] In addition to their proposed role in pathogen trapping and killing,[26] NETs were shown to be associated with various pathological conditions including
thrombosis.[27]
[28]
To address the question whether proteolytic processing of TSP-1 may alter its haemostatic
properties and hence affect thrombus formation, we (1) identified the proteases mediating
TSP-1 fragmentation in co-cultures of neutrophils with platelets or ECs. We (2) characterized
the generated TSP-1 isoform at the molecular level and (3) compared the original full-length
protein to the smaller isoform regarding their potency to promote platelet adhesion
and string formation.
Materials and Methods
Cells and Cell Culture
Human microvascular ECs were isolated from foreskin samples and purified as previously
described.[29] ECs were grown in EGM-2 MV medium (Lonza/Clonetics, Walkersville, Maryland, United
States) supplemented with 5% foetal calf serum (FCS), hydrocortisone, ascorbic acid,
basic human fibroblast GF, insulin-like GF 1, epidermal GF and gentamicin/amphotericin
GA-1000 (concentrations not specified). Attachment to cell culture dishes was promoted
by fibronectin at 1 µg/mL (EMD Millipore Corporation, Temecula, California, United
States). For all experiments, medium was switched to medium without serum to avoid
contamination with bovine TSP-1.
Human neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from
ethylenediaminetetraacetic acid (EDTA) whole blood using Histopaque density gradient
separation (#10771, #11191, Sigma-Aldrich, St. Louise, Missouri, United States). After
centrifugation, the neutrophil layer was aspirated and washed with Dulbecco's phosphate-buffered
saline Ca2+/Mg2+-free (PBSdef) which was followed by lysis of red blood cells and re-establishment
of isotonicity with 3% sodium chloride. Neutrophils were then re-suspended in cell
culture medium and only isolates with more than 90% purity as determined by the Sysmex
XN-350 haemocytometer (Sysmex, Kobe, Japan) were used for experiments. Platelets were
isolated via size exclusion with a Sepharose 2B (Sigma-Aldrich) column from whole
blood collected in citrate, theophylline, adenosine and dipyridamole tubes. Platelets
were washed with PBS and re-suspended in culture medium (containing 1 mM CaCl2, but without serum or GFs) for co-culture and stimulation by A23187 (Sigma-Aldrich)
or thrombin receptor activator peptide 6 (TRAP-6, BACHEM, Basel, Switzerland). To
inhibit platelet activation, prostaglandin E1 (Santa Cruz, Dallas, Texas, United States)
was added.
Co-Culture of Endothelial and Blood Cell Populations
ECs were seeded in 2 mL EGM-2 MV to reach confluence in 6 wells (5 × 105 cells/well) within 1 day. Cells were then washed twice with 2 mL PBSdef and 1 mL
EGM-2 MV w/o GFs and FCS was added. The cells were allowed to release TSP-1 into the
supernatant for 48 hours and subsequently a co-culture was performed. Per well, 20 × 106 platelets, 2 × 106 PBMCs or 2 × 106 neutrophils were applied. A total of 500 µL of the conditioned medium were removed
from the 6-well and replaced with platelets, PBMCs or neutrophils suspended in 500
µL EGM-2 MV w/o GFs and FCS for co-culture. Moreover, 500 µL of these cell isolates
were added to the retrieved 500 µL conditioned medium or to 500 µL EGM-2 MV w/o GFs
and FCS for control. Samples were either left untreated or were supplied with activating
stimuli. Platelets were activated with 10 µM TRAP-6, PBMCs with 1 µg/mL lipopolysaccharide
(LPS, Sigma-Aldrich) and neutrophils with 100 ng/mL phorbol myristate acetate (PMA,
Sigma-Aldrich). Alternatively, platelets (4 × 106) were co-cultured with neutrophils (4 × 105) without additional stimulus. The supernatant was collected after 0.5, 1, 2 and 4
hours for immunoblotting.
Furthermore, co-cultures were supplemented with inhibitors for the neutrophil proteases
cathepsin G and/or elastase (#219372, #324744, Merck KGaA, Darmstadt, Germany). Note
that 1 × 105 ECs or 4 × 106 platelets were combined with 4 × 105 neutrophils. Elastase inhibitor II (2.1 mM) and cathepsin G inhibitor I (0.1 mM)
were added either separately or combined. The supernatant was harvested after 30 minutes
of incubation.
TSP-1 Processing with Purified Neutrophil Proteases
Serum-free, conditioned EC supernatant containing 185 kDa TSP-1 was supplied with
purified, human neutrophil elastase at 10 to 50 mU/mL (SERVA Electrophoresis GmbH,
Heidelberg, Germany) or cathepsin G at 2 to 50 mU/mL (Merck KGaA) for 30 minutes to
4 hours at 37°C.
Immunoblotting
For sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, samples (culture
supernatants without further concentration or dilution) were mixed with loading buffer
containing 11.5% β-mercaptoethanol, 0.4 M dithiothreitol for reducing conditions,
but lacking β-mercaptoethanol and dithiothreitol under non-reducing conditions. Proteins
were subsequently transferred to a polyvinylidene fluoride membrane (Merck KGaA) overnight
at 20 or 35 V. Membranes were blocked and then incubated overnight at 4°C with primary
antibodies: mouse anti-TSP-1 Ab11 (D4.6 + A6.1 + MBC200.1, Thermo Fisher Scientific,
Waltham, Massachusetts, United States), C-terminus specific mouse anti-TSP-1 Ab4 (clone
A6.1, Thermo Scientific) and N-terminus specific goat anti-TSP-1 N20 (Santa Cruz)
in a 1:300 dilution or rabbit anti-histone H3 (citrulline R2 + R8 + R17) (Abcam, Cambridge,
United Kingdom) at 1:1,000 dilution. Thereafter, membranes were incubated for 1 hour
at 1:50,000 dilution of species-specific horseradish peroxidase-conjugated secondary
antibody (Thermo Fisher Scientific). After addition of the chemiluminescent reagent
(Lumigen, Inc., Southfield, Michigan, United States), membranes were exposed to X-ray
film and images were processed with Adobe Photoshop CS6.
NET Formation
NET experiments were based on a co-culture of neutrophils (1 × 105) and platelets (1 × 107). Cells were activated by the calcium ionophore A23187 (4 µM) in the absence or presence
of the NETosis inhibitor GSK484 at 2 mM (Cayman Chemicals, Ann Arbor, Michigan, United
States). Where indicated, 50 µM cathepsin G inhibitor and 1 mM elastase inhibitor
were added (concomitantly with GSK484), then incubated for 30 minutes before stimulation
with A23187. NETs generated by activated neutrophils were digested with 500 mU/mL
micrococcal nuclease (Sigma-Aldrich) for 10 minutes and culture supernatant was retrieved
by centrifugation at 5,000 × g for 90 seconds for immunoblotting of TSP-1 and citrullinated histone H3 (citH3) or
for elastase activity assays (BioVision, Milpitas, California, United States) conducted
according to the manufacturer's instructions.
DNA Release Assay
Neutrophils and platelets were seeded into black 96-well tissue culture plates. Prior
to activation with A23187, the cells were pre-incubated for 30 minutes with the PAD4
inhibitor GSK484 at 2 mM. After addition of 4 µM A23187 and 5 µM Sytox Green (Thermo
Fisher Scientific), the released, cell-free DNA was measured at 405 nm in a plate
reader (Varioskan Flash, Thermo Fisher Scientific) at intervals of 15 minutes over
a period of 6 hours.
In Vitro Platelet Aggregation and Adhesion Under Flow
Parallel flow chambers (µ-slide VI 0.4 Luer) of the ibidi pump system (ibidi, Munich, Germany) were coated with collagen (moeLab GmbH, Langenfeld, Germany) overnight.
Before the experiment, the flow chambers and the perfusion set were pre-perfused for
1 minute with mouse plasma 1:10 diluted with Tyrode's Hepes (TH) buffer supplemented
with 1 mM CaCl2, 0.5% bovine serum albumin and 10 mg/mL fibrinogen. Heparinized blood was drawn from
the vena cava of 8- to 12-week-old mice. A total of 500 µL of blood were either left
untreated or were supplied with 2 or 0.5 µg purified 160 or 185 kDa TSP-1 for 10 minutes.
Platelets were labelled by the addition of fluorescent anti-mouse GPIbβ antibody (emfret
Analytics, Eibelstadt, Germany) at 1:300 prior to 1:3 dilution of the whole sample
with TH buffer. Mouse blood was perfused through parallel flow chambers at a shear
rate of 7 dyne/cm2 for 7 minutes. Platelets were visualized with an IX83 microscope (Olympus, Tokyo,
Japan) and 10 images per sample were taken within 2 minutes with an Orca Flash 4.0
camera (Hamamatsu, Bridgewate, New Jersey, United States) using the CellSens Dimensions software (Olympus). Images were analysed for the number of adherent platelets, the
total area of platelet aggregates in 0.1 mm2 and for the number and mean length of platelet strings (after manual labelling of
the strings in red) using the Fiji-version of the ImageJ software: Composite colour images were split into red, green and blue channels and
the red channel was subjected to automated thresholding using the MaxEntropy algorithm followed by the ‘analyse particles’ routine using a minimum size of 50
pixels. The analysis of platelet strings is further illustrated in [Supplementary Fig. S7], available in the online version.
Mice deficient in TSP-1 and of C57Bl/6J background (B6.129S2-Thbs1 tm1Hyn/J) were
obtained from Charles River Laboratories, Sulzfeld, Germany. Wild-type (WT) mice (C57BL/6J)
were used for comparison.
Results
Platelets and ECs Release Full-Length 185 kDa TSP-1 but do not Generate the 160 kDa
Isoform
When human plasma samples are analysed for the presence of TSP-1 isoforms immediately
prior to and 1 day after surgery (resection of liver metastases), two distinct TSP-1
protein variants are detected ([Fig. 1A] and [Supplementary Fig. S1A], available in the online version) as we have previously reported.[6] While both the 185-kDa full-length protein and a smaller 160-kDa TSP-1 isoform are
present before surgical intervention, a predominance of the 160-kDa TSP-1 protein
is observed in the post-operative period involving haemostasis and the initiation
of wound healing.
Fig. 1 Thrombospondin-1 (TSP-1) isoforms circulating in human plasma as compared with TSP-1
proteins secreted by isolated platelets and endothelial cells (ECs). (A) Blood samples of two colorectal cancer patients were retrieved immediately before
(pre) and 1 day after (post) resection of liver metastases and plasma was processed
as previously described.[6] (B) Human platelets were isolated from whole blood of healthy volunteers by size exclusion
chromatography in the presence of 100 nM prostaglandin E1 (PGE1). They were then either left untreated (w/o), further inhibited with 100 nM PGE1 or activated with different concentrations A23187 (1–40 µM) for 30 minutes at 37°C
before collection of the supernatant. (C) TSP-1 released into the culture medium of human dermal microvascular endothelial
cells after 24 or 48 hours was analysed for confluent and untreated, tumor necrosis
factor-α (TNFα) (100 ng/mL) or lipopolysaccharide (LPS) (1 µg/mL) stimulated cultures
and compared with untreated sub-confluent (proliferating) ECs. Shown are representative
immunoblots (Ab11) with reduced protein samples of culture supernatants (without further
concentration or dilution). Please refer to [Supplementary Fig. S1] (available in the online version) for quantitation of immunoblots. Experiments were
repeated 3 to 4 times. M, biotinylated protein marker.
To identify the source of the smaller 160 kDa TSP-1 variant, isoform generation and
secretion of TSP-1 was investigated in platelets and ECs which constitute the main
producers of TSP-1 in blood. Isolated human platelets showed a dose-dependent release
of 185 kDa TSP-1 ([Fig. 1B] and [Supplementary Fig. S1B], available in the online version) when stimulated with increasing concentrations
of calcium ionophore A23187. Platelets also consistently secreted the full-length
185 kDa TSP-1 protein in response to other agonists such as adenosine diphosphate
or TRAP-6 (data not shown).
With respect to human microvascular ECs, a similar observation was made. Confluent
and sub-confluent cultures were compared, and confluent cells were activated with
tumor necrosis factor-α or LPS or were left untreated. Comparable to platelets, ECs
selectively released full-length 185 kDa TSP-1 into the supernatant which was increased
after pro-inflammatory stimulation of cells ([Fig. 1C] and [Supplementary Fig. S1C], available in the online version). Moreover, proliferating (sub-confluent) cells
also produced 185 kDa TSP-1 molecules. The generation of a 160-kDa TSP-1 isoform was
not observed under any of the experimental conditions.
The 160 kDa TSP-1 Isoform is Generated in Co-Cultures of Neutrophils with ECs or Platelets
We continued to investigate whether TSP-1 might be processed to the 160-kDa isoform
upon co-culture with other blood cell types. Addition of freshly isolated human platelets
or PBMCs to ECs and incubation for up to 4 hours consistently yielded the 185-kDa
TSP-1 protein, with increasing intensity throughout the incubation period ([Fig. 2A] and [B] and [Supplementary Fig. S2A] and [S2B], available in the online version). In contrast, when neutrophils were combined with
ECs or platelets a 160-kDa TSP-1 molecule was generated over time while the amount
of the full-length 185 kDa protein decreased ([Fig. 2C] and [D] and [Supplementary Fig. S2C] and [S2D], available in the online version). Addition of selected stimuli to activate blood
cells in the various co-cultures did not alter the type of TSP-1 isoform generated.
However, TSP-1 processing from 185 to 160 kDa was enhanced, when EC–neutrophil co-cultures
were stimulated with PMA, while platelet TSP-1 was instantly processed to 160 kDa
when neutrophils were added (presumably due to neutrophil activation in platelet co-culture)
and no further neutrophil stimulus was required. Incubation of the various blood cell
types with conditioned EC medium (supernatant of ECs grown for 48 hours) as opposed
to direct cell contact, yielded comparable results (data not shown). Concomitant control
cultures of isolated leukocytes for up to 4 hours revealed that PBMCs and neutrophils
(with or without cell activation) did not release substantial amounts of the TSP-1
glycoprotein during the incubation period ([Supplementary Fig. S4A] and [S4B], available in the online version).
Fig. 2 Thrombospondin-1 (TSP-1) isoforms generated in co-cultures of vascular cell populations.
Confluent endothelial cell (EC) cultures grown in serum-free medium for 48 hours were
either left untreated (ECs) or were supplied with freshly isolated human (A) peripheral blood mononuclear cells (PBMCs), (B) platelets or (D) neutrophils. Where indicated (+) a stimulus was added to activate PBMCs (lipopolysaccharide
[LPS] at 1 µg/mL), platelets (thrombin receptor activator peptide 6 [TRAP-6] at 10
µM) or neutrophils (phorbol myristate acetate [PMA] at 100 ng/ml). (C) Platelets were also combined with neutrophils or kept separately for control. Supernatant
was collected after 0.5, 1, 2 and 4 hours of culture. Reduced protein samples were
analysed by immunoblots with Ab11. Each experiment was repeated four times with blood
drawn from different donors. Please refer to [Supplementary Fig. S2] (available in the online version) for quantitation of immunoblots. M, biotinylated
protein marker.
The 160 kDa TSP-1 Fragment is Proteolytically Generated by Neutrophil-Derived Elastase
and Cathepsin G and Corresponds in Size to the Shorter Plasma Isoform
To test whether neutrophil-derived proteases are mediating TSP-1 processing to the
smaller isoform, co-cultures of neutrophils with ECs or platelets were performed in
the presence of inhibitors for the neutrophil proteases elastase and cathepsin G ([Fig. 3A] and [B] and [Supplementary Fig. S3A] and [S3B], available in the online version). When the inhibitors were given separately, processing
was partially inhibited leading to the concurrent appearance of both TSP-1 variants
(185, 160 kDa). Concomitant treatment with both inhibitors had a co-operative inhibitory
effect on proteolysis of the 185-kDa protein to the 160-kDa molecule.
Fig. 3 Thrombospondin-1 (TSP-1) processing by neutrophil-derived proteases. Freshly isolated
human neutrophils were added to (A) confluent, serum-free cultures of endothelial cells or (B) platelets isolated from the same donor and incubated for 30 minutes at 37°C in the
absence or presence of elastase inhibitor II (2.1 mM) and/or cathepsin G inhibitor
I (0.1 mM). (C–E) Serum-free endothelial cell (EC) supernatant containing 185 kDa TSP-1 (w/o) was
incubated with the purified proteases (C) cathepsin G at 10 mU/mL or (D) elastase at 20 mU/mL for 30 minutes to 4 hours, or (E) was exposed to increasing concentrations of elastase (10–50 mU/mL) and cathepsin
G (2–50 mU/mL) for 30 minutes at 37°C. (F) The 160-kDa TSP-1 proteins generated either by co-culture of ECs with neutrophils
or by 30-minute elastase digest of EC supernatant were compared with the smaller TSP-1
isoform prevalent in human plasma post-surgery. TSP-1 protein was detected in supernatants
by immunoblotting with Ab11. Experiments were performed at least three times with
blood from different donors. Please refer to [Supplementary Fig. S3] (available in the online version) for quantitation of immunoblots. M, biotinylated
protein marker.
Fig. 4 Molecular characterization of the 160-kDa thrombospondin-1 (TSP-1) isoform. Serum-free
endothelial cell (EC) supernatant was either left untreated (w/o) or was digested
with elastase (20 mU/mL) or cathepsin G (10 mU/mL) for 30 minutes at 37°C. (A) TSP-1 was detected in reduced protein samples by immunoblotting with two distinct
antibodies, specific for the N-terminal or the C-terminal domain of TSP-1. (B) Reduced and non-reduced protein samples were compared on a gradient gel (4–20%)
and TSP-1 was immunostained with Ab11, a combination of three monoclonal antibodies
covering N- and C-terminal epitopes. (C) Illustration of the cathepsin G cut site (arrow) in the TSP-1 protein sequence as
determined by mass spectrometry and Edman sequencing refers to amino acid numbering
of secreted TSP-1 (not including the signal peptide); asterisks mark the cysteines
involved in inter-chain disulphide bonds for trimerization. TSP-1 cleavage by cathepsin
G results in the release of the monomeric N-terminal HBD (25 kDa) and a trimeric C-terminal
core fragment of 160 kDa chains. (D) Silver staining of a synthetic peptide (comprising TSP-1 amino acids 208–247, bold
letters in panel C) without or with digest by cathepsin G (50 mU/mL for 45 minutes).
In the next step, processing of TSP-1 by neutrophil-derived proteases was confirmed
with purified enzymes. EC supernatant containing full-length TSP-1 was incubated with
cathepsin G or elastase revealing fragmentation of 185 to 160 kDa TSP-1 by the neutrophil
proteases in a time-dependent ([Fig. 3C] and [D] and [Supplementary Fig. S3C] and [S3D], available in the online version) and dose-dependent ([Fig. 3E] and [Supplementary Fig. S3E], available in the online version) manner. While cathepsin G consistently yielded
the 160-kDa TSP-1 isoform, elastase additionally generated 140 kDa TSP-1 fragments
after prolonged incubation or at high enzyme concentrations. Of note, when 160 kDa
fragments generated by protease digest or produced in co-culture of ECs with neutrophils
were compared with a post-surgical human plasma sample, proteins were found to match
in molecular weight ([Fig. 3F] and [Supplementary Fig. S3F], available in the online version).
Neutrophil-Mediated Proteolysis of TSP-1 Removes the N-Terminus and Yields a Trimeric
Core Fragment of 160 kDa Chains
To elucidate which domains of TSP-1 were removed by neutrophil-derived proteases,
immunoblotting was performed with antibodies specific to N-terminal or C-terminal
epitopes of TSP-1. While the antibody specific for the C-terminus detected both the
185- and 160-kDa TSP-1 isoform, the N-terminus specific antibody revealed the full-length
TSP-1 and a smaller 25 kDa N-terminal fragment which was removed after processing
of TSP-1 with elastase or cathepsin G ([Fig. 4A] and [Supplementary Fig. S4C], available in the online version). Furthermore, TSP-1 isoforms were analysed with
intact or reduced disulphide bonds ([Fig. 4B]). In reduced samples, the original 185 kDa protein was confirmed for EC supernatant
which was processed to 160 kDa when cathepsin G was added. Under non-reducing conditions,
both the untreated and cathepsin G-treated samples showed high molecular weight molecules
migrating at the upper edge of the resolving gel with an estimated molecular weight
of > 400 to 450 kDa, thus pointing to trimeric complexes. Based on these analyses,
we concluded that the 160-kDa TSP-1 fragments resulting from cleavage by neutrophil
proteases were lacking the N-terminus and likely to form a trimeric core molecule
encompassing the inter-chain disulphide bonds.
To identify the exact cut-site of cathepsin G in TSP-1, the 160-kDa TSP-1 fragment
generated after protease digest was analysed by mass spectrometry and by Edman protein
sequencing. Mass spectrometry was able to narrow the potential cut site of cathepsin
G to the area of amino acids 213 to 244 (of the secreted TSP-1 molecule, not including
the signal peptide sequence) which is N-terminal of the connecting region. Edman sequencing
revealed the specific cut site to be located between amino acid R237 and T238 ([Fig. 4C]). Moreover, the cut site was confirmed by cathepsin G cleavage of a synthetically
generated 40 amino acid peptide covering the TSP-1 region of interest ([Fig. 4D]).
NET Formation Promotes the Controlled Processing of TSP-1 by Neutrophil-Derived Proteases
Since NET formation has recently been implicated in thrombosis, we further assessed
whether the release of NETs by activated neutrophils may have an impact on the proteolytic
processing of TSP-1. Thus, co-cultures of platelets and neutrophils were either left
untreated or were activated with the calcium ionophore A23187, a potent trigger of
NET formation. Furthermore, the NETosis inhibitor GSK484 (interfering with histone
citrullination by PAD4) was added to the co-cultures prior to addition of the stimulus
to allow for comparable neutrophil activation in the absence of NET formation. Release
of neutrophil DNA was assessed in a time course by Sytox Green incorporation, showing
a strong increase of free DNA in the supernatant after co-culture treatment with A23187
([Fig. 5A]). Addition of GSK484 was able to block DNA release to a level close to untreated
co-cultures. To test whether the NETosis inhibitor GSK484 still allows for the activation
of neutrophils and thus the release of neutrophil proteases, elastase was measured
in culture supernatant by activity assay ([Fig. 5B]). While A23187 triggered substantial elastase release, an even higher amount of
elastase activity was detected in the presence of the NETosis inhibitor thus reflecting
potent neutrophil activation and degranulation with release of soluble substances
into the supernatant.
Fig. 5 Impact of neutrophil extracellular trap (NET) formation on neutrophil-mediated proteolysis
of thrombospondin-1 (TSP-1). Co-cultures of neutrophils and platelets were left untreated
or stimulated with 4 µM A23187 in the absence or presence of the NETosis inhibitor
GSK484 at 2 mM. (A) Release of neutrophil deoxyribonucleic acid (DNA) was assessed by incorporation
of Sytox Green dye and measurement of relative fluorescence units (RFUs) over 5 hours.
(B) Elastase was evaluated by activity assay. Supernatant was retrieved after 0, 30,
60 and 120 minutes. (C) Occurrence of TSP-1 isoforms (upper panel) and citrullination of histone H3 (lower
panel) was determined at 120 minutes by immunoblotting. (D) Co-cultures seeded on cover slips were left untreated (w/o), stimulated by 4 µM
A23187 for NET formation or exposed to A23187 in the presence of 2 mM GSK484 to block
NETosis. After fixation and permeabilization, cultures were stained for TSP-1 (red),
the granulocyte marker CD66b (green) and DNA (blue). Scale bar: 20 µm. All experiments
were repeated at least three times with cells isolated from different human donors.
Immunoblots and fluorescence images depict one representative experiment, while DNA
release and elastase activity data are given as mean and standard deviation of three
independent experiments.
The occurrence of TSP-1 isoforms and citH3, as a marker of NETosis, was determined
in the supernatant of co-cultures by immunoblotting ([Fig. 5C]). Citrullination of histone H3 was not detectable for untreated neutrophils or platelets
that were cultured separately. Neutrophils in co-culture with platelets produced low
amounts of citH3 which was effectively increased by A23187 stimulation. This effect
was blocked through the addition of the NETosis inhibitor GSK484. Immunoblot results
were further confirmed ([Supplementary Fig. S5A], available in the online version) with a citH3 enzyme-linked immunosorbent assay.[30]
Regarding TSP-1 isoforms, platelets secreted the full-length 185 kDa molecule, which
was processed to 160 kDa in the presence of neutrophils as we had previously observed.
Activation by the calcium ionophore A23187 and thus NET formation was able to promote
TSP-1 release and processing resulting in increased levels of the 160 kDa TSP-1 isoform.
Of interest, inhibition of NETosis by GSK484 led to the total loss of TSP-1 over 2
hours ([Fig. 5C]), presumably due to degradation by the massive release of proteases from activated
neutrophils in the absence of NETosis ([Fig. 5B]). This notion was confirmed when we limited GSK484 exposure to 60 minutes and observed
increased TSP-1 proteolysis which was efficiently blocked by inhibitors against cathepsin
G and elastase ([Supplementary Fig. S5B], available in the online version). To address whether these effects would also hold
true for other, more physiological stimuli of NETosis and for other TSP-1 sources,
we investigated the fate of TSP-1 processing when NETs were comparably induced in
platelet–neutrophil co-cultures by LPS ([Supplementary Fig. S6A] and [S6B], available in the online version) or in endothelial–neutrophil co-cultures by A23187
([Supplementary Fig. S6C] and [S6D], available in the online version). We confirmed that NETs were induced (DNA was
released) which was associated with the enhanced secretion and controlled processing
of TSP-1 to the 160-kDa isoform, while NET blockade resulted in TSP-1 loss.
To investigate the localization of TSP-1 during NET formation, co-cultured platelets
and neutrophils were immunostained for the neutrophil marker CD66b, the platelet marker
CD41 and for TSP-1 to be analysed by confocal fluorescence microscopy ([Fig. 5D] and [Supplementary Fig. S5C], available in the online version). DNA in neutrophil nuclei and NET structures was
visualized by Hoechst 33342 dye. Unstimulated neutrophils showed intact lobulated
nuclei and CD66b cell surface staining. TSP-1 was mostly found to co-localize with
CD41 positive platelets which were randomly distributed and clearly separated from
unstimulated neutrophils. Neutrophils activated by calcium ionophore flattened out
showing less lobulated, enlarged nuclei and decondensed chromatin as well as formation
of NETs with extracellular DNA. A23187 treatment triggered local enrichment of TSP-1
in NET structures. When the inhibitor GSK484 was added prior to stimulation of co-cultures
with A23187, neutrophil nuclei appeared intact and no NET structures were apparent.
In this setting, TSP-1 was not associated with neutrophils but found in/on remaining
platelets. Whether TSP-1 was partly degraded could not be deduced from the analysis,
since the applied antibody was raised against a short TSP-1 peptide sequence.
The Proteolytically Processed 160 kDa TSP-1 Isoform has Enhanced Potency to Promote
Platelet Adhesion and String Formation Under Flow
Ultimately, it was pertinent to assess possible functional consequences of neutrophil-mediated
TSP-1 processing from the full-length 185 kDa molecule to the 160 kDa isoform regarding
the haemostatic properties of TSP-1. Whole blood retrieved from TSP-1 knockout (KO)
mice (as compared with WT mice) was analysed with respect to platelet adhesion, aggregation
and string formation under in vitro flow conditions ([Fig. 6A], [Supplementary Fig. S7 ] and [Supplementary Videos A–D], available in the online version). In line with the previously reported TSP-1 functions
in promoting platelet attachment to collagen and protecting vWF strings from degradation,
the number of adherent platelet aggregates and the mean length of formed platelet
strings were significantly higher for WT than for TSP-1 KO mice ([Fig. 6B] and [C]). To be able to compare the two TSP-1 isoforms in function, commercially obtained,
platelet-purified 185 kDa TSP-1 was treated with cathepsin G to yield the 160-kDa
fragment, and the reaction was subsequently stopped by adding cathepsin G inhibitor.
Conversely, when the inhibitor was given prior to the addition of cathepsin G, TSP-1
remained intact. Substitution of TSP-1 KO blood with the two purified TSP-1 isoforms
at high concentration (4 µg/mL) could restore platelet adhesion and string formation
to the WT situation. However, at lower concentrations (1 µg/mL) the processed 160
kDa isoform was significantly more potent than the original 185 kDa molecule, showing
a higher number of adherent platelet aggregates and longer platelet strings. These
results were confirmed with recombinantly generated TSP-1 isoforms ([Supplementary Fig. S8], available in the online version).
Fig. 6 Comparison of thrombospondin-1 (TSP-1) isoforms in promoting platelet adhesion and
string formation on collagen under flow. Blood retrieved from wild-type (WT) or TSP-1
knockout (KO) mice was supplied with anti-GPIbβ antibody to fluorescently label platelets
and was perfused over collagen-coated slides in an ibidi flow chamber for 7 minutes at 7 dyne/cm2. TSP-1 KO blood was either left untreated or was substituted with 1 or 4 µg/mL of
purified 160 or 185 kDa TSP-1 protein. (A) Representative microscopic images of the different treatment groups were taken after
7 minutes (under continuing flow). (B) The number of adherent platelet aggregates and (C) the mean length of formed platelet strings were analysed with Fiji software as outlined in [Supplementary Fig. S7 (] available in the online version). Boxplots illustrate the data distribution of 5
to 9 independent experiments (representing blood from individual mice); statistically
significant differences between groups were assessed by Mann–Whitney U test (SPSS
23.0).
Supplementary Videos
Comparison of thrombospondin-1 (TSP-1) isoforms in promoting platelet adhesion and
string formation on collagen under flow. Blood retrieved from wild-type (A) or TSP-1 knockout (KO) mice was supplied with anti-GPIbβ antibody to fluorescently
label platelets and was perfused over collagen-coated slides in an ibidi flow chamber for 7 minutes at 7 dyne/cm2. TSP-1 KO blood was either (B) left untreated or was substituted with 1 µg/mL of purified (C) 160 kDa or (D) 185 kDa TSP-1 protein. Time series were recorded with an Olympus IX83 inverted microscope.
Images were captured every 10 seconds over 5 minutes in total, using a 40× objective
and the Hamamatsu Orca Flash 4 sCMOS camera. Online content including video sequences
viewable at: https://www.thieme-connect.de/products/ejournals/html/10.1055/s-0038-1675229
Discussion
The extracellular matrix protein TSP-1 is susceptible to processing by various proteases
generating fragments of different length and function. For example, the matrix metalloproteinase
ADAMTS-1 was found to cleave TSP-1 between E293 and L294 (amino acid numbering without
signal peptide) releasing a trimeric N-terminus of 36 kDa and monomeric C-terminal
fragments of 110 to 125 kDa with enhanced anti-angiogenic properties.[16] Bonnefoy and Legrand reported in 2000 that TSP-1 secreted by human umbilical vein
ECs is processed upon the addition of plasmin, cathepsin G and elastase to fragments
of comparable size.[21] In this study, we were able to extend the observations by Bonnefoy and Legrand identifying
cathepsin G and elastase as being responsible for TSP-1 processing from a full-length
185 kDa protein to 160 kDa fragments in a co-culture setting of ECs or platelets with
neutrophils. Moreover, processing was blocked by inhibitors for the neutrophil proteases.
The processed 160 kDa TSP-1 fragments were found to be trimeric, lacking the 25-kDa
N-terminal HBD, and matched in size with TSP-1 molecules found in post-operative human
plasma. The exact cut site of cathepsin G was identified to reside between R237 and
T238.
Of interest, TSP-1 has previously been shown to act as a competitive inhibitor of
plasmin, elastase and cathepsin G, blocking their proteolytic activity upon their
binding to the TSP-1 type 3 domain.[31] While these investigations elucidated the effects of TSP-1 on protease activity,
our study focused on the impact of proteolytic processing on TSP-1 function, in particular
in haemostasis. TSP-1 is known to contribute to platelet aggregation by forming a
bridge between platelet-bound fibrinogen and integrin and by protecting vWF from cleavage
by ADAMTS-13, thus promoting thrombus formation.[17]
[19] More recently, Kuijpers et al showed that TSP-1 binding to CD36 supports platelet
adhesion and thrombus stability on collagen.[32] While these studies did not evaluate TSP-1 fragmentation during haemostasis, earlier
investigations by Rabhi-Sabile et al reported that smaller (165 kDa) TSP-1 molecules
lacking the N-terminus remained platelet-bound after cathepsin G induced platelet
aggregation.[20] Based on the application of blocking antibodies, they concluded that these TSP-1
fragments retained function in platelet aggregation, but they did not address or reveal
a functional gain of the shorter isoform. We have now compared the full-length TSP-1
molecule and the processed 160 kDa protein for their potency to promote platelet adhesion
and string formation. The experimental design was generally based on whole blood or
purified platelets from WT versus TSP-1 KO mice, to be reconstituted with purified
TSP-1 isoforms for functional comparison. However, in commonly applied assays such
as rotational thromboelastometry, a cone and plate analyser (Impact-R) or light transmission
aggregometry (PAP8, moeLab GmbH, [Supplementary Fig. S5], available in the online version), platelet aggregation did not differ significantly
between blood samples from WT versus TSP-1 KO mice and hence could not be rescued
by the addition of purified TSP-1 isoforms. These data are in accordance with Lawler
et al who showed that platelets of TSP-1 KO mice had normal aggregation.[33] However, Isenberg et al reported in 2008 that TSP-1 acts as an antagonist of NO
signalling to promote platelet aggregation which necessitates the addition of NO donors
during in vitro investigations.[34] Thus, we added the NO donor diethylamine NONOate in light transmission aggregometry
experiments and could confirm the delaying effect of NO on the thrombin-induced aggregation
of WT as well as TSP-1 KO platelets ([Supplementary Fig. S9], available in the online version). Of note, there was no difference between WT and
KO, and the addition of purified TSP-1 had no impact.
We then switched from static to flow conditions and evaluated adhesion, aggregation
and string formation of platelets in an in vitro flow chamber. In this setting, platelets
from TSP-1 KO mice showed severely impaired function pointing to a predominant role
of TSP-1 in promoting platelet adhesion on collagen and protecting vWF strings under
flow. Moreover, we could restore these functions by the addition of purified TSP-1
to blood samples of TSP-1 KO mice. Importantly, the 160-kDa fragment was significantly
more potent than the full-length 185 kDa protein at limiting TSP-1 concentrations
of 1 µg/mL, whereas both molecules restored platelet adhesion and string formation
at higher protein levels (4 µg/mL). While constitutive TSP-1 plasma levels are comparably
low and range at 20 to 40 ng/mL, the local concentration may be expected to increase
substantially after platelet activation, as indicated by TSP-1 serum values of 1 to
10 µg/mL after complete in vitro platelet activation.[7] Thus, neutrophil-mediated TSP-1 proteolysis may serve to enhance the haemostatic
properties of platelet- and EC-released TSP-1 under early or limiting conditions during
thrombus formation.
Since both the number of adherent platelet aggregates on collagen and the average
length of formed platelet strings differed significantly between blood samples reconstituted
by 160 versus 185 kDa TSP-1 protein, the processing of TSP-1 may affect CD36-mediated
platelet binding to collagen as well as vWF protection from degradation. Of note,
both functions are attributed to the type I repeats and might be facilitated by more
accessible domain interactions after HBD removal. Alternatively, the loss of the N-terminal
domain rather than the molecular change of the remaining core molecule may account
for the functional difference between isoforms, since the TSP-1 HBD is known to provide
a variety of interaction sites for platelet surface molecules.[35] Thus, TSP-1 may be more stable after removal of the N-terminus, which has previously
been implicated in protein internalization and degradation of TSP-1 by binding to
the low-density lipoprotein receptor-related protein.[36]
In addition to fostering vWF strings, TSP-1 is known to have a propensity for self-polymerization
and cross-linking to other proteins by thiol-disulphide exchange.[31]
[37] Since this might constitute another mechanism for enhanced platelet adhesion and
string formation, we triggered TSP-1 polymerization by calcium depletion as previously
described[38] and then compared TSP-1 multimers by SDS-agarose discontinuous gel electrophoresis
([Supplementary Fig. S10], available in the online version). Interestingly, TSP-1 polymerization could be
induced for the platelet-purified 185 kDa TSP-1 protein, but not for the cathepsin
G-generated 160 kDa isoform. Comparably, recombinantly produced 160 or 185 kDa TSP-1
did not undergo polymerization upon EDTA treatment, eliminating this TSP-1 feature
as the potential mechanism accounting for the enhanced haemostatic properties of the
160-kDa isoform.
We further extended our analysis to the role of NETs in TSP-1 proteolysis, since NET
formation was discovered to affect haemostasis and play a particular role in thrombosis.[27] When we induced NET formation in co-cultures of platelets and neutrophils, TSP-1
was highly released and entirely processed to 160 kDa. In contrast, inhibition of
NETosis resulted in rapid proteolysis and complete degradation of TSP-1 which is likely
due to the excessive release of neutrophil-derived proteases, as it was blocked by
inhibitors of cathepsin G and elastase. These data suggest that NET formation promotes
processing of TSP-1 to 160 kDa in a controlled manner, protecting TSP-1 from further
degradation by neutrophil proteases in the NET environment which might be of specific
relevance in pathophysiological settings. Of note, NET-guided TSP-1 proteolysis also
occurred in the presence of human plasma (data not shown) which is a central regulatory
element of protease activity under physiological conditions.
In summary, the contact or concomitant activation of neutrophils and platelets/ECs
results in the release of TSP-1 molecules which are processed by neutrophil proteases
to a trimeric 160 kDa molecule lacking the N-terminal domain. NET formation supports
the generation of the smaller TSP-1 isoform and protects the protein from further
degradation. Importantly, the 160-kDa TSP-1 fragment shows enhanced potency to promote
platelet adhesion on collagen and string formation under flow. This finding reveals
a novel mechanism by which neutrophils may support thrombus formation at the site
of vessel injury and provides first evidence for the impact of TSP-1 proteolysis on
its haemostatic properties. Furthermore, neutrophil-mediated processing of TSP-1 might
also have clinical implications, in particular with respect to thrombosis. A gene
polymorphism (N700S) of TSP-1 has previously been identified to be associated with
an increased risk for myocardial infarction[39] and the S700 TSP-1 variant was found to be more susceptible to in vitro digest by
the protease trypsin and to exhibit enhanced platelet aggregation properties when
compared with the N700 protein.[40] Of interest, the proposed binding site of TSP-1 for cathepsin G and elastase[41] is in proximity to TSP-1 amino acid 700 which led us to compare the N700 and S700
TSP-1 variants for their susceptibility to proteolytic cleavage by neutrophil proteases
and the functional impact on platelet string formation. The recombinant, full-length
185 kDa TSP-1 molecule engineered to carry serine in position 700 was markedly more
susceptible to cathepsin G digest and also more potent than the N700 variant in promoting
platelet adhesion and string formation in whole blood, comparable to the processed
160 kDa isoform ([Supplementary Fig. S8], available in the online version). This may indicate that excessive neutrophil-mediated
proteolysis of TSP-1 might favour pathological processes such as thrombosis and warrants
further investigation.
Limitations of the Study
While the applied cathepsin G inhibitor (Merck #219372) is highly selective and only
weakly inhibits other tested proteases such as plasmin, elastase or proteinase 3 (as
reflected in 1,000-fold higher IC50 values), the elastase inhibitor (Merck #324744) has a broader spectrum of affected
enzymes. It has poor reactivity with cathepsin G but may inhibit neutrophil proteinase
3.[42]
[43] Hence, we cannot entirely exclude an additional contribution by proteinase 3 to
TSP-1 processing, but would like to emphasize that purified cathepsin G or elastase
were sufficient to generate the 160-kDa isoform.
Regarding measurement of platelet adhesion and string formation under flow, we gave
preference to heparin over citrate or EDTA for anticoagulation of mouse blood, because
the structure and function of TSP-1 are known to be dependent on calcium.[44] Of note, the 185- and 160-kDa TSP-1 isoforms exhibited comparable affinity for heparin
in vitro (data not shown) which may possibly relate to complex formation between TSP-1,
cathepsin G and heparin or may be conferred by the heparin binding sites within the
type I repeats[45] which are retained in both the 160- and 185-kDa TSP-1 isoform and constitute the
TSP-1 region mediating platelet adhesion to collagen as well as vWF protection. However,
we cannot exclude the possibility that the presence of heparin in mouse blood selectively
limits the function of full-length 185 kDa TSP-1 in platelet adhesion and string formation
by binding to the N-terminal domain (not present in the shorter 160 kDa isoform).
What is known about this topic?
-
The full-length 185 kDa TSP-1 protein is released by activated platelets and ECs as
a homotrimer.
-
It promotes platelet aggregation as well as platelet adhesion to collagen and protects
von Willebrand factor strings from degradation.
What does this paper add?
-
TSP-1 is rapidly processed to a shorter 160 kDa isoform by neutrophil-derived proteases
which is significantly more potent than the original 185 kDa TSP-1 protein in promoting
platelet adhesion to collagen and formation of platelet strings.
-
This is the first study showing an impact of controlled TSP-1 proteolysis on its haemostatic
properties and reveals a novel mechanism by which neutrophils promote primary haemostasis.
