From the Institute for Medicine and Engineering, Department of Chemical Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, Philadelphia, Pennsylvania 19104
Received for publication, November 22, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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Human neutrophil proteases cathepsin G and
elastase can directly alter platelet function and/or participate in
coagulation cascade reactions on the platelet or neutrophil surface to
enhance fibrin formation. The clotting of recalcified platelet-free
plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn
trypsin inhibitor (to shut down contact activation) was studied in
well-plates or flow assays. Inhibitors of cathepsin G or elastase
significantly delayed the burst time (t50) of
thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced
neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets
under flow conditions. In flow assays, purified cathepsin G was a far
more potent activator of platelet-dependent coagulation
than elastase. Anti-tissue factor had no effect on neutrophil
protease-enhanced thrombin formation in PRP. The addition of cathepsin
G (425 nM) or convulxin (10 nM) to PRP
dramatically reduced the t50 of thrombin
generation from 53 min to 17 or 23 min, respectively. In contrast, the
addition of elastase to PRP left the t50
unaltered. Whereas perfusion of PFP ( The triggered display of platelet procoagulant activity is the
major requirement for effective blood coagulation. In the absence of
tissue factor or collagen (i.e. biomaterial thrombosis or
dysfunctional endothelium), the neutrophil and its released proteases
human neutrophil elastase and cathepsin G (Cat
G)1 may help initiate
platelet-dependent thrombin generation by various potential
mechanisms. Both Cat G and human neutrophil elastase are serine
proteinases of the chymotrypsin family.
Cat G can cleave the plasma zymogens factor V and factor X as well as
activate spread human platelets via protease-activated receptor 4 with
consequent calcium mobilization (1-3). Cat G treatment of platelets
increases surface presentation of GPIV, P-selectin, and active
GPIIb/IIIa (4). Elastase can cleave factor V (1) and potentially
facilitate de-encryption of tissue factor in blood (5) by cleaving
tissue-factor pathway inhibitor (6). De-encrypted tissue factor may
function on the platelet surface to initiate coagulation (7).
Additionally, Cat G can degrade neutrophil P-selectin glycoprotein
ligand-1 (8) and platelet GPIb without an effect on platelet P-selectin
(9, 10).
The sequence of platelet capture, translocation, and arrest to a
surface through interactions mediated by glycoproteins GPIb-V-IX, GPIIb/IIIa, and GPIa/IIa (11-13) is followed by spreading and
activation. GPVI, which is associated with the Fc receptor Neutrophil-platelet interactions during adhesion and heterotypic
aggregation have been associated with the enhancement of thrombosis.
Using a Dacron graft thrombosis model, Palabrica et al. (20)
observe that diminished neutrophil accumulation in the presence of
anti-P-selectin was also accompanied by reduced fibrin deposition.
Circulating levels of neutrophil-platelet complexes have been found to
increase in hip arthroplasty patients (21), a group prone to deep vein
thrombosis. In a different study, recombinant P-selectin glycoprotein
ligand immunoglobulin reduced experimentally induced venous thrombosis
(22).
However, the relative roles of human neutrophil elastase and Cat G and
the prioritization of various mechanisms through which these proteases
promote coagulation under flow conditions has not been well studied.
Although neutrophil elastase and cathepsin G can activate platelets
and/or participate in coagulation cascade reactions directly, their
activity may be limited by the presence of antiproteases present in
plasma (23). Our previous studies (24) have focused on the effects of
neutrophil-platelet interactions on subsequent fibrin formations under
venous flow conditions. We demonstrated that individual neutrophils
alone can accelerate fibrin deposition if factor XIIa is present due to
contact activation by a CD18-dependent mechanism attenuated
with inhibitors of elastase or cathepsin G. In the presence of corn
trypsin inhibitor (CTI) to block factor XIIa, neutrophils promote
fibrin formation on fibrinogen-adherent platelets through pathways
attenuated by inhibitors of human neutrophil elastase and Cat G.
In this study, we investigated the mechanisms by which neutrophil
proteases alter platelet function. Cathepsin G, more so than elastase,
plays the major role in turning fibrinogen-adherent platelets
procoagulant. Cathepsin G does so by elevating the activation state of
fibrinogen-adherent platelets rather than by cleavage of plasma
zymogens factor X and factor V in plasma. Relative rates of thrombin
generation for various coagulation scenarios have been measured under
static conditions to complement the videomicroscopy-imaged flow assays
aimed at understanding the role of neutrophils in thrombosis under
venous hemodynamic conditions.
Materials--
Human neutrophil elastase, human cathepsin G,
human thrombin, and calf skin collagen (Calbiochem), human serum
albumin (Golden West Biologicals, Temecula, CA),
N-formyl-Met-Leu-Phe (fMLP), bovine brain
L- Cell Isolation--
Human blood was collected from healthy adult
donors by venipuncture and anticoagulated with sodium citrate (9 parts
blood to 1 part sodium citrate). Neutrophils were isolated by
centrifugation over neutrophil isolation medium (Robbins Scientific) as
previously described (24, 25). Platelet-rich plasma (PRP) was obtained by centrifugation of anticoagulated whole blood at 130 × g for 15 min. Platelet singlets were prepared by gel
filtration (24). After isolation, neutrophils or platelets were diluted
to final concentrations of 106 or 108
cells/ml, respectively.
Microcapillary Flow Chambers--
Rectangular glass capillaries
(Vitrocom, Mountain Lakes, NJ) with a cross-section of 0.2 × 2.0 mm, a length of 7 cm, and a wall thickness of 0.15 mm were used as flow
chambers as previously described (24, 25). To enable adhesion of
neutrophils or platelets microcapillary flow chambers were incubated
with human fibrinogen solution (100 µg/ml) for 120 min at room
temperature or with calf skin collagen (100 µg/ml) for 4 h at
4 °C. The chambers were rinsed, and cells were allowed to adhere
under no-flow conditions as described previously (24). In selected
experiments, adhesion of platelets was preceded by their treatment with
human neutrophil elastase (10 µg/ml) or cathepsin G (10 µg/ml).
Platelet-free plasma treated with CTI (50 µg/ml) was perfused into
the flow chambers containing defined surface compositions at a
controlled flow rate using a syringe pump (Harvard Apparatus). The wall
shear stress ( Fluorogenic Measurement of Thrombin Generation--
Human
neutrophil elastase (11.5 µg/ml; 425 nM), cathepsin G (10 µg/ml; 425 nM), factor VIIa (2 nM),
anti-tissue factor (50 µg/ml), anionic phospholipid vesicles (50 µM), and/or convulxin (10 nM) were mixed with
recalcified citrated platelet-rich plasma supplemented with factor X
(170 nM) and CTI (50 µg/ml) in a 96-well plate.
Fluorogenic substrate for thrombin (tboc-Val-Pro-Arg-MCA; 20 µM) was added to the reaction mixture (150 µl/well) for
kinetic measurements of thrombin generation. In selected experiments, isolated neutrophils (106 cells/ml), fMLP (20 µM), and elastase inhibitor (100 µM,
MeOSuc-Ala-Ala-Pro-Ala-CMK) or cathepsin G inhibitor (100 µM, Z-Gly-Leu-Phe-CMK) were added to the reaction
mixture. To prepare vesicles,
L- Fibrin Formation on Fibrinogen-adherent Platelets; Role of Platelet
Activation--
Recalcified citrated PFP containing CTI (contact
pathway inhibitor of factor XIIa (24, 26)) was perfused over three
different surfaces, fibrinogen-adherent platelets, convulxin-treated
fibrinogen-adherent platelets, and collagen-adherent platelets (Fig.
1) at a wall shear rate of 62.5 s Enhancement of Platelet Procoagulation; Role of Neutrophil
Proteases--
When fibrin formation (
To quantify the effect of neutrophil-platelet interactions on platelet
procoagulant activity under static conditions, thrombin (factor IIa)
activity in recalcified citrated PRP (supplemented with factor X and
CTI) was compared in the absence or presence of neutrophils. Also, the
roles of fMLP-stimulation and inhibition of elastase or cathepsin G on
the dynamics of thrombin generation were examined (Fig.
3). Consistent with the flow experiments, peak thrombin generation (t50, the time to reach
50% thrombin substrate conversion) by PRP was sped up by neutrophils
(13 min faster) and was significantly delayed when a specific peptide inhibitor of cathepsin G (10-min lag) or elastase (28-min lag) was
added to fMLP-stimulated neutrophils in PRP. The 13-min difference in
t50 from the columns labeled N and
No N (Fig. 3) was not statistically different (at
n = 3). However, this is due to the large standard deviation in the mean t50 in the absence of
neutrophils, primarily induced by the t50 value
from the last trace. To clarify the situation, we repeated the
experiment and observed that introduction of more data points (not
shown) along with donor variation alters the difference between mean
t50 in columns labeled N and No
N to 11 min, which is statistically significant (p < 0.1, n = 6). Thrombin generation in PRP with
neutrophils present was not further influenced by fMLP. Interestingly,
inhibition of cathepsin G, although delaying the time to reach maximal
thrombin activity, caused a significant 3-fold increase in the maximum
thrombin production rate, presumably due to protection of GPIb from
degradation (9, 10).
In separate experiments, the addition of either cathepsin G inhibitor
or elastase inhibitor to neutrophils, which were not treated with fMLP,
in PRP delayed thrombin production by 14 min (p < 0.05) and 18 min (p < 0.025), respectively. The mean
t50 in the presence of neutrophils alone,
neutrophils with cathepsin G inhibitor, and neutrophils with elastase
inhibitor (all in PRP, no fMLP) were 57 ± 9 (n = 6), 71 ± 9 (n = 3), and 75 ± 8 (n = 3), respectively. Preincubation of neutrophils
with adherent platelets for 45 min in the absence of plasma (which
contains Effect of Cathepsin G and Elastase on Fibrin Deposition under Flow
Conditions--
To test which of the neutrophil proteases (elastase or
cathepsin G) played the most critical role in turning platelets
procoagulant, fibrin formation on fibrinogen-adherent platelets
(
To examine if cathepsin G turns platelets procoagulant through cell
activation or through zymogen cleavage of plasma factors on the
platelet surface, platelets were treated with different concentrations
of cathepsin G (10, 1, or 0.1 µg/ml) in separate flow chambers for a
short duration of 5 min. Cathepsin G treatment of platelets was then
followed immediately by 5 min of buffer perfusion with the peptide
inhibitor of cathepsin G (100 µM) and subsequent
perfusion of recalcified PFP/CTI supplemented with the peptide
inhibitor (100 µM). Although no fibrin formation could be
observed on fibrinogen-adherent platelets without cathepsin G treatment
(control) at 45 min, the cathepsin G-treated platelets supported
abundant fibrin deposition. Fig. 4C shows fibrin formation on platelets treated with the lowest cathepsin G concentration (0.1 µg/ml). Identical results were seen for 1.0 and 10 µg/ml Cat G
pretreatment of platelets (not shown). Formation of fibrin on platelets
treated with a low concentration of cathepsin G and then rinsed with a
cathepsin G inhibitor indicated that platelet activation is the
dominant mechanism through which cathepsin G turns fibrinogen-adherent
platelets procoagulant.
Effect of Cathepsin G and Elastase on Thrombin Production--
To
verify that cathepsin G is a more potent activator of platelet
procoagulant activity, thrombin generation in recalcified citrated
PRP/CTI was compared with that in the presence of cathepsin G,
elastase, or both proteases. Although the addition of cathepsin G to
PRP reduced the t50 of thrombin generation from
53 to 18 min, the addition of elastase had no effect (Fig.
5). Treatment of PRP with both neutrophil
proteases resulted in a t50 of 17 min,
indicating that this promptness in thrombin production was mainly due
to cathepsin G activity on platelets with no cooperativity with human neutrophil elastase.
In a separate set of experiments, the possible role of tissue factor in
protease-mediated activation of coagulation (6, 7) was assessed by
comparing thrombin generation in PRP treated with cathepsin G and
elastase in the presence or absence of a tissue factor antibody. The
presence of this antibody did not significantly alter thrombin
production (Fig. 6). It is interesting to
note that t50 for thrombin production due to
elastase and cathepsin G activity on platelets was comparable with
t50 for thrombin produced as a result of
platelet stimulation by the potent GPVI agonist, convulxin (no
proteases). As a control, progress curves were obtained to analyze
thrombin generation in PFP (no platelets) in the presence of both
cathepsin G and elastase. An extremely low rate of thrombin generation
(Fig. 6) implied that thrombin production seen in all earlier
experiments was solely platelet-dependent and that even high concentrations of neutrophil proteases do not result in
significant thrombin production in plasma lacking platelets.
We investigated the role of anionic phospholipid surface as a cofactor
for neutrophil-enhanced thrombin generation. The production of thrombin
in PFP (no platelets) was measured in the presence or absence of PSPC
vesicles (Fig. 7). PSPC addition to PFP
produced little thrombin, indicating that mere phosphatidylserine
exposure during platelet activation is not sufficient for coagulation
of the plasma. When neutrophil elastase was added to PFP containing PSPC, thrombin generation was also minimal. However, a detectable level
of slow thrombin production was observed when cathepsin G or
elastase/cathepsin G was added to PFP/CTI containing PSPC. This is the
first report of PSPC enhancing cathepsin G-mediated thrombin production
in PFP treated with CTI, potentially due to protection of lipid bound
cathepsin G from inhibition. These results indicated that platelet
phospholipid exposure alone is not sufficient for the cathepsin
G-enhanced coagulation seen in Figs. 2, 4, 5, and 6. As a control when
a progress curve for thrombin generation in PFP containing both
elastase and cathepsin G was obtained, negligible thrombin production
(also seen earlier in Fig. 6) was detected.
We demonstrated that adherent neutrophils enhanced thrombin
generation and fibrin formation on fibrinogen-adherent platelets through released proteases cathepsin G and elastase either in flow
assays or well-plate assays. Among these two proteases, cathepsin G was
far more potent than elastase in activating
platelet-dependent coagulation of CTI-treated PRP.
Cathepsin G does so by elevating the activation state of platelets
rather than by cleaving coagulation factors in plasma or on the
platelet anionic surface (Fig. 8). The
increased thrombin production of neutrophil protease-treated PRP is not
altered by anti-tissue factor, suggesting that Cat G activity does not
necessarily result in detectable de-encryption of tissue factor. We
show that direct Cat G activation of fibrinogen-adherent platelets can
also turn platelet procoagulant to levels comparable with that of
convulxin-stimulated platelets or collagen-adherent platelets.
w = 62.5 s
1) over fibrinogen-adherent platelets did not result in
fibrin formation until 50 min, massive fibrin could be observed on
cathepsin G-treated platelets even at 35 min. Cathepsin G addition to
corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of
fibrinogen-adherent platelets rather than by cleaving coagulation factors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain
(14), mediates the signaling response to collagen (15-17). Once
activated, platelets participate in the assembly of the tenase
(IXa/VIIIa) and prothrombinase (Xa/Va) complexes by providing anionic
phospholipid binding sites for prothrombin, factor X, factor V, and
factor XI to help accelerate coagulation (18, 19).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-phosphatidyl-L-serine, and bovine brain
L-
-phosphatidylcholine (Sigma), CTI, human fibrinogen,
factor X, and anti-human tissue factor monoclonal antibody TFE (Enzyme
Research Labs, South Bend, IN), and convulxin (Centerchem, Norwalk, CT)
were stored following the manufacturers' recommendations.
MeOSuc-Ala-Ala-Pro-Ala-CMK (human neutrophil elastase inhibitor) and
Z-Gly-Leu-Phe-CMK (cathepsin G inhibitor) were from Enzyme Systems
Products (Livermore, CA). The fluorogenic substrate for thrombin,
tboc-Val-Pro-Arg-MCA (boc-VPR-MCA), was obtained from Peninsula
Laboratories (San Carlos, CA).
w) imposed on the surface was
calculated from the solution of the Navier-Stokes equation for laminar
flow of a Newtonian fluid;
w = (6 Qµ)/(B2W), where
Q represents the flow rate (cm3/s), µ represents the viscosity (0.01 poise at room temperature), B
represents the total plate separation (0.02 cm), and W
represents the width (0.2 cm). Consequently, the wall shear rate,
w (s
1), can be calculated as
w = 6Q/B2W. A flow rate of 50 µl/min corresponded to a shear stress of 0.625 dyne/cm2
and a wall shear rate of 62.5 s
1. To activate
fibrinogen-adherent platelets, convulxin (10 nM) was
perfused over surface-adherent platelets for 10 min. During flow
experiments, the microcapillary flow chambers were mounted on a Zeiss
Axiovert 135 microscope, and a 63× (NA 1.40) oil immersion objective
lens (Plan Apochromat) was used to conduct differential interference
contrast microscopy.
-phosphatidyl-L-serine (PS) and
L-
-phosphatidylcholine (PC) were mixed (1:1 wt %) and dried under nitrogen. The dry film was hydrated with buffer (20 mM HEPES, 150 mM NaCl) and sonicated for 15 min. Using dynamic light scattering (DynaPro 99), the PSPC vesicle
diameter was determined to be 158 ± 52 nm. Fluorescence in the
96-well plate was measured using a Fluoroskan Ascent fluorometer
(excitation 390 nm; emission 460 nm). All well plate experiments were
performed at 37 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 for t = 35 min. Fibrin deposition on
fibrinogen-adherent platelets was essentially undetectable, but
substantial fibrin deposition was observed on the latter two surfaces.
Although it is known that collagen-adherent platelets are more
activated and more procoagulant than fibrinogen-adherent platelets (15,
27, 28), these observations demonstrate that direct
convulxin activation of platelets on a fibrinogen-coated surface can
stimulate fibrin formation to levels comparable with that found on
collagen-adherent platelets. Because convulxin, a C-type lectin
isolated from Crotalus durissus terrificus venom, is a
GPVI-specific platelet activator, this is the first report of
activation of platelet GPVI receptor in turning platelets procoagulant
under venous flow conditions for platelets maintaining adhesion via
GPIIb/IIIa binding to adsorbed fibrinogen.
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Fig. 1.
Role of platelet activation in fibrin
deposition on adherent platelets. Recalcified citrated PFP was
perfused at a shear rate of 62.5 s 1 for 40 min over
fibrinogen-adherent platelets, convulxin-treated (10 nM, 10 min) fibrinogen-adherent platelets, and collagen-adherent platelets.
Massive fibrin accumulation was observed only on the latter two
surfaces, demonstrating that direct stimulation of the GPVI receptor on
fibrinogen-adherent platelets promotes fibrin formation to the levels
supported by collagen-adherent platelets. Flow is from right
to left.
w = 62.5 s
1) on a cell mixture of fibrinogen-adherent neutrophils
and platelets was compared with that over surfaces coated with either
neutrophils or platelets alone, extensive fibrin formation was
visualized on the surface coated with both neutrophils and platelets
(Fig. 2), demonstrating the role of
neutrophil-platelets interactions in promoting fibrin generation. In
addition, this fibrin accumulation was attenuated by specific peptide
inhibitors against cathepsin G (Z-Gly-Leu-Phe-CMK; 100 µM) or, to a lesser extent, elastase
(MeOSuc-Ala-Ala-Pro-Ala-CMK; 100 µM), indicating the role
of these neutrophil proteases in the interaction.
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Fig. 2.
Neutrophil-augmented fibrin formation on
platelets was attenuated by inhibitors of elastase or cathepsin G. Although perfusion of recalcified citrated PFP ( w = 62.5 s
1) over neutrophils alone or platelets alone caused no
fibrin formation, dramatic levels were accumulated on the
neutrophil-platelet surface. This heavy fibrin deposition was inhibited
by specific peptide inhibitors of cathepsin G (100 µM
Z-Gly-Leu-Phe-CMK) or elastase (100 µM
MeOSuc-Ala-Ala-Pro-Ala-CMK). Flow is from right to
left.
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Fig. 3.
Inhibitors (INH) of elastase
or cathepsin G attenuate neutrophil-enhanced thrombin generation in
unstirred PRP. Progress curves for thrombin production in
recalcified citrated PRP supplemented with CTI (50 µg/ml) and factor
X (170 nM) were obtained in the absence of neutrophils
(No N), in the presence of neutrophils (N), or in
the presence of fMLP-activated neutrophils. Thrombin generation was
significantly delayed when a specific peptide inhibitor of cathepsin G
or elastase was added to fMLP-stimulated neutrophils. All experiments
were conducted in triplicate ( , not significant; *, p < 0.05; **, p < 0.005, compared with neutrophils
alone). HNE, human neutrophil elastase;
dF/dt, rate of thrombin generation.
1-antitrypsin) before the start of the flow
experiment (Fig. 2) may explain the different "clotting times"
between Figs. 2 and 3. Also, the close adhesion (and surface spreading)
of neutrophils and platelets in Fig. 2, which favors delivery of
Cat G from neutrophils to platelets (and adhesion-potentiated
signaling), is not achieved in static assay where mass transfer-
and gravity-induced heterotypic aggregation in PRP is insufficient.
w= 62.5 s
1) was compared with that over
platelets treated with either 10 µg/ml elastase or cathepsin G. We
also tested if both proteases can act in a combined manner to amplify
coagulation. Fibrin fibers started appearing on untreated
fibrinogen-adherent platelets (control) only after 50 min (Fig.
4A). At 35 min of perfusion of
CTI-treated PFP, untreated fibrinogen-adherent platelets showed no
fibrin formation, and elastase-treated platelets showed a trace of
fibrin deposition, whereas the platelets treated with either cathepsin G alone or both proteases demonstrated massive fibrin formation (Fig.
4B). These observations indicate that cathepsin G is more potent than elastase in turning platelets into coagulating structures. Moreover, a coupled activity by both proteases is not a prerequisite to
achieve full activation of fibrinogen-adherent platelets.
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Fig. 4.
Elastase or cathepsin G promotes fibrin
deposition on fibrinogen-adherent platelets. When recalcified
citrated PFP was perfused ( w = 62.5 s
1) over
fibrinogen-adherent platelets, fibrin fibers started appearing on
platelets only after 50 min (A). In contrast, when platelets
were pretreated for 40 min with neutrophil elastase (10 µg/ml) or
neutrophil cathepsin G (10 µg/ml), fibrin formation on platelets
could be observed even by 35 min; this fibrin deposition was
considerably greater on cathepsin G-stimulated platelets
(B). Fibrin formation on platelets treated with both
proteases was not substantially different from fibrin on platelets
treated with cathepsin G alone. When PFP containing a peptide inhibitor
of cathepsin G (100 µM) was perfused over platelets that
had been treated with a low concentration of cathepsin G (0.1 µg/ml)
and rinsed with the cathepsin G inhibitor (Inh, 100 µM), fibrin formation on the cells could still be
observed (C). Flow was from right to
left.
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Fig. 5.
Neutrophil cathepsin G enhances thrombin
generation in PRP. Progress curves of fluorescence intensity for
fluorogenic thrombin substrate (boc-VPR-MCA) conversion in recalcified
citrated PRP supplemented with CTI (50 µg/ml) and factor X (170 nM) were obtained in the presence of elastase, presence of
cathepsin G, presence of both proteases, or in the absence of both
elastase and cathepsin G. Although the addition of cathepsin G to PRP
dramatically reduced the t50 of thrombin
generation, the addition of elastase had no effect. All experiments
were conducted in triplicate ( , not significant; *, p < 0.05; **, p < 0.005, compared with no protease).
HNE, human neutrophil elastase.
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Fig. 6.
GPVI activation enhances thrombin generation
on platelets to levels supported by protease-mediated activation.
Measurements of thrombin generation in recalcified citrated PFP
supplemented with CTI and factor X were made in the presence of both
cathepsin G and elastase, in the presence of both proteases and a
tissue factor antibody (anti-TF; 50 µg/ml), and in the
presence of convulxin alone. Although the tissue factor antibody did
not appear to play a role, a similarity in kinetic behavior of thrombin
production on convulxin-stimulated platelets and protease-activated
platelets was observed. Negligible thrombin production was observed
when neutrophil proteases were added in PFP (no platelets). All
experiments were conducted in triplicate. HNE, human
neutrophil elastase.
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Fig. 7.
Thrombin production in plasma by cathepsin G
and elastase; role of phospholipid. Thrombin production in
recalcified citrated PFP supplemented with CTI (50 µg/ml) and factor
X (170 nM) was measured in the presence of anionic
phospholipid vesicles (PSPC). The presence of PSPC did not
affect thrombin generation. Although addition of neutrophil elastase to
PFP containing PSPC did not enhance thrombin generation, a slight
promotion was observed when either cathepsin G or both elastase and
cathepsin G were added. Negligible thrombin production was observed
when both neutrophil proteases were present in PFP with no PSPC.
HNE, human neutrophil elastase.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Enhancement of platelet procoagulant activity
by neutrophil cathepsin G in the absence of tissue factor, collagen,
and contact activation. Shown is a schematic of pathways through
which cathepsin G may promote platelet-dependent
coagulation, including cleavage of Mac-1-bound factor X on neutrophils
(2, 15), activation of platelets via protease-activated receptor 4, and
to a lesser extent, cleavage of plasma zymogens, such as factors X and
V. fbg, fibrinogen; Psel, P-selectin;
N*, activated neutrophil; P*, activated platelet;
EC*, activated endothelial cell; IIb/IIIa,
glycoprotein IIb/IIIa; LFA-1, leukocyte function-associated
antigen-1; PSGL-1, P-selectin glycoprotein ligand-1;
ICAM-1, intercellular adhesion molecule-1; XIIa,
factor XIIa; vWF, von Willebrand factor.
The addition of PSPC alone to CTI-treated platelet free plasma demonstrated that the presence of a negatively charged phospholipid surface is not sufficient to initiate coagulation. Cathepsin G is cationic (29), and the slight increase in thrombin levels on the addition of PSPC supports the view that cathepsin G-mediated cleavage of coagulation factors on a negatively charged surface may help support, albeit in a secondary role, cathepsin G-mediated platelet activation. In comparison to cathepsin G-enhanced thrombin generation in PFP containing PSPC, a five times higher thrombin production in PRP (presence of platelets) indicates that platelets have additional means, beyond merely presenting negative phospholipid, through which they respond to cathepsin G stimulation. These results are in agreement with studies by Sumner et al. (30), which show that PS exposure does not correlate with factor Xa or thrombin production on platelets and suggest that surface participants other than PS are involved in coagulation (31). Fibrin accumulation under the conditions of Fig. 4C supports the view that cathepsin G is predominantly activating platelets rather than cleaving zymogens on the platelet surface.
Neutrophil-enhanced platelet-dependent coagulation (under factor XIIa inhibition by CTI) could not be attenuated by an antibody against tissue factor. It remains possible that after an initial production of minuscule amounts of thrombin through a platelet-supported factor VIIa pathway, factor XI was being activated to XIa on activated platelets by thrombin to trigger the intrinsic pathway in the absence of the upstream contact pathway protein factor XIIa (32-34). It also remains a possibility that surface-bound Cat G activates platelet-released factor V.
Although the addition of a cathepsin G inhibitor to neutrophils in PRP attenuated thrombin generation in plasma, it unexpectedly increased the maximum rate of thrombin generation by 3.7 times (as seen in the difference in slopes on the progress curves in Fig. 3). This may be attributed to the degradation of platelet GPIb by cathepsin G. The surface expression of GPIb, which has been found to be the counter-receptor for neutrophil Mac-1 (35), is expected to increase after the interaction of platelets with neutrophils. However, it is known that neutrophil cathepsin G can degrade GPIb (9, 10). In the light of these studies, whereas the addition of cathepsin G inhibitor to PRP containing neutrophils may not have prevented neutrophil-assisted platelet GPIb expression, it may have blocked the degradation of GPIb by neutrophil cathepsin G. Additionally, GPIb, when left intact, would allow factor XI binding to it where activation of factor XI by a minimal amount of thrombin can accelerate thrombin production via the intrinsic coagulation pathway (33). As expected, this increased rate of thrombin generation is not observed in the absence of neutrophil-platelet interactions due to a possible lack of increased GPIb expression. However, an overall delay in thrombin production in the presence of cathepsin G inhibitor was most likely the result of a decline in platelet activation levels due to cathepsin G inhibition.
Under flow conditions, mass transfer by dispersion and convection can
alter the concentration of local factors (36). The burst in thrombin
production is vital to make fibrin under flow conditions, and this
fibrin is critical for clot stabilization. The close contact of
activated platelets and activated neutrophils mediated by P-selectin
glycoprotein ligand-1 in the absence of tissue factor or collagen may
allow for the function of Cat G. Transfer of Cat G to platelet anionic
phospholipid may also facilitate its activating function in the
presence of plasma inhibitors such as 1-antitrypsin (37,
38).
In summary, we show that neutrophil proteases, most notably cathepsin
G, can promote thrombin generation and subsequent fibrin formation on
platelets under static or venous flow conditions. Cat G does so by
elevating the activation state of platelets. This platelet activation
and resultant procoagulant activity involves additional factors beyond
anionic phospholipid exposure by activated platelets. These events may
be clinically relevant in situations void of collagen or tissue factor
such as those involving biomaterial thrombosis or endothelial
dysfunction and neutrophil activation during deep vein thrombosis.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grant RO1 HL 56621.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A National Institutes of Health predoctoral fellow (Grant T32
HL 07954-02).
§ An Established Investigator of the National American Heart Association. To whom correspondence should be addressed: Institute for Medicine and Engineering, Dept. of Chemical Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104. Tel.: 215-573-5702; Fax: 215-573-2093; E-mail: sld@seas.upenn.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M211956200
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ABBREVIATIONS |
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The abbreviations used are: Cat G, cathepsin G; CTI, corn trypsin inhibitor; fMLP, N-formyl-Met-Leu-Phe; GP, glycoprotein; PFP, platelet-free plasma; PRP, platelet-rich plasma; PS, phosphatidylserine; PC, phosphatidylcholine; PSPC, phosphatidylserine/phosphatidylcholine; Va, factor Va; VIIIa, factor VIIIa; IXa, factor IXa; Xa, factor Xa; XIa, factor XIa; CMK, chloromethyl ketone.
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