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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Medicine, Jagellonian University
School of Medicine, Cracow, Poland; and Department of Biochemistry,
University of Vermont, Burlington.
The sequence of coagulant reactions in vivo following vascular
injury is poorly characterized. Using quantitative immunoassays, the
time courses were evaluated for activation of prothrombin, factor (F)V,
FXIII, fibrinogen (Fbg) cleavage, and FVa inactivation in bleeding-time
blood collected at 30-second intervals from 12 healthy subjects both
before and after aspirin ingestion. Prothrombin decreased at a maximum
rate of 14.2 ± 0.6 nM per second to 10% of initial values at the
end of bleeding. Significant amounts of Hemostasis is triggered in vivo when vascular
damage exposes membrane-bound tissue factor (TF) constitutively present
in the subendothelium.1 The proteolytically active complex
of TF and circulating 2-chain factor (F)VIIa, the extrinsic tenase,
activates FX and FIX. FIXa and FXa, complexed with FVIIIa and FVa,
respectively, form 2 enzyme-cofactor complexes Generation of Blood coagulation has been studied extensively in purified
systems,8-10 plasma,11 and anticoagulated and
nonanticoagulated whole blood12,13 in vitro. A model of
microvascular injury, introduced by Thorngren and
coworkers,14 has also been applied to study coagulant
events triggered by standardized bleeding-time skin
incisions.15 This flowing blood system provides insight into the intricately interwoven hemostatic process involving tissue damage, plasma coagulation reactions, the activation of platelets, the
endothelium, and subendothelial tissue.16,17 Weiss and Lages17 found in patients with deficiencies of FV or FX
that the production of FPA in the wounds depends on activation of FX by
the complex TF-FVIIa and is closely coupled with platelet activation, reflected by a rapid release of platelet factor 4 (PF4). Previous studies undertaken to quantitate thrombin generation in bleeding-time blood focused on the measurement of FPA, thrombin-antithrombin III
complexes (TAT), and prothrombin fragment 1.2 (F1.2)
concentrations.17-20 We wondered whether the model of
microvascular injury could also be used to monitor other coagulant
reactions that regulate thrombin formation, or are catalyzed by this
enzyme (ie, activation of FV and FXIII or inactivation of FVa).
Aspirin has been proven effective in secondary prevention of coronary
artery disease (CAD). Its efficacy has been ascribed to its
antiplatelet action through inhibiting thromboxane
A2-mediated platelet aggregation.21,22 In
various experimental settings aspirin has been shown to reduce thrombin
generation.12,23,24 Using the model of microvascular
injury, it has been demonstrated that aspirin at a dose from 30 to 500 mg down-regulates thrombin formation in healthy
subjects15,25 and patients with CAD.19,26
The purpose of the present study is to describe major coagulant events
during blood clotting at the site of microvascular injury and to
evaluate the effect of low-dose aspirin on the activation of
prothrombin, FV, FXIII, and Fbg, and inactivation of FVa by APC.
Materials
Subjects
Model of microvascular injury The TF-initiated coagulation was evaluated in samples of bleeding-time blood, as described previously.25 Briefly, after compressing the upper arm with a sphygmomanometer cuff to 40 mm Hg, 2 incisions were made on the lateral aspect of a forearm parallel to the antecubital crease using a Simplate II device (Organon Teknika, Durham, NC). The blood shed was collected at 30-second intervals (on average, 10 samples) using heparinized capillary tubes (Kabe Labortechnik, Nümbrecht-Elsenroth, Germany) and then expressed into Eppendorf tubes. An anticoagulant cocktail, provided by an FPA assay (Diagnostica Stago, Asnieres, France), contained sodium citrate, aprotinin, chloromethyl ketone, and heparin (vol/vol, 1:10). Volumes of all the samples and bleeding time were recorded. The procedure was performed by the same investigator. Soluble and insoluble material was immediately centrifuged at 4°C at 2000g for 20 minutes; then the supernatants were removed, aliquoted, and stored at 80°C
for further analysis.
Gel electrophoresis and Western blotting The supernatant aliquots were separated on 5% to 15% linear gradient sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels under reducing (1% 2-mercaptoethanol) or nonreducing conditions according to modified Laemmli procedures.13,31 Transfer from the gels to nitrocellulose membranes (Bio-Rad, Hercules, CA) was performed as described by Towbin and coworkers.32 The membranes were blocked with 5% nonfat dry milk in HEPES buffered saline, pH 7.4 (HBS) at room temperature for 1 hour. After washing, the blots were incubated for an additional hour with the primary antibodies (in Tris buffered saline + 0.01% Tween 20, pH 7.4 [TBST]): -prethrombin-1 (7.5 µg/mL),
-FVaHC no.17 (7.5 µg/mL), -FVaLC no.9
(7.5 µg/mL), -FXIII (5 µg/mL), and -Fbg 3A (1.5 µg/mL). This was followed by a 60-minute incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000-10 000
dilution): for FVa and Fbg goat -mouse; for prothrombin and its
activation productsgoat -horse; for FXIIIgoat -rabbit. The
proteins were visualized by the addition of a chemiluminescence reagent
to the membranes. The blots were developed in a Kodak XOMAT as
described by Rand and colleagues.13 FVa light- and heavy-chain generation, prothrombin activation, Fbg cleavage, and FXIII
activation as a function of time were determined and quantified by
densitometry of immunoblots on a Hewlett-Packard Scanjet 4C/T.
Concentrations were estimated from serial dilutions of purified
internal standard proteins by horizontal comparison of sample band
density. Relative concentrations were determined by normalizing the
data with regard to the maximum. Changes in the reaction rates, as
reflected by the concentrations of immunoreactive fragments from the
immunoblots, were analyzed using IGOR Pro Version 3.1 software
(Wavemetrics, Lake Oswego, OR). A mean value was calculated from the
compilation of 30-second interval densitometric analysis. On the basis
of concentrations and volumes of blood samples, the absolute amounts of
the proteins studied were calculated and analyzed.
In addition to immunoblotting, F1.2 and TAT concentrations in
bleeding-time blood were measured by enzyme-linked immunosorbant assay
(ELISA) using commercially available kits (Enzygnost F1.2 and Enzygnost
TAT, Dade Behring). Concentrations of both markers were also measured
in citrated plasma of peripheral venous blood (vol/vol, 1:9).
Prothrombinase concentration was also calculated at each 30-second
interval using an equation introduced by Rand and
colleagues13: [prothrombinase] = v
(Km + [S])/kcat [S], where [S]
denotes free prothrombin concentration, determined by densitometry of
immunoblots. A value of v was estimated by densitometric analysis of
Statistical analysis Data are presented as mean ± SEM. Measurements before and after aspirin administration were compared with the Wilcoxon signed-rank test. To evaluate correlations between variables, Spearman coefficient was calculated. P less than .05 was considered statistically significant.
The initial bleeding time was 344.6 ± 18.4 seconds and aspirin at 75 mg/d caused a significant increase to 110.2 ± 14.1 seconds (P = .001). Blood volume increased to a maximum value at 150 seconds then declined until bleeding ceased. The total volume of blood collected from bleeding-time wounds was 124.2 ± 8.7 µL and 169.0 ± 13.5 µL (P = .007) before and after aspirin administration, respectively. At the first 8 time points, the volumes of samples were significantly larger (by about 20%) after aspirin than before treatment. Although no correlation was found between the volume of the samples and concentrations of the parameters measured, we decided to express levels of the products studied in bleeding-time blood both as a concentration and as a total amount in the 30-second interval, because the extent to which vessels are injured by incisions is standardized and consistent from sample to sample. When posttreatment levels of the products are compared with those calculated before aspirin ingestion using both concentration and total methods, significant differences were observed using both analyses. Markers of thrombin generation Prior to aspirin, the mean plasma TAT concentration was 0.026 ± 0.006 nM and F1.2 was 0.72 ± 0.05 nM. There was no change in either parameter following low-dose aspirin (0.021 ± 0.004 and 0.67 ± 0.08 nM, P > .05, respectively). These values were within the normal range, according to the manufacturer of both ELISA kits.The time courses for TAT and F1.2 formation in the 30-second
bleeding-time blood samples are depicted in Figure
1, with open symbols representing values
before aspirin treatment. The data are plotted as concentration
(triangles; nM, left y-axis) or total amount (squares; fmol, right
y-axis) versus time (seconds). The generation of both thrombin markers
(Figure 1A,B) follows a similar pattern of 2 phases: a 60- to 90-second
"initiation" phase and a subsequent "propagation" phase, as
seen in other models of blood coagulation.9,11,12,25
Prothrombin F1.2 (Figure 1A, squares) is generated at a rate of
6.13 ± 0.5 fmol/s with total yields reaching 1250 fmol by 240 seconds (P = .03). In contrast TAT (Figure 1B, circles)
complexes are formed at a much lower rate (1.43 ± 0.04 fmol/s) with
total amounts reaching more than 210 fmol by 180 seconds. Overall, F1.2
was produced much more rapidly than TAT (P = .004). The
velocity of the increase of F1.2 (triangles) concentrations was also
significantly higher than that of TAT (diamonds) concentrations (at
maximum, 0.673 ± 0.05 versus 0.11 ± 0.008 nM per second;
P = .002). Levels of F1.2 and TAT were positively
correlated (r = 0.79; P = .004).
Comparison of the time courses for TAT and F1.2 generation before (open symbols) and after aspirin (closed symbols) ingestion showed that low-dose aspirin greatly impairs the formation of both thrombin markers at all the 30-second intervals except for the first time point (Figure 1). Maximum rates for F1.2 and TAT (expressed as the total amounts of both parameters) were reduced to 1.11 ± 0.08 fmol/s and 0.53 ± 0.03 fmol/s, respectively, which corresponds to 70.1% (P = .009) and 67.1% (P = .013) of the baseline values, respectively. Likewise, the velocity of F1.2 (triangles) and TAT (diamonds) formation, when expressed as a concentration in the bleeding-time blood samples, was significantly decreased by 31.4% (P = .007) and 30.3% (P = .019) of the initial values, respectively. Prothrombin activation products The Western immunoblot depicted in Figure 2A displays fragments reactive to a polyclonal serum against human-prethrombin-1 under reducing
conditions. Thirty-second time points before (1A-8A) and after aspirin
treatment (1B-10B) are illustrated above the blot. The bottom bands
(Mr = 30 000; residues 321-579) correspond to
-thrombin B chain, derived from -thrombin and meizothrombin, which are not distinguishable using this polyclonal
antibody.13 The middle Mr = 36 000 bands
have similar mobility to those reported for prethrombin 2 (residues
272-579 in prothrombin). The upper bands most likely represent the
product of prothrombin activation, F1.2 (residues 1-271). The same
triplet patterns of prothrombin activation products are seen on
immunoblots following aspirin administration.
Before aspirin treatment, all 3 products of prothrombin activation
appeared rapidly and simultaneously after an initial 60- to 120-second
lag phase, defined as a time when its concentration, assessed by
densitometry of immunoblots, is below 10 nM (Figure 2A, lanes
1-8A). Following aspirin ingestion (arrow ASA) the bands were
detectable approximately 150 seconds (P = .04) later than before treatment (lanes 1-8B). The kinetics of prethrombin 2 and thrombin B chain, estimated by densitometry of Western blots, showed a
striking resemblance (data not shown). Prior to the ingestion of
aspirin, a maximum rate of Comparing relative amounts of prothrombin activation products in bleeding-time blood, we found that the sum of thrombin B-chain and prethrombin 2 concentrations constituted about 50% to 70% of the F1.2 levels, determined by ELISA. Because TAT concentration accounts for 80% of the surplus, we conclude that other products of thrombin with other inhibitors including thrombomodulin, fibrin, and HCII probably represent the remaining 20%. Aspirin ingestion resulted in a significant reduction (27.2%,
P = .022) in the maximum rate (to 0.163 ± 0.02 nM per
second) of Prothrombin migrating at Mr = 72 000 was no longer
detectable by approximately 210 seconds (Figure
3A, lane 8A). Before aspirin administration, an initial concentration of 1.32 µM for
prothrombin was detected and it disappeared from bleeding-time blood at
a maximum rate of 14.5 ± 0.6 nM per second (Figure 3B, open
circles). Following aspirin administration the rate of prothrombin
removal was slowed by 25.4% (10.82 ± 0.4 nM per second at maximum;
P = .03) and the protein could not be detected to the end
of bleeding in 3 of 12 subjects (Figure 3A, lanes 1-8B and
Figure 3B, closed circles). Absolute amounts of prothrombin were
calculated for each 30-second interval (Figure 3B, diamonds). Analysis
of such maximum rates of prothrombin removal showed that aspirin
induced a significant decrease in the velocity of this process (closed diamonds) by 29.3% (0.266 ± 0.014 versus 0.188 ± 0.011 nmol/s; P = .034).
Using the Michaelis-Menten equation and results of densitometry of
immunoblots for prothrombin and Factor V/Va Factor V, derived from circulating blood and activated platelets (up to 20% of the total amount), could be seen on nonreduced gels, migrating at Mr = 330 000 (data not shown). This band, which is immunoreactive toward a monoclonal antibody raised against FVa heavy chain, disappeared slowly over the bleeding time. From these immunoblots, an initial concentration of FV was estimated to be 18.2 ± 2.1 nM and decreased to only 14.0 ± 1.1 nM by the last interval (P = .03). The maximal rate of disappearance of FV was calculated to be 0.022 ± 0.005 nM per second and was reduced (by 26%; P = .04) following aspirin administration (data not shown).Active FV (FVa), derived from activation by
The loss of cofactor activity by FVa results from APC-mediated peptide bond cleavages in the heavy chain at Arg306, Arg506, and Arg679.28 On nonreduced gels, the complete inactivation product of FVa heavy chain, a fragment of Mr = 30 000 (residues 307-506; Figure 4D) was first seen as early as 90 seconds after incision (lane 3A). These bands migrated identically with the previously described inactivation products of FVa heavy chain.13 The density of the Mr = 30 000 bands tended to increase with some fluctuations over bleeding time. Factor Va inactivation was not influenced by a 7-day aspirin treatment (Figure 4D, lanes B). A Western immunoblot in Figure 4E shows that the Mr = 97 000 fragment of FVa heavy chain (residues 1-643/644), which can be visualized under reducing conditions, is seen in 3 of 12 subjects who also displayed high concentrations of thrombin B-chain (> 38 nM). In all 3 subjects significant amounts of thrombin B chain as well as FVa heavy chain were detected as early as 60 seconds. By comparison with standards (not shown), the mobility of this band is identical to that observed with cultured human umbilical vein endothelial cells (HUVECs) in the presence of thrombin.33 The formation of this FVa inactivation product is independent of APC activity. No relationship was identified between the velocity of APC inactivation of FVa and the Mr = 97 000 fragment release. Density of the Mr = 97 000 band increased slightly over bleeding time (Figure 4E). Aspirin had no effect on the kinetics of the Mr = 97 000 fragment formation in bleeding-time blood (Figure 4E). Fbg Fibrinogen in the fluid phase of bleeding-time blood was detectable by immunoblotting as a Mr = 340 000 band over the first 3 minutes (Figure 5A, lanes 1A-6A). Fbg was depleted rapidly from solution at a peak rate of 0.047 ± 0.02 µM/s (open circles, Figure 5B). The initial concentration of Fbg was estimated by densitometry to be 7.31 µM (2.48 g/L); close to the mean Fbg level determined nephelometrically (7.68 µM). Prior to aspirin ingestion Fbg was completely depleted at the end of bleeding in 9 of the 12 subjects studied, in contrast to only 3 of 12 subjects after aspirin treatment. A significant delay (60 seconds) in Fbg depletion from solution/fibrin formation was associated with aspirin ingestion (closed circles, P = .01). The rate of Fbg removal from bleeding-time blood after aspirin ingestion (closed circles) did not exceed 0.036 ± 0.006 µM/s, which corresponds to 30.5% ± 3.2% of the initial value (P = .002). The maximum rate of decrease in total amounts of Fbg (closed triangles, Figure 5B), estimated in each 30-second interval, was significantly lower by 24.4% following aspirin ingestion (P = .008). There was a significant correlation between the rate of Fbg depletion and that of-thrombin B-chain formation (r = 0.52;
P = .042). Surprisingly, the detection times at which
-thrombin B-chain is first observed and Fbg disappears were not
significantly correlated (P = .17).
Factor XIII/XIIIa The Western immunoblot seen in Figure 5C displays the disappearance over time of a Mr = 80 000 band that migrated identically with the purified subunit A of FXIII (FXIIIA). This fragment was not detected, on average, after 150 seconds of bleeding (range, 90-210 seconds). The activated form of subunit A (FXIIIAa) has an Mr = 74 000 and appears at 120 seconds (lanes 4A-7A) with its concentration increasing gradually. Comparison with standard amounts of FXIIIA gives approximately 86 ± 5 nM prior to aspirin ingestion. Densitometric analysis showed a rapid decrease of FXIIIA concentration in bleeding-time blood (0.784 ± 0.04 nM per second at maximum) to below 10% of the initial value (Figure 5D). After treatment, the concentration of FXIIIA was estimated approximately at 83 ± 3 nM and did not differ from the pretreatment value. Aspirin administration delayed FXIII activation by approximately 60 seconds (lane 5B, P = .031) and decreased the maximum rate of disappearance of FXIIIA, determined by densitometry of immunoblots, to 0.56 ± 0.05 nM per second (P = .033). The rate of increase in total amounts of FXIII also decreased significantly after aspirin ingestion (data not shown). A correlation between the detection time of-thrombin B chain and FXIIIAa was
significant (r = 0.72; P = .015). This corresponds to a
rapid generation of -thrombin B chain, the major activator of FXIII
in vivo.30 The rates of FXIII activation and -thrombin
formation were also significantly correlated (r = 0.49;
P = .024).
The model of microvascular injury presented here provides insights into the complex coagulant reactions following tissue damage with attendant disruption of blood vessels, which ultimately lead to formation of the hemostatic plug and cessation of bleeding. Morphologic studies show that the hemostatic plugs, formed in wounds at the end of small arterioles and venules, consist of aggregated platelets surrounded by fibrin, present both in central areas and abundantly at the periphery close to collagen fibers.34 Thrombin formation via the extrinsic pathway of the coagulation system, as evidenced by a rapid fibrin formation,34 occurs as early as 30 seconds after injury. Therefore, this flowing blood system is a valuable tool to evaluate a series of events resulting in thrombin formation at the site of injury. Previous studies on blood coagulation at sites of hemostatic plug formation, however, have been limited to measurements of FPA, TAT, or F1.2 by commercially available ELISAs.18-20 In the present study, we have extended these observations to the determination of the kinetics of prothrombin, FV, and FXIII activation as well as Fbg consumption and inactivation of FVa using quantitative immunochemical techniques. The principal conclusions from our analyses, performed in bleeding-time
blood, are the following: (1) Prothrombin is rapidly and almost
completely removed at 3 to 4 minutes of bleeding. (2) Thrombin B
chain and prethrombin 2 appear at 60 to 120 seconds of bleeding and are
produced in similar amounts. Their maximum concentrations reach
approximately 35 to 40 nM at the end of bleeding. (3) Activation of FV
is incomplete and at the end of bleeding FV level is about 75% of its
initial concentration. FVa heavy chain is detectable at 120 seconds
after vascular injury and appears somewhat faster than that of the
light chain. (4) FVa inactivation products associated with the
APC-mediated proteolysis of FVa heavy chain are observed simultaneously
with the generation of FVa heavy chain. The products associated with
the inactivation of FVa through thrombin and endothelial cells are also
observed. (5) The maximum prothrombinase concentration (about 22 pM) is
achieved by 150 seconds and is limited by the factor Xa concentration.
(6) Fbg is removed very rapidly from the bleeding-time blood and is
below detection limits by 3 minutes of bleeding. (7) Significant
activation of FXIII is observed by 120 seconds. The extent of the
activation process is correlated with Because the same antibodies have been used in the current bleeding-time
blood model and the whole-blood system, described by Rand and
colleagues,13 comparative evaluation of both models appears to be helpful and validated. In the present model, the amount
of any protein in a volume unit downstream is determined by 3 factors There are some interesting similarities and differences between our model and that of Rand and colleages,13 which deserve special comments. At the site of microvascular injury, we detect prethrombin 2 (Figure 2A), a prothrombin activation product, which has been observed in the purified systems35 and in experiments in blood or plasma in vitro.13,36,37 The concentrations of thrombin B chain and prethrombin 2, observed at the end of bleeding (on average, 5 minutes), are almost identical to those found during clotting of whole blood at 10 minutes on addition of relipidated TF.13 Enhanced thrombin generation could be attributed to the potentiating effect of fibrin formed quickly at sites of microvascular injury.38 The amounts of Activation of FV is faster but incomplete at sites of hemostatic plug
formation when compared to the whole-blood model.13 As
evidenced by a positive correlation between concentrations of FVa and
Comparing FVa inactivation by APC in the current system and the whole-blood model,13 we have found that release of APC-mediated FVa inactivation products is much faster at the site of microvascular injury (Figure 4D). Lower concentrations of thrombomodulin, derived mostly from platelets,39 most likely account for a slow inactivation of FVa by APC in the whole-blood model.13 In the microvasculature, the protein C pathway plays a key role in anticoagulant mechanisms, due to high concentrations of thrombomodulin on the endothelial cells relative to the small volumes of blood.7 Our study convincingly supports this conclusion. It is of note that despite concomitant FVa proteolysis, amounts of FVa increase significantly in the consecutive blood samples, resulting in a rapid thrombin and finally fibrin formation. Furthermore, molar concentrations of FVa heavy chain are underestimated because APC-mediated cleavage of this chain lowers amounts of FVa detected by immunoblotting. We identified the Mr = 97 000 fragment of FVa heavy
chain at sites of microvascular injury (Figure 4E). Its mobility was
identical to the product observed by Hockin and
colleagues33 in the thrombin inactivation of FVa on
HUVECs. Because significant amounts of this fragment were observed only
at The bleeding-time blood model is also characterized by a rapid
consumption of Fbg and prothrombin from blood collected from skin
wounds (Figures 3A and 5A) when compared to the situation in the
whole-blood model.13 Apart from Fbg cleavage by thrombin and activation of prothrombin, other mechanisms might contribute to the
relatively high rates of both processes. It has been reported that Fbg
can bind to hyaluronic acid,40 platelet
thrombospondin,41 and endothelial cells via a process,
which is enhanced by thrombin produced in loco,42 while
prothrombin can attach to the subendothelium and platelets via
Collectively, the differences reported here provide evidence for the concept that kinetics of coagulation reactions under flow conditions cannot be extrapolated from static, closed systems. Moreover, cells of the vessel walls at the site of microvascular injury, as well as activated platelets and blood cells, provide membranes necessary for optimal function of most components of coagulation reactions known to be surface dependent4 and consequently, rates of most reactions are relatively high. The model, used in the current study, has some limitations: (1) a possible contact activation in test tubes is not eliminated, unlike in the whole-blood model,13 in which this pathway is suppressed by corn trypsin inhibitor, a selective inhibitor of FXIIa44; (2) our study focuses on early coagulant events (up to 5-7 minutes) in vessels less than 25 µm in diameter and therefore, later stages of blood coagulation, along with a process triggered by an injury of diseased large arteries, escape evaluation; (3) the bleeding-time technique used here requires expertise to achieve an appropriate reproducibility of results; and (4) volumes of the blood samples collected from skin wounds show a distinct interindividual variability. Nevertheless the present flowing blood model is a valuable tool in the in vivo coagulation studies. In the current study, we demonstrated that thrombin generation is impaired by low-dose aspirin given for 7 days in healthy subjects. This finding is in keeping with most,15,23,25,26 though not all previous studies.24 In contact pathway-inhibited whole-blood coagulation studied in vitro, high-dose aspirin administered in vivo had no effect on exclusively TF-induced coagulation.45 This suggests that the aspirin effect in the present model may be tissue- or shear-dependent. In addition, our results provide the first evidence that at the site of vascular injury FV and FXIII activation, along with the removal of Fbg from the blood shed, are delayed, most likely as a consequence of impaired thrombin generation following aspirin ingestion. The mode of action of aspirin on hemostasis has not been fully elucidated. It is well known that aspirin inhibits platelet function by irreversible acetylation of a serine at position 529 in the platelet cyclooxygenase.21 This, however, does not explain antithrombin properties of this drug,12,15,23-25 which appear to be related to platelet reactivity,46 through possible alterations in the exposure of negatively charged phospholipids on platelet surfaces47 or in membrane fluidity.48 It has also been shown that aspirin is able to acetylate some proteins, which are involved in the coagulation reactions, such as ATIII49 and Fbg.50 The existence of such phenomena has not been convincingly demonstrated in vivo. Another intriguing possibility results from observations showing that aspirin impairs activation of platelet glycoprotein GPIIb-IIIa.51 Studies on various GPIIb-IIIa receptor antagonists indicate that inhibition of this Fbg receptor leads to a significant suppression of thrombin generation.52 Recently it has been reported that these agents reduce platelet surface FV/Va binding and phosphatydylserine expression in whole blood taken from healthy volunteers.53 Because aspirin prolonged the lag phase of thrombin generation and the FVIIa-TF complex determines the duration of the lag phase in reconstituted systems,54 one might speculate that aspirin down-regulates TF expression. In fact, aspirin has been reported to inhibit synthesis of TF in human monocytes55 and to reduce TF expression in human atherosclerotic plaques.56 In addition, we found that aspirin decreases the maximum rate of thrombin generation in the propagation phase. This suggests that aspirin may affect other coagulant reactions, possibly via altered functions of the protein C system, regulating the propagation phase. Further studies are warranted to evaluate aspirin's impact on different stages of the TF-initiated blood coagulation. Taken together, our results provide convincing evidence for the utility of the model of microvascular injury in the qualitative and quantitative analysis of the TF-initiated coagulation process in vivo. The present study extends the work of others showing a very rapid activation of prothrombin, FV, FXIII, and Fbg conversion to fibrin, along with FVa inactivation, which can be mediated by APC and other processes. The current model also enables monitoring of drug-induced alterations in the coagulation cascade. We have demonstrated a significant decrease in the rates of the activation of prothrombin, FV, FXIII, and Fbg cleavage following administration of low-dose aspirin. However, it remains to be clarified whether aspirin exerts similar effects on coagulation at sites of the injury in patients with CAD and if higher doses of aspirin produce more pronounced anticoagulant alterations. This issue is presently under investigation.
We thank Jan Brozek, Jed Pauls, and Grzegorz Guzik for their expert technical assistance, and Marek Grzywacz for performing ELISAs.
Submitted March 5, 2001; accepted June 5, 2001.
Supported by National Institutes of Health (NIH) grant HL-46703 (to K.G.M.) and NIH training grant T32 HL-07594 (to K.B. and K.G.M.). A.U. is a recipient of a Fulbright Fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth G. Mann, University of Vermont, Department of Biochemistry, Given Bldg, Rm E407, Burlington, VT 05405; e-mail: kmann{at}zoo.uvm.edu.
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1988;71:629-635 18. Eichinger S, Wolzt M, Nieszpaur-Los M, et al. Effects of a low molecular weight heparin (Fragmin) and of unfractionated heparin on coagulation activation at the site of plug formation in vivo. Thromb Haemost. 1994;72:831-835[Medline] [Order article via Infotrieve].
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Inhibition of thrombin generation by aspirin is blunted in hypercholesterolemia.
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Patrono C.
Aspirin as an antiplatelet drug.
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Monoclonal antibodies to human coagulation factor V and factor Va.
Blood.
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Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay.
Blood.
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30.
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An integrated study of fibrinogen during blood coagulation.
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Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci U S A.
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Protein C activation and factor Va inactivation on human umbilical vein endothelial cells.
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A membrane-mediated catalytic event in prothrombin activation.
J Biol Chem.
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Studies of prothrombin activation pathway utilizing radioimmunoassays for the F2/F1+2 fragment and thrombin-antithrombin complex.
Blood.
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Prothrombin fragment 1.2.3, a major product of prothrombin activation in human plasma.
J Biol Chem.
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The influence of fibrinogen and fibrin on thrombin generation
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Functionally active thrombomodulin is present in human platelets.
J Biochem.
1988;104:628-633
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Human fibrinogen specifically binds hyaluronic acid.
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1986;261:12586-12592 41. Leung LLK, Nachman RL. Complex formation of platelet thrombospondin with fibrinogen. J Clin Invest. 1982;70:542-549. 42. Hatton MWC, Moar SL, Richardson M. Enhanced binding of fibrinogen by the endothelium after treatment of the rabbit aorta with thrombin. J Lab Clin Med. 1990;115:356-364[Medline] [Order article via Infotrieve].
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Networking in the hemostatic system: integrin alpha IIb 3 binds prothrombin and influences its activation.
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45.
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Antiplatelet agents in tissue factor-induced blood coagulation.
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2001;97:2314-2322 46. Musial J, Radwan J, Szczeklik A. Aspirin delays thrombin generation in vitro through interaction with platelet phospholipids. Thromb Res. 1997;85:367-368[Medline] [Order article via Infotrieve]. 47. Hasa M, Jackson SK, Siclair AJ. Aspirin-induced conformational changes in platelet membrane in subjects with stroke. Gerontology. 1995;41:212-219[Medline] [Order article via Infotrieve]. 48. Watala C, Gwozdzinski K. Effect of aspirin on conformation and dynamics of membrane proteins in platelets and erythrocytes. Biochem Pharmacol. 1993;45:1343-1349[CrossRef][Medline] [Order article via Infotrieve]. 49. Villanueva GV, Allen N. Acetylation of antithrombin III by aspirin. Semin Thromb Hemost. 1986;12:213-215[Medline] [Order article via Infotrieve].
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Björnsson TD, Schneider DE, Berger H Jr.
Aspirin acetylates fibrinogen and enhances fibrinolysis: fibrinolytic effect is independent of changes in plasminogen activator levels.
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1989;250:154-161 51. Dominguez-Jimenez C, Diaz-Gonzalez F, Gonzalez-Alvaro I, Cesar JM, Sanchez-Madrid F. Prevention of alphaII(b)beta3 activation by non-steroidal antiinflammatory drugs. FEBS Lett. 1999;446:318-322[CrossRef][Medline] [Order article via Infotrieve]. 52. Reverter JC, Beguin S, Kessels H, Kumar R, Hemker HC, Coller BS. Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody: potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis." J Clin Invest. 1996;98:863-874[Medline] [Order article via Infotrieve]. 53. Furman MI, Krueger LA, Frelinger AL III, et al. GPIIb-IIIa antagonist-induced reduction in platelet surface factor V/Va binding and phosphatydylserine expression in whole blood. Thromb Haemost. 2000;84:492-498[Medline] [Order article via Infotrieve].
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Evaluation of the initial phase of blood coagulation using ultrasensitive assays for serine protease.
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1997;272:21527-21533 55. Osnes LTN, Foss KB, Joo GB, et al. Acetylosalicylic acid and sodium salicylate inhibit LPSinduced NF-kB/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alpha (TNF-alpha) in human monocytes. Thromb Haemost. 1996;76:970-976[Medline] [Order article via Infotrieve].
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© 2001 by The American Society of Hematology.
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  P. H. Desai, D. Kurian, N. Thirumavalavan, S. P. Desai, P. Ziu, M. Grant, C. White, R. C. Landis, and R. S. Poston A Randomized Clinical Trial Investigating the Relationship Between Aprotinin and Hypercoagulabilityin Off-Pump Coronary Surgery Anesth. Analg., November 1, 2009; 109(5): 1387 - 1394. [Abstract] [Full Text] [PDF]
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 T. S. Kickler Dr William W. Duke: Pioneer in Platelet Research JAMA, June 3, 2009; 301(21): 2267 - 2269. [Full Text] [PDF]
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 A. Undas, K. Szuldrzynski, K. E. Brummel-Ziedins, W. Tracz, K. Zmudka, and K. G. Mann Systemic blood coagulation activation in acute coronary syndromes Blood, February 26, 2009; 113(9): 2070 - 2078. [Abstract] [Full Text] [PDF]
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 A. Undas, I. Wiek, E. Stepien, K. Zmudka, and W. Tracz Hyperglycemia Is Associated With Enhanced Thrombin Formation, Platelet Activation, and Fibrin Clot Resistance to Lysis in Patients With Acute Coronary Syndrome Diabetes Care, August 1, 2008; 31(8): 1590 - 1595. [Abstract] [Full Text] [PDF]
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 P. A. Gurbel, R. C. Becker, K. G. Mann, S. R. Steinhubl, and A. D. Michelson Platelet Function Monitoring in Patients With Coronary Artery Disease J. Am. Coll. Cardiol., November 6, 2007; 50(19): 1822 - 1834. [Abstract] [Full Text] [PDF]
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A. Undas, K. E. Brummel-Ziedins, and K. G. Mann Antithrombotic properties of aspirin and resistance to aspirin: beyond strictly antiplatelet actions Blood, March 15, 2007; 109(6): 2285 - 2292. [Abstract] [Full Text] [PDF]
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 A. Undas, M. Celinska-Lowenhoff, K. E. Brummel-Ziedins, J. Brozek, A. Szczeklik, and K. G. Mann Simvastatin Given for 3 Days Can Inhibit Thrombin Generation and Activation of Factor V and Enhance Factor Va Inactivation in Hypercholesterolemic Patients Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1524 - 1525. [Full Text] [PDF]
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S. Butenas, B. A. Bouchard, K. E. Brummel-Ziedins, B. Parhami-Seren, and K. G. Mann Tissue factor activity in whole blood Blood, April 1, 2005; 105(7): 2764 - 2770. [Abstract] [Full Text] [PDF]
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A. Undas, K. E. Brummel-Ziedins, and K. G. Mann Statins and Blood Coagulation Arterioscler Thromb Vasc Biol, February 1, 2005; 25(2): 287 - 294. [Abstract] [Full Text] [PDF]
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J. Kawasaki, N. Katori, M. Kodaka, H. Miyao, and K. A. Tanaka Electron Microscopic Evaluations of Clot Morphology During Thrombelastography(R) Anesth. Analg., November 1, 2004; 99(5): 1440 - 1444. [Abstract] [Full Text] [PDF]
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 T. Orfeo, N. Brufatto, M. E. Nesheim, H. Xu, S. Butenas, and K. G. Mann The Factor V Activation Paradox J. Biol. Chem., May 7, 2004; 279(19): 19580 - 19591. [Abstract] [Full Text] [PDF]
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 A. Undas, W. J. Sydor, K. Brummel, J. Musial, K. G. Mann, and A. Szczeklik Aspirin Alters the Cardioprotective Effects of the Factor XIII Val34Leu Polymorphism Circulation, January 7, 2003; 107(1): 17 - 20. [Abstract] [Full Text] [PDF]
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 S. N Nowak and L. A Jaber Aspirin Dose for Prevention of Cardiovascular Disease in Diabetics Ann. Pharmacother., January 1, 2003; 37(1): 116 - 121. [Abstract] [Full Text] [PDF]
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K. G. Mann, S. Butenas, and K. Brummel The Dynamics of Thrombin Formation Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 17 - 25. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-chain, releasing fibrinopeptides A (FPA) and B
(FPB), respectively.
-carboxamido and 
-amino groups of glutamyl and lysyl
residues in the 
