261
JACC Vol. 33, No. 1
January 1999:261– 6
Increased Reactivity of Platelets Induced by Fibrinogen Independent
of Its Binding to the IIb-IIIa Surface Glycoprotein:
A Potential Contributor to Cardiovascular Risk
DAVID J. SCHNEIDER, MD, FACC, DOUGLAS J. TAATJES, PHD,* DIANTHA B. HOWARD, MS,
BURTON E. SOBEL, MD, FACC
Burlington, Vermont
Objectives. To determine whether augmented activation (degranulation) of platelets might contribute to the association
between higher concentrations of fibrinogen and risk of myocardial infarction, we characterized adenosine diphosphate (ADP)induced expression of P-selectin by platelets in whole blood as a
function of this exposure to selected concentrations of fibrinogen.
Background. An increased risk of myocardial infarction has
been associated with increased concentrations of fibrinogen.
Methods. Fibrinogen was added to blood anticoagulated with
corn trypsin inhibitor (a specific inhibitor of Factor XIIa without
effect on other coagulation factors). Degranulation of platelets
was identified by flow cytometry.
Results. Addition of fibrinogen to blood did not activate platelets under basal conditions (without ADP). By contrast, a
concentration-dependent increase in ADP and thrombin receptor
agonist peptide (TRAP)-induced activation occurred with increas-
ing concentrations of fibrinogen. Increased ADP-induced degranulation was apparent with the addition of 100 mg/dl of fibrinogen
(p <
2 0.001 for 1.5 mmol/liter ADP, n 5 10 subjects). Inhibition by
abciximab of binding of fibrinogen to the surface glycoprotein
IIb-IIIa did not attenuate the observed augmentation of reactivity
induced by fibrinogen. Augmented degranulation was associated
with uptake of fibrinogen into a-granules without surface binding
despite pretreatment with abciximab as shown by laser scanning
confocal microscopy.
Conclusions. Fibrinogen in blood augments degranulation of
platelets in response to ADP and is accompanied by uptake of
fibrinogen into a-granules. Thus, elevated concentrations of fibrinogen secondary to inflammation implicated in cardiovascular
risk may operate, in part, by increasing reactivity of platelets.
(J Am Coll Cardiol 1999;33:261– 6)
©1998 by the American College of Cardiology
Increased concentrations of fibrinogen in blood have been
associated with an increased risk of cardiovascular events
independent of other known risk factors (1–5). Its positive
predictive value for cardiovascular events exceeded that for
cholesterol in the Northwick Park Heart Study (3). One
proposed mechanism is a predisposition to exuberant thrombosis complicating atherosclerotic plaque rupture because
increased concentrations of fibrinogen have been associated
with an increased deposition of fibrin accompanying activation
of coagulation factors (6,7). In addition, increased fibrinogen
may predispose to aggregation of platelets secondary to surface glycoprotein binding and cross-linking of activated platelets (8).
Activation of platelets is complex and entails 1) a conformational change in the surface glycoprotein IIb-IIIa that
exposes a binding site for fibrinogen on the activated conformer that facilitates aggregation (9); 2) degranulation, involving release of the contents of a-granules and of dense
granules (not necessarily concordantly); 3) a procoagulant
response involving exposure of negatively charged phospholipids on the surface of the platelet that facilitates assembly of
coagulation factor complexes tenase and prothrombinase (10);
and 4) change in the shape of the platelet with pseudopod
extension. The a-granule degranulation occurs by fusion of the
vesicles with the plasma membrane and leads to exposure on
the platelet surface of membrane-bound granular proteins
including P-selectin (11).
We have recently characterized a method to define activation of platelets in whole blood with the use of flow cytometry
(12). We have found that the conditions used facilitate identification of agents that alter the reactivity of platelets. Because
of the possibility that fibrinogen would alter platelet reactivity
independent of its capacity to aggregate already activated
platelets through binding to glycoprotein IIb-IIIa, the present
studies were performed to characterize effects of fibrinogen on
the threshold for activation of platelets. Activation of platelets
was characterized by a-granule degranulation reflected by
surface expression of P-selectin. Our studies were designed to
determine 1) whether elevated concentrations of fibrinogen in
From the Departments of Medicine and *Pathology, The University of
Vermont College of Medicine, Burlington, Vermont. These studies were supported in part by a grant from the National Science Foundation (NSF EPS9703938).
Manuscript received November 18, 1997; revised manuscript received August 10, 1998, accepted September 10, 1998.
Address for correspondence: Dr. David J. Schneider, Cardiovascular Division, Colchester Research Facility, University of Vermont, 55A South Park
Drive, Colchester, Vermont 05446. E-mail: [email protected].
©1998 by the American College of Cardiology
Published by Elsevier Science Inc.
0735-1097/99/$20.00
PII S0735-1097(98)00515-4
262
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PLATELET REACTIVITY AND FIBRINOGEN
Abbreviations and Acronyms
ADP 5 adenosine diphosphate
FITC 5 fluorescein isothiocyanate
PBS 5 phosphate buffered saline
PE
5 phycoerythrin
TRAP 5 thrombin receptor agonist peptide
blood alter reactivity of platelets in addition to the augmentation of aggregation of previously activated platelets, and 2)
whether the threshold for degranulation is lowered despite
inhibition of binding of fibrinogen to the surface glycoprotein
IIb-IIIa by abciximab (ReoPro).
Methods
Human subjects. Blood was obtained from 19 human
subjects that had not taken aspirin or any nonsteroidal antiinflammatory drugs for at least 10 days. All subjects gave
written informed consent before phlebotomy. The study protocol was approved by the Institutional Review Board of the
University of Vermont.
Phlebotomy was performed with the use of a two syringe
technique whereby the first 3 ml of blood was discarded.
Activation of platelets was delineated in blood drawn directly
into a syringe containing 32 mg/ml of corn trypsin inhibitor
(CTI, 9:1 v/v), an agent that inhibits Factor XIIa without
affecting other coagulation factors (12,13). Unlike CTI, other
anticoagulants, particularly chelators of calcium such as citrate,
alter activation of platelets (12). Additional aliquots of blood
were drawn into syringes containing CTI (32 mg/ml) plus
4 mg/ml, 10 mg/ml and 100 mg/ml of ReoPro (Centocor BV,
Leiden, Netherlands, 9:1 v/v). The concentration of fibrinogen
was assayed by the method of Clauss (14) in plasma anticoagulated with sodium citrate (0.129 mol/liter, pH 6.0) that was
stored at 280°C until assay.
Activation of platelets. Fibrinogen (free of plasminogen
and von Willebrand Factor, homogeneous by SDS–PAGE, and
greater than 95% clottable; Enzyme Research, South Bend,
Indiana) was dialyzed exhaustively against phosphate buffered
saline (PBS) to remove citrate. Whole blood was treated with
PBS (control) and with PBS plus fibrinogen to increase the
concentration of fibrinogen in selected aliquots by 50 mg/dl,
100 mg/dl, 200 mg/dl, 300 mg/dl or 500 mg/dl. Albumin and
ferritin (Sigma, St. Louis, Missouri) were resuspended in PBS,
and molar concentrations equivalent to 100 mg/dl, 200 mg/dl
and 500 mg/dl of fibrinogen were added to blood to determine
the specificity of effects of fibrinogen.
Activation of platelets was induced as described previously
(12). Briefly, 5 ml of blood was added to microcentrifuge tubes
containing HEPES-Tyrode’s buffer (5 mmol/liter HEPES,
137 mmol/liter NaCl, 2.7 mmol/liter NaHCO3, 0.36 mmol/liter
NaH2PO4, 2 mmol/liter CaCl2, 4 mmol/liter MgCl2 and 5 mmol/
liter dextrose, pH 7.4), a fluorescein isothiocyanate (FITC)conjugated IgG directed against glycoprotein IIb-IIIa (HP1-
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1D, 4 mg/ml), a phycoerythrin (PE)-conjugated IgG directed
against P-selectin (Becton Dickinson, San Jose, California,
0.6 mg/ml), and selected concentrations of adenosine diphosphate (ADP) (0, 0.2, 0.8 and 1.5 mmol/liter) and thrombin
receptor agonist peptide (20 mmol/liter thrombin receptor
agonist peptide [TRAP], Bachem). The HP1-1D binds to an
activation-independent epitope on glycoprotein IIb-IIIa and
was used to identify platelets. The P-selectin expression was
used identify a-granule degranulation (11). Blood was added
to the reaction tubes within 15 min after phlebotomy.
After addition of blood, the reaction solution was mixed
gently and incubated at room temperature for 15 min. Subsequently, the platelets were fixed, and the red blood cells were
lysed by addition of Optilyse-C (Immunotech, Westbrook,
Maine). Thus, platelets were exposed to the added fibrinogen
for approximately 30 min before fixation. Control tubes (containing FITC-HP1-1D and nonfractionated PE-IgG) were used
for detection of nonspecific antibody association with platelets.
The association of antibodies with platelets was detected with
the use of a fluorescence-activated cell sorter (Becton Dickinson) as previously described (12).
Binding of fibrinogen to platelets was detected with the use
of flow cytometry. Fibrinogen was labeled with FITC as has
been described previously (15). The less stringent labeling
conditions used with celite FITC (Calbiochem, La Jolla, California) preserve functional activity of fibrinogen and do not
alter binding of fibrinogen to activated platelets (15). Binding
of fibrinogen was detected by the addition of 5 ml of blood to
microcentrifuge tubes containing HEPES-Tyrode’s buffer,
FITC-fibrinogen (0.1 mg/ml), a PerCP conjugated IgG
(2.3 mg/ml) directed against glycoprotein IIIa (anti-CD61,
Becton Dickinson), and selected concentrations of ADP (0,
0.2, 0.8 and 1.5 mmol/liter). Binding of anti-CD61 to glycoprotein IIIa is independent of activation and does not interfere
with binding of fibrinogen to platelets (information from
supplier). The population of platelets identified by HP1-1D is
identical to that identified by anti-CD61. After addition of
blood to the reagent tubes, samples were mixed gently and
incubated for 15 min at room temperature. Platelets were
fixed, and red blood cells were lysed with Optilyse C. Control
tubes with albumin-FITC and anti-CD61 PerCP were used to
detect nonspecific association of proteins with platelets.
Confocal laser scanning microscopy. The association with
and localization in platelets of exogenous fibrinogen were
determined in blood that was anticoagulated with CTI or with
CTI spiked with ReoPro (10 mg/ml) and exposed to 300 mg/dl
of FITC-fibrinogen for 15 min. Platelets were fixed and red
blood cells were lysed with Optilyse C. The plasma membranes
of the platelets were rendered permeable by addition of 0.1%
Triton X-100 (Sigma) and anti-CD62 (P-selectin) was added.
The platelet suspension was incubated for 15 min and then
applied to a glass microscopic slide for 15 min. The slide was
then washed and an Alexa 568 conjugated goat anti-mouse IgG
(Molecular Probes, Eugene, Oregon) was applied for 30 min.
After additional washes the samples were air-dried and a
cover-slip was applied with 1% n-propyl gallate (Sigma) in
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SCHNEIDER ET AL.
PLATELET REACTIVITY AND FIBRINOGEN
50% glycerol: 50% PBS. The slides were evaluated with a
BioRad (Hercules, California) MRC 1000 confocal scanning
laser system equipped with a krypton/argon laser mounted on
an Olympus BX50 microscope.
Platelets were imaged with the use of a 100X phase-contrast
oil immersion lens (numerical aperture 5 1.3) and an electronic zoom factor of 2.4. Phase-contrast (nonconfocal) images
were acquired with a transmitted light detector attachment.
The FITC-fibrinogen was visualized with the use of 488-nm
laser excitation, and the Alexa 568 conjugated secondary
antibody localizing P-selectin was visualized with 568-nm laser
excitation. Co-localization analysis was performed with the
multiply function in Macro Programming Language Software
(BioRad). This function multiplies each pixel in the active
display box by the corresponding pixel of a second image. After
multiplication, the resulting image displays color only in areas
where fluorescence was present in both images to co-localize
two fluorochromes.
Analysis of data. Values are means 6 SEM. Repeated
measures analysis of variance (ANOVA) using a mixed model
with block compound symmetry that addressed the heterogeneity of variances across concentrations of ADP was performed to test the effect on P-selectin expression of ADP in
combination with fibrinogen, albumin, ferritin and ReoPro
concentrations. After definition of significance with ANOVA,
Dunnett’s test was used to compare each concentration of
fibrinogen, albumin and ferritin with control. Results with
TRAP were analyzed with the use of paired Student t tests.
Significance was defined as p # 0.05.
Results
Results are from assays from each individual donor with
respect to each concentration of ADP performed in triplicate
in each case. The effect of fibrinogen on the activation of
platelets induced by ADP was determined in blood taken from
19 subjects, 11 male and 8 female normal volunteers ranging in
age from 20 to 55 years and who had no significant medical
history and were not taking medications. The hematocrit
(range 39% to 44%), hemoglobin (range 12 to 15 mg/dl), white
blood count (range 3,700 to 9,000/mm3) and platelet count
(range 140,000 to 290,000/mm3) were normal. The average
concentration of fibrinogen was 257 6 66 (SD).
Compared with control conditions, fibrinogen significantly
increased the activation of platelets in response to ADP (Figs.
1 and 2). A significant interaction (p , 0.001) was observed
between ADP and fibrinogen. No activation (,1%) of platelets was observed after addition of PBS (control) or after
addition of fibrinogen in the absence of ADP. The P-selectin
expression in response to 0.2 mmol/liter ADP was increased
significantly when 300 and 500 mg/dl of fibrinogen were added
to blood; P-selectin expression in response to 0.8 mmol/liter
ADP was increased significantly when 200, 300 and 500 mg/dl
of fibrinogen were added to blood. Also, P-selectin expression
in response to 1.5 mmol/liter ADP was increased significantly
263
Figure 1. The effect of addition of fibrinogen to blood on ADPinduced expression of P-selectin. Blood was anticoagulated with CTI
(32 mg/ml). Degranulation of a-granules was determined with flow
cytometry and based on detection of surface expression of P-selectin.
Values are means 6 SEM determined in blood samples from 10
subjects. A significant (p , 0.001) interaction between fibrinogen and
ADP was observed. The addition of fibrinogen ($100 mg/dl) significantly increased P-selectin expression in response to 1.5 mmol/liter
ADP.
when 100, 200, 300 and 500 mg/dl of fibrinogen were added to
blood.
Fibrinogen increased the extent of degranulation of platelets induced by TRAP. The P-selectin expression in response
to 20 mmol/liter TRAP was nearly doubled after addition of
500 mg/dl of fibrinogen to blood (percentage of platelets
expressing P-selectin, control 5 24.5 6 7.2, addition of
500 mg/dl of fibrinogen 5 42 6 6.6, p 5 0.01, n 5 7 subjects).
To determine whether the effects of fibrinogen were specific, albumin and ferritin were added to blood in molar
concentrations equivalent to 100 mg/dl, 200 mg/dl and 500 mg/dl
of fibrinogen before determination of degranulation in reFigure 2. The effect of addition of fibrinogen to blood on ADPinduced (1.5 mmol/liter) expression of P-selectin. Values are expression of P-selectin expressed as a percentage of control values and are
means 6 SEM determined in blood samples from 19 subjects for
samples with addition of 500 mg/dl, 12 subjects for samples with
addition of 50 mg/dl and 300 mg/dl, and 10 subjects for samples with
addition of 100 mg/dl and 200 mg/dl. Results with the addition of each
concentration of fibrinogen greater than 50 mg/dl showed significant
increases (p , 0.001) compared with control.
264
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PLATELET REACTIVITY AND FIBRINOGEN
Figure 3. The effect of addition of fibrinogen, albumin and ferritin to
blood on ADP-induced (average of results with all concentrations of
ADP used) expression of P-selectin. Values are P-selectin expressed as
a percentage of control values and are means 6 SEM determined in
blood samples from four subjects. Equimolar concentrations of albumin and ferritin were selected to permit determination of the specificity of results with the addition of 100 mg/dl, 200 mg/dl and 500 mg/dl
of fibrinogen.
sponse to ADP (Fig. 3). Although the addition of 6 mmol/liter
albumin (equivalent to 200 mg/dl of fibrinogen) led to significant (p 5 0.04) increase in ADP-induced P-selectin expression,
no change in ADP-induced P-selectin expression was seen with
3 and 15 mmol/liter albumin. The addition of ferritin did not
alter ADP-induced P-selectin expression.
To clarify mechanisms by which fibrinogen led to increased
ADP-induced degranulation of platelets, the binding of fibrinogen to the surface glycoprotein IIb-IIIa was inhibited pharmacologically. We found that the addition of greater than 4
mg/ml of ReoPro to blood inhibited binding of FITCfibrinogen to platelets (Fig. 4). The addition of 4 mg/ml,
Figure 4. The binding of fibrinogen (FITC labeled) to platelets in
response to ADP. Blood was anticoagulated with CTI (32 mg/ml) and
results were compared with those in blood anticoagulated with CTI
and spiked with ReoPro (4, 10 and 100 mg/ml). The binding of 10 mg/dl
of FITC-fibrinogen to platelets was determined with the use of flow
cytometry. Values are means 6 SEM of results of triplicate determination in blood from two representative subjects.
JACC Vol. 33, No. 1
January 1999:261– 6
Figure 5. The effect of ReoPro on a-granule degranulation induced by
ADP after addition of fibrinogen to the sample. Fibrinogen (500 mg/
dl) was added to blood that had been anticoagulated with CTI or
anticoagulated with CTI and spiked with ReoPro (4, 10 and 100 mg/ml).
Values are means 6 SEM of results with blood samples from 10 to 19
subjects. No significant differences were observed when results under
the two sets of conditions were compared.
10 mg/ml and 100 mg/ml of ReoPro to blood did not alter the
augmentation of expression of P-selectin observed when
500 mg/dl of fibrinogen was added to blood followed by the
exposure of the platelets to ADP (Fig. 5).
As can be seen in Figure 4, despite inhibition of binding of
fibrinogen to the surface glycoprotein IIb-IIIa, the exposure of
platelets to 10 mg/dl of FITC-fibrinogen resulted in approximately 5% to 15% of platelets exhibiting associated FITCfibrinogen. Results with confocal scanning laser microscopy
demonstrated that the FITC-fibrinogen was localized in
a-granules (n 5 5 subjects, Fig. 6). No FITC-fibrinogen was
observed on the surface of quiescent platelets that had not
been exposed to ADP. Thus, the exposure of platelets to
fibrinogen led to its uptake into a-granules. When blood was
pretreated with ReoPro (10 mg/ml) before exposure of platelets to FITC-labeled fibrinogen, the FITC-fibrinogen was again
localized in the a-granules (n 5 5 subjects, Fig. 6). Accordingly, inhibition of binding of fibrinogen to the surface glycoprotein IIb-IIIa by ReoPro does not inhibit uptake of fibrinogen into a-granules of nonactivated platelets.
Discussion
In the present study we found that the addition of fibrinogen to whole blood increased degranulation of platelets induced by ADP and TRAP. Fibrinogen, per se, did not activate
platelets under basal conditions (without the addition of an
agonist). Accordingly, fibrinogen is not a direct agonist but
rather a factor that augments a-granule degranulation induced
by agonists such as ADP and TRAP. The increased predisposition to agonist-induced degranulation of platelets induced by
fibrinogen may contribute to the increased risk of cardiovascular events in subjects with increased concentrations of fibrinogen (16).
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PLATELET REACTIVITY AND FIBRINOGEN
265
Figure 6. The association of FITC-fibrinogen
with quiescent platelets in blood anticoagulated
with CTI (32 mg/ml) and exposed to FITCfibrinogen (300 mg/dl) for 15 min (micrographs
on left: A, C, and E). The platelets were fixed in
Optilyse-C and exposed subsequently to 0.1%
Triton X-100 and anti-P-selectin IgG. After
washing, platelets were exposed to a secondary
Alexa-conjugated anti-mouse IgG. The concentration of fibrinogen in blood from this subject
was 360 mg/dl. The total concentration of fibrinogen (endogenous plus FITC-fibrinogen) to
which the platelets were exposed was 330 mg/dl.
The micrographs on the right (B, D, and F) show
results with blood drawn into CTI spiked with
ReoPro (10 mg/ml). For each set of observations, the micrograph on the top (A and B) is a
representative platelet imaged with phase contrast with the transmitted light detector of the
confocal microscope merged with fluorescence
(excitation 488 nm) demonstrating FITCfibrinogen (green signal). The micrograph in the
center (C and D) is the same platelet visualized
with phase-contrast transmitted light and fluorescence (excitation 568 nm) demonstrating
P-selectin expression (red signal). The red signal
localizes a-granules. The micrograph on the
bottom shows the same platelet viewed by multiplying fluorescent images (excitation 488 and
568) and thus the degree of co-localization of
fibrinogen-FITC and P-selectin (yellow color).
Fluorescence in images A and C are multiplied
to create E, and fluorescence in images B and D
are multiplied to create F. Yellow demonstrates
localization of fibrinogen in a-granules. Bar 5
5 mm; all images are same magnification.
Fibrinogen and glycoprotein IIb-IIIa. Binding of fibrinogen to the activated surface glycoprotein IIb-IIIa is responsible
for cross-linking and hence aggregation of platelets (9,17). To
determine whether binding of fibrinogen could potentiate
ADP-induced degranulation of platelets, surface binding was
inhibited by the addition of ReoPro to blood. Addition of
fibrinogen augmented the ADP-induced degranulation of
platelets despite verified inhibition of surface binding of fibrinogen to the activated IIb-IIIa conformer. With the use of
confocal scanning laser microscopy we found that the fibrinogen did not bind to the surface of quiescent platelets. By
contrast, the exposure of platelets to added fibrinogen resulted
in uptake of the fibrinogen into a-granules. This uptake was
not abolished by pretreatment with ReoPro. Thus, uptake into
a-granules of fibrinogen may potentiate degranulation of
platelets.
Fibrinogen and a-granules. One potential mechanism by
which the uptake of fibrinogen into a-granules could potentiate degranulation of platelets is through a mass effect. That is,
an increase in the mass of proteins in the a-granules could
potentiate degranulation. Additional mechanisms could include 1) changes in intra-platelet concentration or localization
of calcium necessary for degranulation, 2) changes in the
intraplatelet concentrations of proteins or nucleotides such as
cAMP or cGMP that alter the reactivity of platelets, and 3)
changes in the expression of surface receptors for agonists.
The protein content of a-granules of platelets is derived
from endogenously synthesized megakaryocyte proteins and
from endocytosis of circulating plasma proteins. Megakaryocytes do not synthesize fibrinogen (18,19). Fibrinogen has been
identified in a-granules of platelets after intravenous injection
(20,21). Our results suggest that the prevailing concentration
of fibrinogen in blood can influence not only uptake of
fibrinogen into a-granules but also agonist-induced degranulation of platelets.
Mechanisms of uptake of fibrinogen. Mechanisms by
which fibrinogen is taken up into a-granules are incompletely
understood. Treatment of guinea pigs with Kistrin, an antagonist of both the integrins glycoprotein IIb-IIIa and avb3,
inhibits the uptake of biotinylated fibrinogen (22). Our data
demonstrate that augmented activation of platelets occurs
after exposure to fibrinogen despite pretreatment with ReoPro
at a concentration that inhibits binding of fibrinogen to platelets.
ReoPro inhibits intracellular trafficking of fibrinogen and
binding of fibrinogen to avb3 (23,24). Thus, three possibilities
may account for the increased ADP-induced degranulation of
platelets produced by fibrinogen: 1) uptake mediated by
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SCHNEIDER ET AL.
PLATELET REACTIVITY AND FIBRINOGEN
binding of fibrinogen to other integrins not inhibited by
ReoPro or mediated by the pool of integrins (IIb-IIIa and
avb3) that are not associated with the added ReoPro; 2) uptake
of fibrinogen into a-granules independent of binding to surface
integrins; or 3) mechanisms independent of uptake of fibrinogen into a-granules.
Clinical implications. The increased ADP-induced degranulation of platelets induced by fibrinogen may contribute,
in part, to the impact of inflammation on risk of myocardial
infarction. Recently, protective effects of aspirin in the primary
prevention Physicians’ Health Study were found to be most
prominent in those subjects who had increased concentrations
in blood of another acute-phase reactant, C-reactive protein
(25). Subjects with evidence of previous infection with chlamydia, cytolomegalovirus, and heliobacter are at increased risk
of cardiac events (26 –29). Any process that causes an increase
in the concentrations in blood of acute-phase reactants is likely
to increase concentrations of fibrinogen and, as judged from
our results, increase the propensity for degranulation of platelets. Thus, for example, pregnancy is associated with both
increased concentrations of fibrinogen and increased reactivity
of platelets (30). Elucidation of mechanisms responsible for
increased propensity for degranulation of platelets induced by
fibrinogen may provide insights useful for development of
novel pharmacologic agents designed to attenuate their sensitivity to increased concentrations of fibrinogen in blood.
The authors thank Paula B. Tracy, PHD, for helpful discussion and review of the
manuscript, and Julie L. Ludko, Colette Charland, Matthew Hofmann, and
Marilyn Wadsworth for expert technical assistance.
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