© 1999 American Heart Association, Inc.
Original Contribution |
Deficient Platelet-Derived Nitric Oxide and Enhanced Hemostasis in Mice Lacking the NOSIII Gene
From the Departments of Pharmacology and Medicine (J.E.F.), Georgetown University Medical Center, Washington, DC; Whitaker Cardiovascular Institute and Evans Department of Medicine (R.S., E.M.B., J.L.), Boston University School of Medicine, Mass; Maine Medical Center Research Institute (K.A., C.K.), South Portland; and Cardiovascular Research Center (P.L.H.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass.
Correspondence to Jane E. Freedman, Med-Dent Bldg, Room NE 403, Georgetown University Medical Center, 3900 Reservoir Rd, NW, Washington, DC 20007. E-mail freedmaj{at}gunet.georgetown.edu
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Abstract |
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Abstract—Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2±3 versus 133.4±3 seconds, P<0.00005). To determine the contribution of endothelium- versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (
bleeding time, -24.6±9.1 and -3.4±5.3 seconds, respectively;
P<0.04). Platelet recruitment was studied by
measuring serotonin release from a second recruitable
population of platelets that were added to stimulated platelets
at the peak of NO production. There was 40.3±3.7% and
52.0±2.1% serotonin release for platelets added to
wild-type or eNOS-deficient platelets, respectively
(P<0.05). In summary, mice that lacked eNOS had
markedly decreased bleeding times even after
endothelial NO production was controlled. These
data suggest that the lack of platelet-derived NO alters in vivo
hemostatic response by increasing platelet recruitment. Thus, these
data support a role for platelet-derived NO production in
the regulation of hemostasis.
Key Words: selectin • mice • platelet • nitric oxide synthase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal hemostatic balance is maintained by tight regulation of platelet activation and recruitment. Adhesion of platelets to the endothelium is prevented by several mechanisms, including endothelial cell production of nitric oxide (NO).1 2 NO inhibits platelet adhesion and aggregation3 4 and prevents thrombosis.5 Exogenous NO inhibits the normal activation-dependent increase in the expression of platelet surface glycoproteins, including P-selectin and the integrin glycoprotein IIb/IIIa complex.6 In addition, we have shown that a functional decrease in exogenous NO can lead to a clinical thrombotic disorder.7 8
In addition to its presence in endothelial cells, constitutive NO synthase (NOS) has been identified in human platelets and megakaryocytic cells.9 10 11 12 Consistent with these observations, studies report NO release from aggregating platelets.13 14 15 Platelet aggregation is enhanced by incubation with inhibitors of NOS and inhibited by incubation with the NOS substrate L-arginine.16 Although platelet-derived NO appears to inhibit the primary aggregation response only modestly, we have recently shown that NO release from activated human platelets markedly inhibits platelet recruitment15 and thus may attenuate the progression of intra-arterial thrombosis. In vivo, systemic infusion of a NOS inhibitor causes a reduction in bleeding time without changing vessel tone17 and enhances platelet reactivity to various agonists,18 which supports the clinical relevance of platelet-derived NO. In addition, it is well established that thrombosis is the usual cause of unstable angina and myocardial infarction19 20 and, more importantly, activated platelets from patients with these acute coronary syndromes produce significantly less NO versus patients with stable coronary artery disease.21 This observation suggests that impaired platelet-derived NO may contribute to the development of acute coronary syndromes by enhancing platelet function or recruitment and subsequently thrombus formation.
Homozygous eNOS-mutant mice have been studied and are known to have impaired endothelium-derived relaxing factor activity,22 increased blood pressure, decreased heart rate, and increased plasma renin concentration.23 In the pulmonary vasculature, eNOS deficiency produces mild pulmonary hypertension.24 Although vascular reactivity has been extensively characterized in these mice, hemostatic and thrombotic responses have not yet been studied. Therefore, eNOS-mutant mice were examined to define the role of platelet-derived NO in platelet function and hemostasis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All studies were approved by the Institutional Animal Care and Use Committee at Boston University. The generation of mice with the NOSIII gene deletion has been previously described in detail.22 Ten-week-old male mice that weighed 22 to 24 g were used for all experiments. Mice deficient in eNOS(-/-) were compared with both SV-129 and c57 wild-type mice (Charles River Laboratories, Cambridge, Mass), which represented the strains from which the eNOS-deficient mice (Massachusetts General Hospital) were derived. Blood was drawn into syringes and anticoagulated with trisodium citrate by inferior vena caval puncture. Bone marrow cells were isolated from euthanized mice by gently perfusing the marrow cavity of isolated femurs with 10 mmol/L sodium phosphate, pH 7.2, and 150 mmol/L NaCl after extirpation of the proximal and distal metaphyses.
Reverse Transcription–Polymerase Chain Reaction for Mouse NOSIII
and P-Selectin
Total RNA was extracted from bone marrow cells with a commercial
solution that contained guanidinium thiocyanate (RNAsol,
Cinna/ Biotecx). Contaminating DNA was digested with RNase-free
DNaseI (Boehringer-Mannheim Biochemicals). Reverse
transcription (RT) was performed with 150 ng of total RNA in 20 µL of
a reaction mixture that contained 50 mmol/L Tris-HCl (pH 8.3),
75 mmol/L KCl, 3 mmol/L MgCl2, 10
mmol/L DTT, 0.5 mmol/L of each dNTP, 200 U of superscript reverse
transcriptase (Gibco BRL), and 10 µg/mL
oligo-dT16. After incubation at 42°C for 1
hour, the reaction was stopped by incubation at 70°C for 5 minutes. A
nested primer polymerase chain reaction (PCR) was performed with 10
µL of the reaction mixture from the RT reaction in 10
mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 200 µmol/L of each dNTP, and 2.5 U
Taq DNA polymerase (Boehringer-Mannheim). Primer
sequences used for the first and second PCR reactions for eNOS were
5'-ggtactactctgtcagttcagcac and 5'-tatttctgggccaggcgggtcaaa, and
5'-gagatccagtgccctgcttcatca and 5'-tgtcacctcctgggtgcgcaatgt,
respectively. Primer sequences used for the first and second PCR
reactions for P-selectin were 5'-tccaggaagctctgacgtacttgg
and 5'-tgtcccctagtaccatctggaggt, and
5'-gcttctacaacaggcctggcagtg and
5'-tgcccaccaacattgctactctgc, respectively. Primers were added at a
final concentration of 1.0 µmol/L. Amplification was performed
in a DNA thermal cycler. A second PCR reaction was performed with 10
µL of the original PCR mixture and the nested primer pairs under the
same conditions. Amplified products were analyzed by 1.5%
agarose gel electrophoresis and visualized under UV illumination after
they were stained with ethidium bromide.
Preparation of Platelets
The blood was centrifuged (150g, 10 minutes,
22°C) and the supernatant, which represented
platelet-rich plasma (PRP), was separated. Gel-filtered
platelets (GFP) were prepared by passing PRP over a Sepharose-2B
column equilibrated with Tyrode's-HEPES buffered saline as previously
described.25 Platelet counts were determined with
a Coulter Counter, model ZM (Coulter Electronics).
Measurement of Platelet NO Production and
Aggregation
We adapted a NO-selective26 microelectrode (Inter
Medical Co Ltd) for use in a standard platelet aggregometer (Payton
Associates) to monitor platelet NO production and
aggregation simultaneously as previously
described.15 Platelet NO production was
quantified as the integrated signal detected by the microelectrode
after platelet activation with 5 µmol/L ADP. Aggregation of
GFPs was monitored with a standard nephelometric technique as
previously described.27 28
Measurement of Platelet Surface P-Selectin Expression by
Flow Cytometry
Resting or epinephrine-activated (100
µmol/L) platelets in whole blood were incubated with CD62 (rabbit
anti–P-selectin antibody) in PBS for 15 minutes at 22°C. This
concentration of epinephrine causes the maximal aggregation
response in mouse platelets. Samples were then fixed in
paraformaldehyde (1% FC) and incubated for 30 minutes
at 22°C. Samples were diluted 10-fold in PBS, and the cells were
collected by centrifugation (1700g, 5
minutes). The samples were incubated with PE–GaRG (BioSource
International) for 15 minutes at 22°C. After the samples were washed
with PBS, they were incubated with FITC-D9 (CD41 rat anti-mouse
antibody) and incubated for 15 minutes at 22°C. Samples were
resuspended in PBS and analyzed in a flow cytometer (FACScan,
Becton Dickinson) with settings for FL1 fluorescence to measure
the FITC D9-labeled platelets. Platelets were gated with a
forward scatter versus FL1 dot plot and a gated histogram of
forward scatter versus FL2 to separate activated from
unactivated platelets.
Measurement of Bleeding Time
Bleeding times were measured by determining the time required
for clotting to occur after a single puncture (2 mm) with a
23-gauge needle to the dorsal tail vein. The bleeding times were
conducted in duplicate in each animal. During the bleeding times, body
temperature was maintained at 36°C to 38°C by placing the mice on
heating pads. Before the mice were killed, blood samples were drawn
from them for measurement of platelet-derived NO and aggregation
studies.
For some experiments, platelets were isolated, pooled, and infused
into eNOS-deficient mice that were rendered thrombocytopenic with a
single intraperitoneal injection of carboplatin
(Bristol-Myers Squibb), a chemotherapeutic agent that causes
thrombocytopenia at a nonlethal dose.29 Carboplatin was
injected intraperitoneally at a dose of 125 mg/kg
to induce thrombocytopenia. By use of this method, platelet counts
in mice decreased by >80% 
12 days after injection. Platelets
were reinfused 12 days after injection, after confirmation of decreased
platelet count. Approximately 1.2x109
platelets were reinfused into each animal. Bleeding times were
determined before and after the platelet infusion.
Platelet Secretion
Platelet secretion was measured with
[14C]-radiolabeled serotonin as
previously described.30 31 GFPs were incubated with
[14C] serotonin at 37°C for 10
minutes. Imipramine was added immediately before the initiation of
secretion to prevent reuptake of secreted serotonin.
Secretion was initiated by the addition of 10 µmol/L ADP and
allowed to proceed for 2 minutes and then terminated by the addition of
ice-cold formaldehyde in 0.05 mol/L EDTA. Samples were spun in a
microfuge at 14 000g for 3 minutes, and radioactivity was
measured in the supernatant.
Thromboxane B2 in Platelets
Platelet isolates (resting and
epinephrine-stimulated) from NO-deficient or control mice were
precipitated with 10% trichloroacetic acid.32
After centrifugation and extraction,
thromboxane B2 levels were measured
by enzyme immunoassay (Cayman Chemicals). The results were expressed as
nanograms of thromboxane B2 per
1x108 platelets.
Statistical Analysis
Differences between groups were determined with an unpaired
Student t test. The effects of the interventions were
analyzed by a paired t test. A statistically
significant difference was assumed with a value of P<0.05.
All data are expressed as the mean±SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Absence of eNOS RNA in Bone Marrow
Previous genetic characterization of eNOS-deficient mice has not examined megakaryocytic cell lines. Therefore, to confirm the absence of eNOS expression in these cells, RNA was extracted from the bone marrow samples of eNOS-deficient and wild-type (c57) animals and amplified by RT-PCR. As shown in Figure 1
, eNOS-deficient animals do not express
NOSIII mRNA in marrow cells (lane 1), although wild-type animals do
(lane 2). This result contrasts with the expression of P-selectin,
which is present in both groups of mice.
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Platelet NO Production
To determine whether the lack of eNOS alters aggregation-dependent
NO production in the mouse platelet, NO production
was measured with a selective electrochemical detector adapted for use
in a platelet aggregometer. GFPs were stimulated with 5
µmol/L ADP, and aggregation and NO release was determined. As can be
seen in the representative tracing (Figure 2A), the eNOS-deficient
platelets do not release measurable NO compared with the stimulated
platelets from both c57 (Figure 2A) and SV-129 (data not
shown) wild-type animals. The consistency of this finding
is demonstrated in the cumulative results shown in Figure 2B.
Comparable levels of platelet NO release were detected with the use
of thrombin and collagen as platelet agonists (data not shown).
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Platelet Surface P-Selectin Expression in Resting and
Stimulated Platelets
Because mice yield insufficient quantities of blood to perform
complete studies of platelet function with standard aggregometry,
the extent of platelet activation was assessed by flow cytometry.
Both resting and stimulated platelets were assessed for surface
expression of P-selectin, a marker of platelet activation, by use
of a rabbit antibody specific for mouse P-selectin. As is shown in
Figure 3, the normal
stimulation-dependent increase in platelet surface expression of
P-selectin is present in both eNOS-deficient and wild-type mice.
Interestingly, there is no significant difference in either of these
levels between the eNOS-deficient and wild-type animals.
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Bleeding Time Measurements in eNOS-Deficient and Wild-Type
Mice
To determine whether the absence of platelet NO release
results in enhanced hemostasis, bleeding times in eNOS-deficient
(NOSIII-/-) and wild-type
(NOSIII+/+) mice were measured. Compared with the
wild-type mice (c57), bleeding times from eNOS-deficient animals were
significantly decreased (bleeding time, 124.6±3 versus 77.2±3
seconds, P<0.00005, Figure 4). This finding was confirmed by use of
SV-129 control mice (126±4 versus 62.6±3 for SV-129 versus
eNOS-deficient mice, respectively; P<0.00005, n=5 for
both).
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The integrity of hemostasis, which was measured by the bleeding time,
is influenced both by endothelial and platelet
function. To determine the contribution of platelet- versus
endothelium-derived NO on the bleeding time and in vivo
hemostasis, platelets from eNOS-deficient or wild-type animals were
transfused into thrombocytopenic eNOS-deficient mice. Bleeding times
were measured before and after the transfusion, and the change in
bleeding time was determined. Bleeding times were decreased in most
animals after platelet infusion. More importantly, mice transfused
with wild-type platelets had less of a decrease in bleeding times
versus those transfused with eNOS-deficient platelets
(bleeding time, -24.6±9.1 versus –-3.4±5.3 seconds;
P<0.04, Figure 5).
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Effect of eNOS Deficiency on Platelet Recruitment
Although the prolonged bleeding times (Figure 5
) suggest
that platelet NO production contributes to hemostasis, no
abnormality was detected in the platelet activation response in
eNOS-deficient animals (Figure 3). With an inhibitor
of NOS, we have previously shown that decreasing platelet-derived
NO modestly attenuates platelet activation but markedly inhibits
platelet recruitment. Therefore, to determine whether the
hemostatic changes detected in the eNOS-deficient animals are due to
alterations in platelet recruitment, we used
[14C] serotonin release, a measure
of platelet-dense granule secretion, as an index of platelet
activation.15 GFPs from wild-type or eNOS-deficient
animals were stimulated with ADP and, at the peak of NO
production, unstimulated
[14C]serotonin-loaded wild-type
GFPs were added and serotonin release measured. When
[14C]serotonin-containing
platelets were added to wild-type or eNOS-deficient platelets,
there was 44.3±2.9% and 52.0±2.1% serotonin release,
respectively (Figure 6;
P<0.05).
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To confirm that the change in serotonin release was due to the absence of NO, this experiment was repeated with platelets from wild-type animals that had been incubated with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). GFPs treated with 300 µmol/L of L-NAME or vehicle control were stimulated with ADP and, at the peak of NO production, unstimulated [14C]serotonin-loaded control GFPs were added and serotonin release was measured. When [14C]serotonin-containing platelets were added to control or NOS-inhibited platelets, there was a 35±1.8% or 47.3±3.1% serotonin release, respectively (P<0.05, n=3).
Effect of eNOS Deficiency on Thromboxane Activity
Although bleeding time measurements determined the
contribution of platelet- versus
endothelium-dependent NO release to hemostasis, these
studies did not explore other hemostatic mechanisms that may be
influenced by the presence or absence of NO. Previously, platelet
thromboxane synthase activity was shown to be attenuated by
NO.33 Therefore, the contribution of
endogenous NO to platelet thromboxane
activity was determined by measuring thromboxane
B2, the chemically stable hydration product
of thromboxane A2. There was no
difference in thromboxane B2
formation in activated platelets from control or
NO-deficient mice (0.55±0.13 versus 0.61±0.16 ng per
108 platelets, respectively; n=4 experiments,
P=ns).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO has been previously shown to be a potent inhibitor of platelet function. Although endothelium-derived NO production is known to both mediate vasorelaxation as well as inhibit platelet adhesion and activation, less is known about the contribution of platelet-derived NO. In this study, which used mice that lacked the NOSIII gene, we demonstrate that platelet-derived NO production contributes to hemostasis in vivo. After confirming the lack of NOSIII expression in the marrow (megakaryocytes) of these animals, stimulated platelets were found to lack stimulation-dependent NO release. These animals were also found to have significantly shortened bleeding times. Also, after controlling for endothelium-dependent NO production by inducing thrombocytopenia and transferring wild-type or eNOS-deficient platelets into these animals, they still had reduced bleeding times, which suggests that NO release by both the endothelium and the platelet contributes to hemostasis.
Although the platelet reinfusion experiment determined the contribution of platelet- versus endothelium-dependent NO release to the bleeding time, it did not control for other hemostatic mechanisms that may be influenced by the presence or absence of NO. Previously, platelet thromboxane synthase activity was shown to be attenuated by NO.33 In our study, there was no significant difference in levels of thromboxane B2 in eNOS-deficient versus control mice. However, previous studies that detected NO-dependent changes in thromboxane activity33 used exogenous NO donors at concentrations that greatly exceed levels of endogenous NO release.
As discussed, platelets normally adhere to the subendothelium after intimal injury, which leads to platelet activation. Once activated, platelets promote thrombus growth and recruit additional platelets to the growing thrombus by the release of ADP and serotonin, production of thromboxane A2, and promotion of surface thrombin generation.34 This process is known as the recruitment phase of platelet activation. Interestingly, lack of NO production by platelets was not associated with alterations in stimulation-dependent surface P-selectin expression. However, deficiency in platelet-NO production was associated with enhanced platelet activation in a "recruitable" platelet population assessed by serotonin release. This is consistent with our previous characterization of NO production by the human platelet.15 In this study, the alteration of NO production in platelets that used NOS inhibitors minimally altered platelet activation. By use of a double-labeling technique, platelet-derived NO markedly inhibited recruitable platelets that were added during the peak of NO release. This study suggested that platelet-derived NO regulated platelet recruitment; however, it did not address the in vivo contribution of platelet-derived NO release.
Although platelet NO release does not have a major effect on the primary aggregation response, platelet-derived NO appears to play an important counterregulatory role after platelet activation by inhibiting the recruitment of platelets to the growing thrombus.15 Because platelet recruitment is a critical component in thrombus propagation, it is reasonable to speculate that platelet-derived NO release may play a role in the regulation of hemostasis. In addition, we have recently shown that aggregating platelets from patients with acute coronary syndromes produce less NO.21 Because platelet aggregation and thrombus formation are implicated in unstable angina and myocardial infarction, impaired platelet-derived NO production may also contribute to the development of acute thrombotic events.
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Acknowledgments |
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Dr Freedman is the recipient of a Grant-in-Aid from the Massachusetts Affiliate of the American Heart Association and NIH grant K08 HL-03556. Dr Loscalzo is supported by NIH grants HL-48743, HL-55993, and HL-58976, as well as by a merit award from the Veterans Administration.
Received October 8, 1998; accepted March 18, 1999.
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 V. W.T. Liu and P. L. Huang Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice Cardiovasc Res, January 1, 2008; 77(1): 19 - 29. [Abstract] [Full Text] [PDF]
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 V. Randriamboavonjy, F. Pistrosch, B. Bolck, R. H.G. Schwinger, M. Dixit, K. Badenhoop, R. A. Cohen, R. Busse, and I. Fleming Platelet Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase and {micro}-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone Circulation, January 1, 2008; 117(1): 52 - 60. [Abstract] [Full Text] [PDF]
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 E. Gkaliagkousi, J. Ritter, and A. Ferro Platelet-Derived Nitric Oxide Signaling and Regulation Circ. Res., September 28, 2007; 101(7): 654 - 662. [Abstract] [Full Text] [PDF]
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C. J. Lowenstein Nitric oxide regulation of protein trafficking in the cardiovascular system Cardiovasc Res, July 15, 2007; 75(2): 240 - 246. [Abstract] [Full Text] [PDF]
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T. M.C. Brunini, A. C. Mendes-Ribeiro, J. C. Ellory, and G. E. Mann Platelet nitric oxide synthesis in uremia and malnutrition: A role for L-arginine supplementation in vascular protection? Cardiovasc Res, January 15, 2007; 73(2): 359 - 367. [Abstract] [Full Text] [PDF]
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 M. D. Iafrati, O. Vitseva, K. Tanriverdi, P. Blair, S. Rex, S. Chakrabarti, S. Varghese, and J. E. Freedman Compensatory mechanisms influence hemostasis in setting of eNOS deficiency Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1627 - H1632. [Abstract] [Full Text] [PDF]
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 C. N. Morrell, K. Matsushita, K. Chiles, R. B. Scharpf, M. Yamakuchi, R. J. A. Mason, W. Bergmeier, J. L. Mankowski, W. M. Baldwin III, N. Faraday, et al. Regulation of platelet granule exocytosis by S-nitrosylation PNAS, March 8, 2005; 102(10): 3782 - 3787. [Abstract] [Full Text] [PDF]
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G. B. Robb, A. R. Carson, S. C. Tai, J. E. Fish, S. Singh, T. Yamada, S. W. Scherer, K. Nakabayashi, and P. A. Marsden Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript J. Biol. Chem., September 3, 2004; 279(36): 37982 - 37996. [Abstract] [Full Text] [PDF]
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U. Landmesser, B. Hornig, and H. Drexler Endothelial Function: A Critical Determinant in Atherosclerosis? Circulation, June 1, 2004; 109(21_suppl_1): II-27 - II-33. [Abstract] [Full Text] [PDF]
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S. C. Tai, G. B. Robb, and P. A. Marsden Endothelial Nitric Oxide Synthase: A New Paradigm for Gene Regulation in the Injured Blood Vessel Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 405 - 412. [Abstract] [Full Text]
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A. Ahluwalia, P. Foster, R. S. Scotland, P. G. McLean, A. Mathur, M. Perretti, S. Moncada, and A. J. Hobbs Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment PNAS, February 3, 2004; 101(5): 1386 - 1391. [Abstract] [Full Text] [PDF]
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P. Clutton, A. Miermont, and J. E. Freedman Regulation of Endogenous Reactive Oxygen Species in Platelets Can Reverse Aggregation Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 187 - 192. [Abstract] [Full Text] [PDF]
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J. Shibata, J. Hasegawa, H.-J. Siemens, E. Wolber, L. Dibbelt, D. Li, D. M. Katschinski, J. Fandrey, W. Jelkmann, M. Gassmann, et al. Hemostasis and coagulation at a hematocrit level of 0.85: functional consequences of erythrocytosis Blood, June 1, 2003; 101(11): 4416 - 4422. [Abstract] [Full Text] [PDF]
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 M. Liu, A. Wallmon, C. Olsson-Mortlock, R. Wallin, and T. Saldeen Mixed tocopherols inhibit platelet aggregation in humans: potential mechanisms Am. J. Clinical Nutrition, March 1, 2003; 77(3): 700 - 706. [Abstract] [Full Text] [PDF]
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 K. Gertz, U. Laufs, U. Lindauer, G. Nickenig, M. Bohm, U. Dirnagl, and M. Endres Withdrawal of Statin Treatment Abrogates Stroke Protection in Mice Stroke, February 1, 2003; 34(2): 551 - 557. [Abstract] [Full Text] [PDF]
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A. J. Prorock, A. Hafezi-Moghadam, V. E. Laubach, J. K. Liao, and K. Ley Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H133 - H140. [Abstract] [Full Text] [PDF]
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B. C Kone Molecular biology of natriuretic peptides and nitric oxide synthases Cardiovasc Res, August 15, 2001; 51(3): 429 - 441. [Abstract] [Full Text] [PDF]
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J. E. Freedman, C. Parker III, L. Li, J. A. Perlman, B. Frei, V. Ivanov, L. R. Deak, M. D. Iafrati, and J. D. Folts Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release Circulation, June 12, 2001; 103(23): 2792 - 2798. [Abstract] [Full Text] [PDF]
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J. Loscalzo Nitric Oxide Insufficiency, Platelet Activation, and Arterial Thrombosis Circ. Res., April 27, 2001; 88(8): 756 - 762. [Abstract] [Full Text] [PDF]
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A.C. Mendes Ribeiro, T.M.C. Brunini, J.C. Ellory, and G.E. Mann Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure Cardiovasc Res, March 1, 2001; 49(4): 697 - 712. [Abstract] [Full Text] [PDF]
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 B. D. Car and V. M. Eng Special Considerations in the Evaluation of the Hematology and Hemostasis of Mutant Mice Veterinary Pathology, January 1, 2001; 38(1): 20 - 30. [Abstract] [Full Text] [PDF]
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U. Laufs, K. Gertz, P. Huang, G. Nickenig, M. Bohm, U. Dirnagl, M. Endres, and C. J. Vaughan Atorvastatin Upregulates Type III Nitric Oxide Synthase in Thrombocytes, Decreases Platelet Activation, and Protects From Cerebral Ischemia in Normocholesterolemic Mice Editorial Comment Stroke, October 1, 2000; 31(10): 2442 - 2449. [Abstract] [Full Text] [PDF]
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 J D Pearson Normal endothelial cell function Lupus, March 1, 2000; 9(3): 183 - 188. [Abstract] [PDF]
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B. Yan and J. W. Smith A Redox Site Involved in Integrin Activation J. Biol. Chem., December 15, 2000; 275(51): 39964 - 39972. [Abstract] [Full Text] [PDF]
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