1

Compensatory Mechanisms Influence Hemostasis in the Setting of eNOS-Deficiency

Mark D. Iafrati, Olga Vitseva, Kahraman Tanriverdi, Price Blair, Sybille Rex,

Subrata Chakrabarti, Sonia Varghese, and Jane E. Freedman

From the From the Department of Surgery, Tufts-New England Medical Center, 750 Washington

Street, Boston, MA 02111(M.D.I.), and the Whitaker Cardiovascular Institute and Evans

Department of Medicine, 700 Albany Street, Boston University School of Medicine, Boston, MA

02118 (O.V., K.T., P.B., S.R., S.C., S.V., J.E.F.).

Address for correspondence: Mark D. Iafrati, Tufts-New England Medical Center, 750

Washington Street, Boston, MA 02111. Miafrati@Tufts-NEMC.org

Hemostasis in eNOS-Deficient Mice

Articles in PresS. Am J Physiol Heart Circ Physiol (November 24, 2004). doi:10.1152/ajpheart.00819.2004

Copyright © 2004 by the American Physiological Society.

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Abstract

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide

(NO) is an important mediator of these processes as it prevents platelet adhesion to the

endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-

deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in

vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a

model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-

type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion as

compared to the wild-type mice (p<0.001). Consistent with this finding, plasma markers

suggesting enhanced fibrinolysis (tissue plasminogen activator (t-PA) activity and antigen and D-

dimer levels) were significantly elevated in eNOS-deficient animals. Vascular tissue expression

of t-PA and platelet activity were not altered. In endothelial cells, t-PA is stored in Weibel-

Palade bodies and exocytosis of these storage granules is inhibited by NO. Thus, in the absence

of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced, an observation

also supported by increased von Willebrand factor (vWF) levels observed in eNOS-deficient

animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases

platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent

inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-

dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially

explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and

normal lifespan seen in animals deficient in a pivotal regulator of vascular patency.

Key words: Platelets, thrombosis, nitric oxide, transgenic mice

3

Introduction

Normal hemostatic balance is maintained by tight regulation of coagulation, fibrinolysis,

and platelet activation. Adhesion of platelets to the endothelium is prevented by several

mechanisms including endothelial cell production of nitric oxide (NO) and prostacyclin (3, 22).

Nitric oxide inhibits platelet adhesion and aggregation (2, 26) and prevents thrombosis (25).

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.

Platelet-derived NO appears to inhibit the primary aggregation response only modestly but NO

release from activated human platelets markedly inhibits platelet recruitment(7) and, thus, may

attenuate the progression of intra-arterial thrombosis. The vascular endothelium, which mediates

vasomotor tone, in part, through NO release, has been extensively characterized. Endothelium-

dependent dilation is impaired in human atherosclerotic coronary arteries as well as patients with

cardiovascular disease (1). Although interventions that are believed to enhanced the systemic

bioavailablity of NO have been clearly shown to enhance peripheral and coronary vascular

relaxation (29), correlative changes in NO production and antithrombotic propensity have not

been directly measured in these studies. In fact, estrogen supplementation, shown in multiple

studies to enhance vascular relaxation (8), was associated with enhanced arterial thrombosis in

prospective clinical studies (11, 18).

Homozygous eNOS-mutant mice are known to have impaired endothelium-derived

relaxing factor activity (9), increased blood pressure, decreased heart rate, and increased plasma

renin concentration (24). In the pulmonary vasculature, eNOS deficiency produces mild

pulmonary hypertension (27). In addition, we previously found that activated platelets from mice

lacking eNOS do not release NO (6) and, although no changes were detected in platelet function,

eNOS-deficiency was associated with shortened bleeding times (6, 19) suggesting enhanced

coagulation. The lack of difference in the platelet activation response in eNOS-deficient animals

suggests that platelet- and endothelial-derived NO deficiency alters the in vivo hemostatic

response by another (non-platelet dependent) mechanism. To further explore this question and

determine if NO alters thrombosis in vivo, these animals were studied using a carotid artery

injury model.

4

Materials and Methods

All studies were approved by the Institutional Animal Care and Use Committee at

Boston University. The generation of mice bearing the NOS III (eNOS) gene deletion has been

previously described in detail (9). Male and female mice weighing 22-24 gm and 10 weeks old

were used for all experiments. Blood was drawn into syringes and anticoagulated with trisodium

citrate by inferior vena caval puncture.

Carotid Thrombosis Model

Mice were subject to a full thickness arterial injury model to determine the rate of

thrombotic vessel occlusion. Briefly, the technique of carotid artery injury(5) was used with

slight modification. After establishing adequate anesthesia, a 1 cm vertical midline incision

was made in the neck and the left common carotid artery was exposed and a disc (0.6mm

diameter) saturated with FeCl3 was applied for 3 minute followed by removal and irrigation.

A mini-Doppler flow probe (model 0.5VB, Transonic Systems) was placed on the distal

carotid artery and monitored on a computerized data acquisition system for time to

thrombotic occlusion. Carotid arteries were then excised and fixed.

Measurements of Fibrinolysis

For determination of plasma t-PA activity, a photometric method using a kit with

chromogenic substrate S-2251 (Coaset® t-PA, Chromogenix AB) was utilized and the

absorbance was measured at 405 nm with a spectrophotometer (UV-160 1PC, Shimadzu).

PAI-1 activity was determined by immunoassays (ELISA; MPAIKTTM, Molecular

Innovations Inc.). Samples were compared with the calibration curve to estimate active PAI-

1 level (ng/ml). The conversion factor for PAI-1 used was PAI-1 IU/ml= 1.34 ng/ml.

D-dimer and fibrinogen were also measured by ELISA. For D-dimer, a 1:5 dilution in

plasma was used (Diagnostica Stago, asserachrom D-Dimer). For fibrinogen, a 1:200

dilution of mouse plasma was used (DiaPharma, Zymutest Fibrinogen).

5

Measurement of Weibel-Palade Body Contents: Plasma Levels

The measurement of t-PA levels has been described above. P-selectin was measured

using sP-selectin immunoassay diluted 1:50 (R&D systems, Inc. Quantikine M). For vWF,

plasma was diluted 1:2 and measured by immunoassay (Corgenix, REAADS von Willebrand

Factor Antigen Test kit). Samples, standards and controls were assayed in triplicate.

Immunofluorescence

Standard immunohistochemical staining procedure was used for frozen sections. Sections

with mouse aorta were fixed in 3.7% formaldehyde for 30 min at RT. The slides were then

washed with TBS and incubated with tPA goat polyclonal antibody at a dilution of 1:50 (Santa

Cruz Biotechnology, Inc.) followed by washing incubation with second mouse anti-goat IgG-

FITC conjugated antibody at a dilution of 1:200 (60 min, 22°C). Sections used for control were

incubated in buffer instead of a primary antibody. The sections were examined using

fluorescence microscopy (Nikon eclipse TE 300) and scanned for intensity.

Expression of t-PA

Aortas were removed from mice and immediately frozen at -80°C until RNA extraction

was performed. Total RNA from aorta samples were isolated by High Pure RNA Tissue Kit

(Roche Applied Sciences). RNA samples were reverse transcribed with 1st Strand cDNA

Synthesis Kit (Roche Applied Science). Real-Time Quantitative PCR was performed for tPA and

G3PD as an internal control by using Assay-on-Demand Gene Expression primers and probes

and TaqMan Universal Master mix (Applied Biosystems) in ICycler Real-Time PCR instrument

(Bio-Rad).

Measurement of Platelet Activation by Flow Cytometry

Blood from wild type and eNOS-deficient mice were isolated from vena cava following

anaesthesia. Murine platelet rich plasma was prepared by centrifugation of whole blood for 20

min at 110 g. The platelets were counted in a Coulter Counter (Coulter Electronics, Miami, FL).

The platelet rich plasma was directly stimulated with thrombin. The samples were immediately

6

fixed with final 1% paraformaldehyde, and immuno-labeled with FITC-conjugated mouse

CD62P, mouse CD41b, or corresponding isotype controls (Pharmingen, BD Biosciences).

Analysis of the FITC-labeled samples was performed with a FACScan flow cytometer

(Becton Dickinson) at an excitation wavelength of 488 nm and a laser power of 15 mW. The

green fluorescence was collected through a 530-nm band-pass filter. The isotypic control was

positioned between fluorescence values of 1 and 10 as reference. For each sample, data from 2 x

105 platelets were recorded in a 1,024-channel distribution showing the logarithmic amount of

green fluorescence. Flow cytometric data were analysed using histogram statistics of the

CellQuest software (Becton Dickinson).

Statistical Analysis

Comparisons between genotypes and genders were performed by unpaired 2-tailed

Student’s t tests. Differences between groups were determined using an unpaired Student’s t test.

The effect of interventions were analyzed using 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.

7

Results

Measurement of Thrombosis in eNOS-Deficient and Wild-Type Mice

Previously, we (6) and others (19) have found that bleeding times are decreased in eNOS-

deficient mice. Bleeding times, however, are primarily dependent on platelet adhesion and

recruitment in the setting of intact endothelium (28). Therefore, to determine the relevance of

vascular NO deficiency on an in vivo model of thrombosis, eNOS-deficient or wild-type mice

underwent a standard characterized murine arterial injury model that is known to induce carotid

artery thrombosis (4). Time to occlusion was measured using a mini-Doppler flow probe in the

distal carotid artery. Using this model, the injury is long relative to the artery diameter. The flow

was monitored and not found to have increased velocities. There was a gradual decrease in

velocity until flow ceased as a result of the thrombosis. Paradoxically, we found that both female

and male eNOS-deficient mice had prolonged measurement of time to occlusion (Figure 1)

suggesting decreased carotid thrombosis. Visual histological examination of cross sectional

analyses of carotid arteries confirmed the limited injury and formation of thrombus as well as the

lysis of thrombus in appropriate specimens.

Measurement of Platelet Activation

We previously found that platelet activation as determined by platelet surface P-selectin

expression in resting and stimulated platelets were not significantly different in the eNOS-

deficient and control animals (6). To confirm and extend these findings, platelet surface

expression of CD41 (GPIIb) and CD62P (P-selectin) was measured in resting and activated

platelets (thrombin 1U/ml) for both male and female animals. For CD41 expression, in resting

platelets, mean fluorescence for eNOS-deficient and wild-type male mice were 58.2 ± 3.4 and

60.8 ± 1.9, respectively (P=ns). For thrombin activated platelets, mean fluorescence for CD41

expression was 71.7 ± 2.7 and 76.9 ± 5.1 for the eNOS-deficient and wild-type male mice,

respectively (P=ns). As represented in Figure 2, flow cytometric analysis of glycoprotein IIb

(CD41) demonstrates no significant change in expression between wild-type and eNOS-deficient

platelets. This lack of difference in GPIIb expression between eNOS-deficient and control

platelets was also seen in the female eNOS-deficient and control groups (data not shown).

Consistent with previous results (6), there was no change in P-selectin expression with

stimulation for the male eNOS-deficient mice as compared to the control male mice. Similar

8

findings (P=ns) for surface P-selectin expression were found for the female eNOS-deficient mice

as compared to the wild-type female mice. As measured by change in light transmittance, there

was also no change in ADP (5µM) induced platelet aggregation (n=3, P=ns).

The Effect of NO Deficiency on Fibrinolysis

Importantly, as seen in Figure 1, time to occlusion for eNOS-deficient animals is

prolonged suggesting decreased thrombosis. However, it is also plausible that these findings are

due to enhanced fibrinolysis. To study this question, potential regulators of fibrinolysis were

measured in plasma from NO-deficient and control animals. Activity of t-PA was found to be

significantly enhanced in female as well as male eNOS-deficient mice as compared to wild-type

controls (Figure 3). Additionally, t-PA activity and antigen were increased in both female and

male eNOS-deficient mice as compared to wild-type mice in the absence of arterial injury (data

not shown). Consistent with this finding was elevated levels of D-dimer, a specific derivative of

cross-linked fibrin, in the eNOS-deficient animals (Figure 4). The change in levels only reached

statistical significance in the female mice. It should be noted that although fibrinogen was not

different between the groups (22.5 ± 1.0 mg/ml and 19.6 ± 2.6 mg/ml for wild-type and eNOS-

deficient animals, respectively), the levels of D-dimer found in plasma (ng/ml) are considerably

lower than fibrinogen (mg/ml). Additionally, it is unlikely that changes in fibrinogen levels

would be seen in the setting of enhanced lysis. Enhanced fibrinolysis can also be seen in the

setting of decreased plasminogen activator inhibitor (PAI-1). As shown in Table 1, neither PAI-1

levels nor PAI-1 activity are altered in either gender of eNOS-deficient mouse.

Expression of t-PA in Vascular Tissue

Endothelial cells are the source of t-PA found in the plasma. Enhanced t-PA in the

plasma may be due to enhanced release with or without increased vascular tissue expression. To

determine whether deficiency of eNOS in the vascular tissue is associated with change in t-PA

expression, aortic tissue was studied by real-time quantitative PCR for t-PA. As seen in Table 2,

there was no difference between eNOS-deficient and wild-type mice. These findings were

confirmed by immunohistochemistry of aortic tissue (Figure 5).

9

Plasma Levels of Weibel-Palade Body Derived Proteins

Vascular endothelial cells contain vesicles known as Weibel-Palade bodies, which serve

as a storage compartment for vWF, P-selectin, tPA, and other proteins. Exocytosis of Weibel-

Palade bodies has been recently shown to be inhibited by NO(19). Thus, in the absence of NO,

release of Weibel-Palade body contents (and t-PA) could be enhanced. To determine if other

substances stored in Weibel-Palade bodies had increased plasma levels, von Willebrand factor

(vWF) and plasma P-selectin levels were measured. P-selectin levels were found to be 51.4 ±

10.3 (ng/ml) and 43.3 ± 9.9 (ng/ml) in eNOS-deficient and control animals, respectively (P<0.05;

Figure 6), although only the male and pooled levels reached statistical significance. This finding

was confirmed with measurement of vWF found to be 57.8 ± 28.1 (%) and 48.3 ± 16.2 (%) in

eNOS-deficient and control animals, respectively (P<0.05).

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Discussion

In the setting of intimal injury, platelets adhere to the subendothelium leading to platelet

activation. Once activated, platelets facilitate thrombus growth by recruitment of additional

platelets and promoting surface thrombin generation (23). In the intact vessel wall, NO is an

inhibitor of platelet function. While endothelial-derived NO production is known to both

mediate vasorelaxation as well as inhibit platelet adhesion and activation, less is known about the

contribution to thrombosis in vivo. Although deficiency in platelet-NO production is associated

with enhanced platelet activation in a “recruitable” platelet population, this failed to correlate

with extent of vessel-occluding thrombus. These are not the first observations to demonstrate

paradoxical findings in the absence of eNOS. A recent study showed that in the absence of

eNOS, mice with experimentally induced portal hypertension developed a hyperdynamic

circulation (12). Although the mechanism was not directly investigated, it was suggested that

compensatory vasodilator molecules were upregulated (12).

Although the extent of in vivo thrombosis was not correlated with degree of primary

platelet activation, it was correlated with fibrinolysis as demonstrated by enhanced t-PA and D-

dimer levels. Expression of t-PA was not altered (Table 2, Figure 5), suggesting enhanced

release of t-PA from the endothelium into the blood. Tissue plasminogen activator is stored in

vascular endothelial cells and contained in vesicles known as Weibel-Palade bodies (10). Upon

activation of endothelial cells, the Weibel-Palade bodies fuse with the plasma membrane and

release their contents into the blood, allowing endothelial cells an additional mechanism for

participating in hemostasis. Interestingly, as recently shown by Matsushita and colleagues (19),

NO inhibits exocytosis of Weibel-Palade bodies by regulating the activity of N-ethylmaleimide-

sensitive factor (NSF). In this study, NO inhibited NSF disassembly of soluble NSF attachment

protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF.

Consistent with our findings, serum levels of vWF were higher in eNOS-deficient mice as

compared to wild-type mice. Importantly, in the study by Matsushita and colleagues,

pharmacologic modulation of eNOS in human endothelial cell culture also induced these changes

suggesting that our findings are not specific to eNOS-deficient mice.

11

With respect to fibrinolysis, there appears to be some divergence between our findings

and previous studies. Chronic infusion of L-nomega-nitro-L-arginine methyl ester (L-NAME)

has been shown to alter PAI-1 levels (13, 15), however L-NAME can be a nonspecific NOS

inhibitor and chronic L-NAME administration has been shown to induce the expression of iNOS

in the aorta (17). Our findings were specific for eNOS and we have found no change in iNOS

expression in the vascular tissue of the eNOS-deficient animals by RT-PCR or

immunohistochemistry (data not shown). Also potentially conflicting are brachial studies

showing that bradykinin infusion is associated with increased levels of t-PA through a NO

synthase-independent pathway, however, these studies themselves are in opposition to direct

examination of this question (19). In addition, this study examined normal subjects and not those

known to have decreased bioavialable NO. Supportive of our findings is a recent study reporting

increased t-PA antigen D-dimer levels in subjects with increased risk of atherothrombosis, a

group with known impaired NO-dependent vascular function (21). Clearly, the clinical

implications of our findings cannot be assumed.

Von Willebrand factor is a plasma protein that plays an essential role in controlling the

adhesion and aggregation of platelets at sites of vascular injury and may be responsible for the

previously observed decrease in bleeding time in the eNOS-deficient animals (6, 19). The

enhanced release of mediators of both platelet adhesion and fibrinolysis may also explain the

conflicting bleeding time and carotid artery occlusion results. It has been shown in other animal

models of arterial thrombosis that changes in carotid occlusion due to thrombus formation may

not be associated with changes in the bleeding time (28). The release from Weibel-Palade bodies

of both t-PA as well as vWF highlights the complex nature of hemostasis. Although seemingly

paradoxical, such release ensures that while hemorrhage due to vascular trauma is contained, the

unmitigated perpetuation of thrombus is limited to prevent eventual occlusion of the vessel.

These observations are also enhanced by the differences in damage done to the vessel

wall in the setting of injury. Bleeding times reflect normal tissue while the carotid artery

occlusion model studies damaged endothelium. Thus, changes in release of NO from the

endothelium and its contribution to hemostasis would be reflected primarily in the bleeding time.

Previous studies utilizing the carotid injury model have specifically studied thrombolysis at sites

of arterial injury (4) and found a specific contribution to fibrinolysis. This observation is

12

confirmed by a study demonstrating that arterial thrombosis is more influenced by blood

components than by elements within the arterial wall (14). Thus, in this setting of carotid injury,

the secondary effect of NO deficiency, namely the enhanced release of tPA from endothelial

Weibel-Palade bodies is of greater relevance than the release of NO from the endothelium. The

clinical relevance of these findings, while not known, are potentially intriguing.

Although not the specific focus of this study, some differences between the male and

female animals were observed. Gender differences in platelet function in wild-type mice have

been shown recently (16). In this study, female mice had enhanced fibrinogen binding and,

similar to our results, no change in GPIIb expression. These studies found differences only in

washed platelets so the direct relevance to our in vivo observations is not clear. Gender

differences (20) have also been assessed by the response to injury by quantitative morphometry

and measuring the intimal to medial (I/M) volume ratio. In wild-type mice, cuff placement

causes pronounced intimal proliferation. Female mice show less intimal response than do males,

although eNOS mutant female mice still have more response than do wild-type females.

Whether these changes are estrogen-dependent or -independent is not known.

In summary, although eNOS deficiency attenuates vascular reactivity and increases

platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent

inhibition of Weibel-Palade body release. It has been assumed that, for cardiovascular disease, a

relative deficiency of bioactive NO is harmful as it leads to attenuated vascular reactivity and

enhanced thrombosis. However, as suggested by these studies, the processes regulating

thrombosis are more complex. As highlighted by the release of both t-PA and vWF from

Weibel-Palade bodies, hemostasis is a delicate balance between occlusion and patency.

Acknowledgements: This work has been supported in part by NIH grants NIH RO1AG08226

(J.F.), NIH RO1HL62267 (J.F.), and an Established Investigator Award from the American

Heart Association (J.F.).

Conflict-of-Interest Disclosure Statement: The authors (M.D.I., O.V., K.T., P.B., S.R., S.C.,

S.V., J.E.F.) have no conflicts-of-interest to disclose.

13

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15

Table 1. Markers of Fibrinolysis

PAI-1 (ng/ml) PAI-1 Activity (IUml) tPA antigen (ng/ml)

Female wild-type 2.7 ± 0.2 2.0 ± 0.1 2.3 ± 0.1

Male wild-type 2.4 ± 0.3 1.8 ± 0.2 2.2 ± 0.1

Female eNOS-

deficient

2.5 ± 0.4 1.8 ± 0.3 3.0 ± 0.1*

Male eNOS-

deficient

2.5 ± 0.2 1.8 ± 0.2 3.1 ± 0.3*

P=ns for PAI-1 and PAI-1 activity. *P<0.01 compared to wild-type. N=5 for each group. Data

are expressed as the mean ± SEM.

16

Table 2. Tissue Plasminogen Activator: Expression in Vascular Tissue

tPA/G3PD (normalized to

G3PD)

tPA (fluorescence

intensity; arb. Units)

Female wild-type 4.0 ± 10-2 99.3 ± 4.3

Male wild-type 6.6 ± 10-3 95.6 ± 3.8

Female eNOS-deficient 4.2 ± 10-2 100.2 ± 7.3

Male eNOS-deficient 6.2 ± 10-3 99.2 ± 6.1

(Tissue Plasminogen Activator (tPA); P=ns for all, n=5 for each group; data are expressed as the

mean ± SEM)

17

Figure Legends

Figure 1. Time to occlusion after carotid artery injury. After carotid artery injury, a doppler

flow probe was inserted and time to occlusion was determined in minutes (n=7 in each group;

*P=0.06 compared to female wild-type mice; **P≤0.05 compared to female wild-type mice;

***P≤0.05 compared to male wild-type mice; data are expressed as the mean ± SEM).

Figure 2. Measurement of surface GPIIb (CD41) expression in resting and thrombin activated

platelets from eNOS-deficient and wild-type male mice. Enhanced CD41 expression is seen with

stimulation but no difference is detected between the eNOS-deficient (black line) and wild-type

(dotted line) mice. Similar results were noted for female mice (Results are representative of n=7

for each group, P=ns).

Figure 3. Tissue plasminogen activator (tPA) activity. TPA activity (IU/ml) was measured in

plasma from female and male wild-type and eNOS-deficient mice (*P≤0.05 compared to wild-

type mice, n=5 for each group; data are expressed as the mean ± SEM).

Figure 4. Plasma D-dimer levels. Levels of D-dimer were determined by ELISA in plasma from

female and male wild-type and eNOS-deficient mice (*P≤0.05 compared to female wild-type

mice, n=5 for each group; data are expressed as the mean ± SEM).

Figure 5. Immunohistochemistry or aortic tissue from eNOS deficient male and female mice as

compared to wild-type. No change was detected between the groups as shown in the

representative figures (representative of n=5 animals in each group, P=ns as compared by

intensity of fluorescence).

Figure 6. Plasma P-selectin levels. Levels of P-selectin were determined by ELISA in plasma

from female and male wild-type and eNOS-deficient mice (*P≤0.05 compared to male wild-type

mice, n=5 for each group; data are expressed as the mean ± SEM).

18

0

10

20

30

40

50

Female wild-type

Male wild-type Female eNOS-deficient

Male eNOSdeficient

Tim

eto

Occ

lusi

on(m

in.)

***

**

*

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

0

10

20

30

40

50

Female wild-type

Male wild-type Female eNOS-deficient

Male eNOSdeficient

Tim

eto

Occ

lusi

on(m

in.)

***

**

*

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

19

Male unstimulated CD41b Dotted wild-type; black eNOS-deficient

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

Male thrombin stimulated (3U/ml) CD41bDotted wild-type; black eNOS-deficient

Male unstimulated CD41b Dotted wild-type; black eNOS-deficient

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

Male thrombin stimulated (3U/ml) CD41bDotted wild-type; black eNOS-deficient

20

0

0.5

1

1.5

2

2.5

3

3.5

4

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

tPA

Act

ivity

(IU

/ml)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

**

0

0.5

1

1.5

2

2.5

3

3.5

4

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

tPA

Act

ivity

(IU

/ml)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

**

21

0

50

100

150

200

250

300

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

D-d

imer

(ng/

ml)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

*

0

50

100

150

200

250

300

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

D-d

imer

(ng/

ml)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

*

22

23

0

10

20

30

40

50

60

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

P-s

elec

tin(n

g/m

l)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

*

0

10

20

30

40

50

60

Female wild-type Male wild-type Female eNOS-deficient

Male eNOS-deficient

P-s

elec

tin(n

g/m

l)

ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�ዊ�

*