Am J Physiol Heart Circ Physiol 288: H1627-H1632, 2005.
First published November 24, 2004; doi:10.1152/ajpheart.00819.2004
0363-6135/05 $8.00
Compensatory mechanisms influence hemostasis in setting of eNOS deficiency
Mark D. Iafrati,1
Olga Vitseva,2
Kahraman Tanriverdi,2
Price Blair,2
Sybille Rex,2
Subrata Chakrabarti,2
Sonia Varghese,2 and
Jane E. Freedman2
1Department of Surgery, Tufts-New England Medical Center, Boston; and 2Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts
Submitted 11 August 2004
; accepted in final form 17 November 2004
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ABSTRACT
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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 compared with 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 levels 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; this observation is also supported by increased von Willebrand factor 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 life span that are seen in animals deficient in a pivotal regulator of vascular patency.
platelets; thrombosis; endothelial nitric oxide synthase; transgenic mice
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). NO 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 intraarterial 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 in patients with cardiovascular disease (1). Although interventions that are believed to enhance the systemic bioavailability 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, which has been shown in multiple studies to enhance vascular relaxation (8), was associated with enhanced arterial thrombosis in prospective clinical studies (11, 18).
Homozygous endothelial NO synthase (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 that lack 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), which suggests 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 (nonplatelet-dependent) mechanism. To further explore this question and determine whether NO alters thrombosis in vivo, these animals were studied using a carotid artery-injury model.
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MATERIALS AND METHODS
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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 (22–24 g; 10 wk old) were used for all experiments. Blood was obtained by inferior vena caval puncture, drawn into syringes, and anticoagulated with trisodium citrate.
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 adequate anesthesia was established, a 1-cm vertical midline incision was made in the neck, the left common carotid artery was exposed, and a disc (diameter, 0.6 mm) saturated with FeCl3 was applied for 3 min and removed. The incision area was then irrigated. 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.
Measurement of fibrinolysis.
For determination of plasma tissue-plasminogen activator (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 a 405-nm wavelength with a spectrophotometer (model UV-160 1PC; Shimadzu). Plasminogen activator inhibitor (PAI)-1 activity was determined by ELISA (MPAIKT active mouse PAI-1 antigen assay; Molecular Innovations). Samples were compared with a calibration curve to estimate active PAI-1 levels (in ng/ml). The conversion factor used was 1 IU of PAI-1/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 (Asserachrom D-Dimer; Diagnostica Stago). For fibrinogen, a 1:200 dilution of mouse plasma was used (Zymutest Fibrinogen; DiaPharma).
Measurement of Weibel-Palade body contents: plasma levels.
The measurement of t-PA levels is described in Measurement of fibrinolysis. P-selectin was measured using an sP-selectin immunoassay diluted 1:50 (Quantikine M; R&D Systems). For von Willebrand factor (vWF), plasma was diluted 1:2 and measured by immunoassay (REAADS von Willebrand Factor Antigen Test Kit; Corgenix). 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 room temperature. The slides were then washed with Tris-buffered saline and incubated with t-PA goat polyclonal antibody at a 1:50 dilution (Santa Cruz Biotechnology), washed again, and incubated with a second mouse anti-goat IgG-FITC-conjugated antibody at a 1:200 dilution (for 60 min at 22°C). Sections used for control were incubated in buffer instead of a primary antibody. The sections were examined using fluorescence microscopy (Eclipse TE300; Nikon) and were 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 was isolated using a High Pure RNA Tissue Kit (Roche Applied Science). RNA samples were reverse transcribed with a First Strand cDNA Synthesis Kit (Roche Applied Science). Real-time quantitative PCR was performed for t-PA and glucose-3-phosphate dehydrogenase as an internal control by using Assay-on-Demand gene expression primers and probes and TaqMan Universal Master Mix (Applied Biosystems) in an ICycler real-time PCR instrument (Bio-Rad).
Measurement of platelet activation by flow cytometry.
Blood from wild-type and eNOS-deficient mice was isolated from vena cavas after administration of anesthesia. 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 fixed with 1% (final) paraformaldehyde and immunolabeled 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 analyzed using histogram statistics of CellQuest software (Becton Dickinson).
Statistical analysis.
Comparisons between genotypes and genders were performed by unpaired two-tailed Student’s t-tests. Differences between groups were determined using an unpaired Student’s t-test. The effects 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 means ± SE.
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RESULTS
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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. With the use of this model, the injury is long relative to the artery diameter. The flow was monitored and was 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 (Fig. 1), which suggests 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 measurements of 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 expressions of CD41 [glycoprotein (GP)IIb] and CD62P (P-selectin) were measured in resting and activated platelets (thrombin, 1 U/ml) for both male and female animals. For CD41 expression, in resting platelets, mean fluorescence values for male eNOS-deficient and wild-type mice were 58.2 ± 3.4 and 60.8 ± 1.9, respectively [P = not significant (ns)]. For thrombin-activated platelets, mean fluorescence values for CD41 expression were 71.7 ± 2.7 and 76.9 ± 5.1 for male eNOS-deficient and wild-type mice, respectively (P = ns). As represented in Fig. 2, flow-cytometric analysis of GPIIb 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).
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Fig. 2. Measurement of surface glycoprotein IIb (CD41) expression in resting and thrombin-activated platelets from endothelial nitric oxide synthase (eNOS)-deficient and wild-type male mice (n = 7 mice/group). Enhanced CD41 expression was observed with stimulation, but no difference was detected between the eNOS-deficient (solid line) and wild-type (dotted line) mice. Similar results were noted for female mice. P = not significant (ns).
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Consistent with previous results (6), there was no change in P-selectin expression with stimulation for the male eNOS-deficient mice compared with the control male mice. Similar findings (P = ns) for surface P-selectin expression were found for the female eNOS-deficient mice compared with the wild-type female mice. As measured by change in light transmittance, there was also no change in ADP (5 µM)-induced platelet aggregation (n = 3; P = ns).
Effects of NO deficiency on fibrinolysis.
Importantly, as seen in Fig. 1, the time to occlusion for eNOS-deficient animals is prolonged, which suggests 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 compared with wild-type controls (Fig. 3). Additionally, t-PA activity and antigen were increased in both female and male eNOS-deficient mice compared with wild-type mice in the absence of arterial injury (data not shown). Consistent with this finding were elevated levels of D-dimer, a specific derivative of cross-linked fibrin, in the eNOS-deficient animals (Fig. 4). The change in levels only reached statistical significance in the female mice. It should be noted that although fibrinogen levels were not different between the groups (22.5 ± 1.0 and 19.6 ± 2.6 mg/ml for wild-type and eNOS-deficient animals, respectively), the levels of D-dimer found in plasma (in ng/ml) were considerably lower than fibrinogen (in 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 PAI-1. As shown in Table 1, neither PAI-1 levels nor PAI-1 activity was altered in either gender of eNOS-deficient mice.
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Fig. 3. Tissue plasminogen activator (t-PA) activity was measured in plasma from female and male wild-type and eNOS-deficient mice (n = 5 mice/group). *P  0.05 compared with wild-type mice.
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Fig. 4. D-dimer levels were determined by ELISA in plasma from female and male wild-type and eNOS-deficient mice (n = 5 mice/group). *P 0.05 compared with female wild-type mice.
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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 (Fig. 5).
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Fig. 5. Immunohistochemistry of t-PA in aortic tissue from eNOS-deficient male and female mice compared with wild-type mice (n = 5 mice/group). A: wild-type females. B: wild-type males. C: eNOS-deficient females. D: eNOS-deficient males. No changes were detected between the groups. P = ns as compared by intensity of fluorescence.
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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, t-PA, and other proteins. Exocytosis of Weibel-Palade bodies was 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 whether other substances stored in Weibel-Palade bodies had increased plasma levels, vWF and plasma P-selectin levels were measured. P-selectin levels were found to be 51.4 ± 10.3 and 43.3 ± 9.9 ng/ml in eNOS-deficient and control animals, respectively (P < 0.05; Fig. 6), although only the male and pooled levels reached statistical significance. This finding was confirmed with measurements 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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Fig. 6. Plasma P-selectin levels were determined by ELISA in plasma from female and male wild-type and eNOS-deficient mice (n = 5 mice/group).*P 0.05 compared with male wild-type mice.
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DISCUSSION
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In the setting of intimal injury, platelets adhere to the subendothelium, which leads to platelet activation. Once activated, platelets facilitate thrombus growth by recruitment of additional platelets and promotion of surface thrombin generation (23). In the intact vessel wall, NO is an inhibitor of platelet function. Although endothelial-derived NO production is known to both mediate vasorelaxation and inhibit platelet adhesion and activation, less is known about the contribution to thrombosis in vivo. Deficiency in platelet NO production is associated with enhanced platelet activation in a "recruitable" platelet population; however, 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 (12) showed that in the absence of eNOS, mice with experimentally induced portal hypertension developed a hyperdynamic circulation. Although the mechanism was not directly investigated, it was suggested that compensatory vasodilator molecules were upregulated (12).
The extent of in vivo thrombosis was not correlated with degree of primary platelet activation; however, it was correlated with fibrinolysis as demonstrated by enhanced t-PA and D-dimer levels. Expression of t-PA was not altered (see Table 2 and Fig. 5), which suggests enhanced release of t-PA from the endothelium into the blood. The t-PA 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 et al. (19), NO inhibits exocytosis of Weibel-Palade bodies by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). In this study, NO inhibited the NSF disassembly of soluble NSF attachment-protein-receptor complexes by nitrosylating critical cysteine residues of NSF. Consistent with our findings, serum levels of vWF were higher in eNOS-deficient mice compared with wild-type mice. Importantly, in the study by Matsushita et al. (19), pharmacological modulation of eNOS in human endothelial cell culture also induced these changes, which suggests that our findings are not specific to eNOS-deficient mice.
With respect to fibrinolysis, there appears to be some divergence between our findings and previous studies. Chronic infusion of N
-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 inducible NOS (iNOS) in aorta (17). Our findings were specific for eNOS, and we have found no change in iNOS expression in vascular tissue of eNOS-deficient animals by RT-PCR or immunohistochemistry (data not shown). Also potentially conflicting are brachial studies that show bradykinin infusion is associated with increased levels of t-PA through an NOS-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 bioavailable NO. Supportive of our findings is a recent study (21) that reported increased t-PA antigen D-dimer levels in subjects with increased risk of atherothrombosis, a group with known impaired NO-dependent vascular function. Clearly, the clinical implications of our findings cannot be assumed.
vWF 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 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, whereas 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 considered thrombolysis at sites of arterial injury (4) and found a specific contribution to fibrinolysis. This observation is confirmed by a study (14) that demonstrates that arterial thrombosis is more influenced by blood components than elements within the arterial wall. Thus in this setting of carotid injury, the secondary effect of NO deficiency, namely, the enhanced release of t-PA from endothelial Weibel-Palade bodies, is of greater relevance than the release of NO from the endothelium. The clinical relevance of these findings, although unknown, is potentially intriguing.
Although not the specific focus of this study, some differences between 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 unclear. Gender differences (20) have also been assessed by the response to injury by quantitative morphometry and measuring of the intimal-to-medial volume ratio. In wild-type mice, cuff placement causes pronounced intimal proliferation. Female mice show less intimal response than males, although eNOS-mutant female mice still have more response than wild-type females. Whether these changes are estrogen dependent or independent is unknown.
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 that regulate 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.
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GRANTS
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This work was supported in part by National Institutes of Health Grants RO1 AG-08226 and HL-62267 and an Established Investigator Award from American Heart Association (to J. E. Freedman).
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FOOTNOTES
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Address for reprint requests and other correspondence: M. D. Iafrati, Tufts-New England Medical Center, 750 Washington St., Boston, MA 02111 (E-mail: Miafrati{at}Tufts-NEMC.org)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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D. E. Handy
Hyperhomocysteinemia: the protein C pathway to thrombosis
Blood,
October 1, 2006;
108(7):
2137 - 2138.
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S. Dayal, K. M. Wilson, L. Leo, E. Arning, T. Bottiglieri, and S. R. Lentz
Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia
Blood,
October 1, 2006;
108(7):
2237 - 2243.
[Abstract]
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N. J. Brown, J. A.S. Muldowney III, and D. E. Vaughan
Endogenous NO Regulates Plasminogen Activator Inhibitor-1 During Angiotensin-Converting Enzyme Inhibition
Hypertension,
March 1, 2006;
47(3):
441 - 448.
[Abstract]
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Copyright © 2005 by the American Physiological Society.
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