Endothelial nitric oxide synthase (eNOS) in platelets:

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Review

Endothelial nitric oxide synthase (eNOS) in platelets:

how is it regulated and what is it doing there?

Voahanginirina Randriamboavonjy, Ingrid Fleming

Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

Correspondence: Ingrid Fleming, e-mail: fleming@em.uni-frankfurt.de

Abstract:

Platelets express the endothelial form of the nitric oxide synthase (eNOS) and generate NO. However, in contrast to eNOS in endothelial cells, eNOS in platelets is largely Ca²⁺-independent and the activity is regulated by phosphorylation. Platelet-derived NO plays an important role in the regulation of platelet aggregation and secretion. Changes in the activity of platelet eNOS are responsible for the abnormal platelet activation encountered in different pathological situations (e.g. hypertension and diabetes). In this review, we will summarize the current knowledge of the role of platelet eNOS and the regulation of its activity as well as the fate of platelet-derived NO in physiological and pathological situations.

Key words:

platelets, signal transduction, eNOS, ATP/adenosine

Abbreviations: AMPK – AMP-activated protein kinase, ATP – adenosine triphosphate, [Ca²⁺]_i– intracellular calcium concentration, CaM – calmodulin, CaMKII – Ca²⁺ calmodulin- dependent kinase II, cyclic AMP – 3’,5’-cyclic adenosine monophosphate, cyclic GMP – cyclic guanosine monophosphate, eNOS – endothelial nitric oxide synthase, Hsp – heat- shock protein, iNOS or NOS II – inducible nitric oxide synthase, nNOS – NOS I or bNOS – neuronal nitric oxide synthase, NO – nitric oxide, NOSIP – eNOS interacting protein, NOSTRIN – eNOS traffic inducer, O₂– superoxide anions, PKA – protein kinase A, PKC – protein kinase C, sGC – soluble guanylyl cyclase, t-SNARE – target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor, VAMP-3 – vesicle-associated membrane protein-3, VEGF – vascular endothelial growth factor

Introduction

Since the identification of nitric oxide (NO) as the pri- mary endothelium-derived relaxing factor, an increas-

ing body of evidence has accumulated to show that NO is more than a vasodilator as it exerts anti-platelet actions and plays a crucial role in the regulation of vascular homeostasis. NO is synthesized from the amino acid L-arginine by a family of enzymes called the NO synthases (NOS). The ‘endothelial’ (eNOS or NOS III) and ‘neuronal’ (nNOS, NOS I or bNOS) NOS isoforms were named after the tissues in which they were first identified, are expressed constitutively and are generally regulated by Ca²⁺/calmodulin (CaM) as well as by phosphorylation. The inducible NOS isoform (iNOS or NOS II), on the other hand, is not generally expressed in unstimulated cells (although exceptions to this rule of course exist), binds CaM so tightly that it is essentially Ca²⁺-independent, and releases NO in larger quantities during inflamma- tory or immunological defence reactions. There are only a few intracellular mechanisms that regulate iNOS activity which is usually determined by its expression level [15].

Pharmacological Reports 2005, 57, suppl., 5965 ISSN 1734-1140

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reproductive organs, cardiac myocytes, megakaryo- cytes, and in platelets.

The characterization of the NOS isoform found in platelets began in 1990 following a report describing the presence of an L-arginine/NO pathway which was able to regulate collagen-induced aggregation [48].

Since then, a number of different studies have shown directly or indirectly the presence of a NOS isoform in platelets [45]. There has even been a report of a distinct platelet-specific constitutive NOS isoform in cy- tosolic fractions generated from washed human platelets. The latter enzyme appeared as a single band (80 kD) under denaturing conditions and the native enzyme was suggested to be a dimer since a molecular mass of about 150 kD was estimated by gel filtra- tion. The same group also showed that the activity of the enzyme was dependent on L-arginine, NADPH and tetrahydrobiopterin as well as on calmodulin [43].

Although further studies confirmed the existence of a pathway for NO synthesis in platelets, the identification of the platelet NOS isoform as eNOS was made in the mid 90’s. However, at approximately the same time as Sase and Mitchel [52] reported that the isoform present in platelets is eNOS, a second group of researchers described the presence of both the eNOS and iNOS isoforms [38]. While there is evidence of iNOS in platelets [6], the majority of reports are related to an enzyme that can be detected by eNOS- specific antibodies.

Regulation of the activity of platelet eNOS

Role of calcium

eNOS was originally classified as a Ca²⁺/calmodulin(CaM)-dependent enzyme but it is now evident that eNOS activation does not necessarily require an increase in the intracellular calcium concentration ([Ca²⁺]i). In fact, in contrast to vasoactive agonists which normally elevate the [Ca²⁺]i in endothelial cells, stimuli such as fluid shear stress and 17b-estradiol increase NO production predominantly by altering the phosphorylation of the enzyme [13, 26].

In platelets, theb2-adrenoceptor – mediated activation of NOS is adenylyl cyclase-dependent but is not

thrombin-induced aggregation but is correlated with a decrease in [Ca²⁺]_isuggesting that platelet eNOS is largely Ca²⁺-independent [17].

Role of phosphorylation

Phosphorylation is one of the most important mechanisms for post-translational regulation of proteins.

The activity of eNOS has been shown to depend tightly on its phosphorylation state to a greater extent than the other NOS isoforms. It is now well estab- lished that the phosphorylation of two residues;

Ser¹¹⁷⁷ in the reductase domain and Thr⁴⁹⁵ in the calmodulin-binding domain, plays a crucial role in regulating eNOS activity in response to different stimuli.

Ser1¹⁷⁷(bovine sequence Ser¹¹⁷⁹)

In unstimulated, cultured endothelial cells, Ser¹¹⁷⁷is not phosphorylated but is rapidly phosphorylated after the application of fluid shear stress [3, 8, 23], estrogen [26], vascular endothelial growth factor (VEGF) [22, 40], insulin [22, 30], or bradykinin [16]. The kinases involved in this process vary with the stimuli applied.

For example, while shear stress elicits the phosphorylation of Ser¹¹⁷⁷by activating the protein kinase B or Akt and protein kinase A (PKA), insulin, estrogen and VEGF mainly phosphorylate eNOS in endothelial cellsvia Akt. The bradykinin-, Ca²⁺ionophore- and thapsigargin-induced phosphorylation of Ser¹¹⁷⁷, on the other hand, is mediated by CaMKII [16, 55]. The phosphorylation of eNOS Ser¹¹⁷⁷ increases NO production 2- to 3-fold above basal levels, an effect that can be attributed to an increase in the flux of electrons through the reductase domain [37].

Thr⁴⁹⁵(bovine sequence Thr⁴⁹⁷)

This residue is constitutively phosphorylated in all of the endothelial cells investigated to-date and is a negative regulatory site i.e. phosphorylation is associated with a decrease in enzyme activity [16, 25, 40].

The link between phosphorylation and NO production can be explained by interference with the binding of CaM to the CaM-binding domain, and in endothelial cells stimulated with agonists, such as bradykinin, histamine or a Ca²⁺ ionophore, substantially more

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CaM binds to eNOS when Thr⁴⁹⁵is dephosphorylated [16]. Analysis of the crystal structure of the eNOS CaM-binding domain with CaM indicates that the phosphorylation of eNOS Thr⁴⁹⁵not only causes elec- trostatic repulsion of nearby glutamate residues (Glu⁷ and Glu¹²⁷) within CaM but may also affect eNOS Glu⁴⁹⁸ and thus induce a conformational change within eNOS itself [2]. Recently the dephosphorylation of Thr⁴⁹⁵ has been linked to eNOS uncoupling (i.e. O₂^– production by eNOS) [33] however, it re- mains to be determined whether this occurs in vivo and whether or not the actual cause of the uncoupling is a decrease in H₄B and/or L-arginine availability as a consequence of prolonged activation of the enzyme.

The constitutively active kinase which phosphorylates eNOS Thr⁴⁹⁵is most probably protein kinase C (PKC) [16, 35, 40], a finding which could account for the fact that protein kinase inhibitors and the down- regulation of PKC markedly increase endothelial NO production [7, 27]. Moreover amlodipine, which inhibits PKC activity in endothelial cells, is able to enhance NO production by attenuating eNOS Thr⁴⁹⁵ phosphorylation [31].

Changes in Thr⁴⁹⁵ phosphorylation are generally associated with stimuli (e.g., bradykinin, histamine and Ca²⁺ ionophores) which elevate endothelial [Ca²⁺]_i and increase eNOS activity by 10 to 20 fold over basal levels. In response to such agonists, the activity of eNOS is not simply determined by the forma- tion of a Ca²⁺/CaM complex and its unregulated association with the enzyme, but rather by simultaneous changes in Ser¹¹⁷⁷and Thr⁴⁹⁵phosphorylation and re- sulting changes in the accessibility of the CaM- binding domain to CaM. Stimulation of endothelial cells with growth factors/hormones such as estrogens do not appear to result in a marked change in the phosphorylation of Thr⁴⁹⁵, rather these agonists appear to increase NO production by exclusively increasing the phosphorylation of Ser¹¹⁷⁷.

Although the activation of eNOS is linked to simultaneous changes in the phosphorylation of Ser¹¹⁷⁷ and Thr⁴⁹⁵ (see below) there are certainly additional eNOS phosphorylation sites. Indeed, the eNOS im- munoprecipitated from unstimulated cultured endothelial cells is serine phosphorylated [13, 39], however the residue(s) that is constitutively phosphorylated under these conditions is not Ser¹¹⁷⁷.

Thus while, the regulation of eNOS in endothelial cells is largely understood, much less is known about the mechanisms determining its activity in platelets.

The importance of phosphorylation in the regulation of platelet eNOS was highlighted in studies aimed at addressing the differential sensitivity of endothelial eNOS and platelet eNOS to insulin. Indeed, although insulin elicits the acute phosphorylation of eNOS in endothelial cells this is not generally associated with an increase in NO production or the relaxation of endothelium-intact isolated arteries [12, 49]. In platelets however, insulin is known to increase NOS activity and to attenuate thrombin-induced platelet aggregation by decreasing the thrombin-induced Ca²⁺transient [50, 56]. Although insulin induced the phosphorylation of eNOS in both cell types the kinases involved are distinct, with Akt being held responsible for the insulin-induced phosphorylation of Ser¹¹⁷⁷in endothelial cells but with AMP-activated protein kinase (AMPK) playing a prominent role in the insulin- induced activation of eNOS in platelets [17].

PKA may also play a role in the regulation of platelet eNOS as substances that enhance the intracellular concentration of 3’,5’-cyclic adenosine monophosphate (cyclic AMP), such as catecholamines,b-adrenoce- ptor agonists [46] and adenosine [1] initiated platelet NO production. Indeed, the activation of platelet eNOS has been linked to its phosphorylation on Ser¹¹⁷⁷by PKA [51]. To-date, there have been no reports describing the phosphorylation of platelet eNOS on sites other than Ser¹¹⁷⁷.

Interaction with other proteins: the eNOS signalosome

The production of NO and the activation of the soluble guanylyl cyclase (sGC) were initially thought to take place within the cell cytosol and that the NO generated simply diffuses to its target molecule. While this may be the case for NO when it acts as a neuro- transmitter, it is now clear that cells suffer from molecular crowding and that it is more likely that genera- tor and effector enzymes in a given signalling pathway exist in a large molecular complex or signalosome. The eNOS signalosome is perhaps the best characterized of the three enzymes since it has been clear for quite a few years that the association with calmodulin and caveolin has profound effects on the intracellular localization and activity of eNOS and that the enzyme can be phosphorylated by a series of kinases and can

Regulation of eNOS in platelets

Voahanginirina Randriamboavonjy et al.

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activity is also determined by its association with the heat shock protein 90 (Hsp90). More eNOS- associated proteins have been identified (NOSIP, NOSTRIN, dynamin, etc.). Thus, it has become in- creasingly clear that either by influencing eNOS subcel- lular localization or by directly acting on its catalytic process, protein-protein interactions provide a particularly versatile way to modulate eNOS function.

eNOS and Hsp90

Heat-shock proteins (Hsp’s) are molecular chaperones that are constitutively expressed [58]. They play funda- mental roles in cell function, ranging from protein fold- ing and transmembrane protein movement, to serving as scaffolds for the assembly of enzyme signalling complexes. The 90-kD heat shock protein (Hsp90) is required for the coordination of the trafficking and regulation of diverse signalling proteins, of which eNOS is one [24]. The association of Hsp90 with eNOS increases NO generation in endothelial cells [24], an effect that can be attributed to its function as an allosteric enhancer as well as to the promotion of eNOS Ser¹¹⁷⁷phosphorylation by Akt. Hsp90 can directly bind to Akt to protect it from protein phosphatase 2A-mediated dephosphorylation [53]. Thus, the interaction of Akt with Hsp90 might play an important role in regulating Akt kinase activity [19]. Re- cently, it has been shown that the activator role of Hsp90 on eNOS is also due to the stabilization of the 3-phosphoinositide-dependent kinase 1 (PDK1) [57]

which is known to activate Akt. Hsp90 also plays an essential role in maintaining eNOS in a coupled state i.e. preventing the uncoupling of the enzyme so that it generates oxygen-derived free radicals rather than NO. In proliferating endothelial cells, the association of Hsp90 with eNOS is markedly increased and the inhibition of Hsp90 results in an increase in the generation of superoxide anions (O₂^–) by eNOS [44].

Although Hsp90 is reported to be expressed in platelets [28, 29], evidence indicating a role in the regulation of the activity of platelet eNOS is purely circumstantial. For example, geldanamycin which in- terferes with Hsp90 binding to eNOS, attenuates the insulin-induced production of NO in platelets and po- tentiates thrombin-induced platelet aggregation [18].

In addition, in female pigs estrogen therapy was asso-

Physiological role of platelet eNOS

Role of the NO/cyclic guanosine monophos- phate (GMP) system in platelet function

Although eNOS can be detected in human platelets, the capacity of platelets to produce NO as well as the importance of platelet-derived NO in influencing platelet recruitment remain a matter of debate and contradictory findings have been reported concerning the role of NO on platelet activity.In vitro, NO produced by stimulated platelets has been shown to only modestly inhibit platelet activation by collagen or thrombin but to markedly inhibit additional platelet recruitment [20]. Studies performed using platelets from eNOS-deficient mice showed that the lack of platelet-derived NO alters the hemostatic responsein vivo by increasing platelet recruitment supporting a role for platelet-derived NO in the regulation of hemostasis [21]. Interestingly, recent evidence indicates that rather than preventing platelet activation and aggregation, platelet-derived NO may actually stimulate platelets [32]. The NO effector, cyclic GMP elicits a biphasic response in platelets consisting of an initial transient stimulatory response (that promotes platelet aggregation) and a subsequent inhibitory response that limits the size of thrombi. Additional evidence indicates that low concentrations of NO promote a dis- crete platelet degranulation. When stimulated with insulin to generate NO, there is a cyclic GMP- and protein kinase G-dependent association of the dense granule-bound protein vesicle-associated membrane protein-3 (VAMP-3) with the target membrane solu- bleN-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) syntaxin 2, which leads to the release of adenine nucleotides from dense granules [49]. Indeed, insulin elicits the release of enough ATP/adenosine from platelets to elicit the relaxation/vasodilatation of porcine coronary arteries. The adenine nucleotides released are metabolized by vascular ecto-nucleotidases to the potent vasodilator adenosine accounting for the insulin-mediated vasodilatation.

However, the consequences of platelet-derived NO may depend on its concentration as higher concentrations of NO inhibit the exocytosis of dense granules, lysosomal

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granules, anda-granules from human platelets, an effect that is related to the S-nitrosylation of the N-ethylmaleimide-sensitive factor [41].

Pathological modulation of eNOS activity

Blood platelets contribute not only to normal homeostasis but also to thrombotic disorders. Although different pathological situations such as diabetes, athero- sclerosis, obesity, and hypertension are associated with abnormal platelet function, reports relating to the modulation of platelet NOS expression/activity have so far been limited to diabetes and hypertension.

Platelet eNOS and diabetes

Both insulin-dependent (type 1) and non-insulin- dependent diabetes mellitus (type 2) are characterized by enhanced platelet activation. So far, endothelial dysfunction caused by uncoupling of endothelial NOS has been described to be the leading cause of platelet activation in diabetes mellitus. Also, an acute decrease in the bioavailability of systemic NO causes platelet activation. For example, platelet function in diabetes can be rescued by the restoration of endothelium-derived NO [54]. However, since NO produced by platelet NOS is sufficient to inhibit platelet activation by increasing cytoplasmic cyclic GMP levels, an impairment of the NO/cyclic GMP pathway in platelets could contribute to the hyperactivation of platelets found in diabetic patients. Indeed, in platelets from patients with diabetes, platelet NOS activity was significantly lower than that measured in platelets from healthy subjects, suggesting that the decreased NOS activity might play a role in the pathogenesis of diabetic vascular complications [34, 47].

There is evidence suggesting thatin vitro hyperglycemia [10] as well as type 2 diabetes in human subjects [11] result in the modification of Ser¹¹⁷⁷ by O-linked N-acetylglycosylation. Proteins modified in this manner tend to be under-phosphorylated relative to unglycosylated proteins and it has been suggested that O-GlcNAc glycosylation may obscure phosphorylation sites and thus interfere with signalling mechanisms and, in the case of eNOS, attenuate NO production. However, an increased activity of eNOS and an enhanced NO and peroxynitrite production were measured in platelets from women with gestational diabetes mellitus compared with healthy preg- nant women [36]. Regardless of whether the activity

of NOS in platelets is decreased or increased, platelets from diabetic patients are characterized by a reduced NO bioavailability.

Platelet eNOS and hypertension

Hypertension is characterized by an increased risk of thrombo-embolism. Abnormalities in hemostasis and platelet function can account for the pathogenesis of thrombosis in hypertension. Several studies have demonstrated that an impairment of platelet NOS activity can account for the enhanced platelet activation associated with hypertension [5, 42]. Hypertension has also been linked with the inhibition of the L-arginine transportvia system y + L in both humans and animals. The inhibition of this carrier system leads to a decrease in the availability of L-arginine and an impaired NO production [42]. It has been suggested that the endogenous L-arginine analogues (i.e.

asymmetric dimethylarginine), which inhibit NOS activity are implicated in the platelet activation detected in subjects with hypertension [4, 42]. The situation could however be more complicated as eNOS activity and NO production are not necessarily one and the same.

Platelet eNOS, like the enzyme in endothelial cells, can also be uncoupled and be converted (particularly in the absence of adequate tetrahydrobiopterin levels) into an O₂^–- and/or peroxynitrite-generating enzyme [9, 42].

Concluding remarks

Although the last 15 years have witnessed substantial progress in our understanding of the consequences of platelet-derived NO, current knowledge regarding the regulation of the activity of platelet eNOS and its modulation during the progression of cardiovascular diseases is still limited.

Acknowledgement:

Experimental work performed in the authors own laboratory was supported by the Deutsche Forschungsgemeinschaft (SFB 553, B5).

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Received:

September 2, 2005.