Elsevier

Clinical Biochemistry

Volume 30, Issue 7, October 1997, Pages 509-515
Clinical Biochemistry

Reviews
Diabetes-Induced Alterations in Platelet Metabolism

https://doi.org/10.1016/S0009-9120(97)00094-5Get rights and content

Abstract

Objectives: This review summarizes the recent findings on some aspects of platelet metabolism that appear to be affected as a consequence of diabetes mellitus. The metabolites include glutathione, l-Arginine/nitric oxide, as well as the ATP-dependent exchange of Na+/K+ and Ca2+.

Conclusions: Several aspects of platelet metabolism are altered in diabetics. These metabolic events give rise to a platelet that has less antioxidants, and higher levels of peroxides. The direct consequence of this is the overproduction platelet agonists. In addition, there is evidence for altered Ca2+ and Na+ transport across the plasma membrane. Recent evidence indicates that plasma ATPases in diabetic platelets are not damaged instead their activities are likely to be modulated by oxidized LDL. Finally, platelet inhibitory mechanisms regulated by NO appear to be perturbed in the diabetes disease-state. The combined production of NO and superoxide by NOS isoforms in the platelet could be a major contributory factor to platelet pathogenesis in diabetes mellitus.

Introduction

Platelets are the first line of defence upon loss of vascular integrity. Interaction of platelet membrane receptors with such injury related factors as collagen, microfibrils, and von Willebrand factor, induce adhesion to other platelets and to the vessel wall. The adhered platelets rapidly change their shape from smooth disks to burr-like spheres with multiple pseudopods. Concomitant with the shape change, aggregating agents such as ADP and serotonin are released from their storage granules. Other aggregating agents such as thromboxane A2 (TXA2) and seretonin are synthesized de novo and extruded from the platelet. The release of these agents induce platelets arriving at the site of injury to undergo the same processes, thus increasing the size of the platelet plug. Induction of platelet aggregation by exogenous substances such as collagen is termed first-phase aggregation. When substances secreted from platelets induce aggregation the process is termed second-phase aggregation. The sequence of events can be summarized as follows: First-phase aggregation → Release reaction→ Second-phase aggregation

The aggregation response appears to be altered in diabetics as their platelets display hypersensitivity to aggregating agonists (hyperaggregation) in vitro 1, 2, 3, 4, 5.

Platelet aggregation results as the consequence of a series of reactions initiated by the receptor mediated activation of a G protein and membrane associated phospholipase C producing inositol 1,4,5-trisphophate (IP3) and 1,2-diacylglycerol (1,2-DAG) [6]. IP3 then mobilizes Ca2+ from intracellular stores, and 1,2-DAG activates protein kinase C (PKC) [7]. Both the rise in [Ca2+]intracellular and PKC activation is associated with platelet release reaction and second phase aggregation. The elevations in intracellular Ca2+ levels also results in the activation of Ca2+-calmodulin-dependent processes. Platelets reportedly contain ∼0.5 pg of calmodulin (CaM) per cell. This translates to an intracellular concentration that has been estimated to be ∼30 μM [8].

The sodium pump or Na+/K+ ATPase is the enzyme that allows the coupled active transport of Na+ and K+ across the animal cell membrane. The enzyme transduces the chemical energy, derived from the release of phosphate, in the coupled efflux of 3Na+ and influx of 2K+, against their electrochemical gradients. The maintenance of the transmembrane gradients of Na+ and K+ is at the basis of physiological cell functions such as the regulation of cell volume, excitability, modulation of transport of other ions and molecules coupled to the Na+ and K+ gradients [9]. Lipids are required for the full enzymatic activity [10].

The number of Na+/K+ ATPases per cell varies from tissue to tissue. About 25,000 Na+/K+ ATPase molecules are present in the platelet plasma membrane [11]. Marks et al. [12]linked volume regulation, the expression of fibrogen receptors and sensitivity of platelets to agonists to the activity of the enzyme. Bork and Mrsny [13]demonstrated that the number of accessible molecules of the enzyme increased at the platelet surface during activation.

The homeostasis of intracellular calcium is mainly regulated by a balance between membrane influx and efflux: the influx is due both to voltage-dependent and receptor-operated channels, while the main mechanism of efflux is the active transport by the Ca2+-ATPase. Moreover, Ca2+-transport is strictly integrated with the regulation of sodium transport, as the Na+/Ca2+-exchange pathway is driven by the electrochemical potential of sodium and maintained by the activity of the Na+ pump [14]. Furthermore, altered Na+ and Ca2+ transport have been described in some pathological conditions [15].

A plasma membrane Ca2+-ATPase has been identified in human platelets 16, 17. De Metz et al. [18]demonstrated that plasma membrane. Ca2+-ATPase, like many other membrane associated enzymes, require phosphatidylserine and phosphatidylinositol for full activity in vitro.

Calmodulin (CaM) regulates both the stimulation and inhibition of the release reaction. The stimulatory activity of CaM is related to its regulation of platelet myosin light chain kinase (MLCK). CaM-dependent phosphorylation of myosin leads to the formation of the actomyosin complex. The force generated during the contraction of the actomyosin complex initiates the release reaction.

CaM-dependent regulation of the inhibition of the release reaction is also initiated upon platelet activation. Concomitant with platelet activation intraplatelet Ca2+ levels rise. Ca2+-calmodulin then activates the enzyme nitric oxide synthase (NOS) which produces nitric oxide (NO). NO inhibits platelet aggregation by stimulating the activity of guanylate cyclase thus elevating cGMP. cGMP is then postulated to bind at the G-protein/phospholipase C interface, inhibiting 1,2-DAG and IP3 production thus preventing the rise in [Ca2+]intracellular and PKC phosphorylation [19].

The dual role of CaM in the release reaction, is thought to be analogous to a threshold-sensitive gate: if the stimulatory response is below the threshold, NO-pathway inhibits aggregation. However, if the stimulatory response is above the threshold, the inhibitory effect via the NO-pathway is overridden or inhibited and the release reaction proceeds.

NO is an important factor in vascular homeostasis as it prevents platelet activation, limits leukocyte and platelet adhesion to the endothelium, and regulates myocardial contractility. In normal tissues NO is implicated in the attenuation of smooth muscle proliferation through cGMP-dependent activation of cAMP kinase [20]and by preventing the direct and indirect release of growth factors [21]. In addition, NO reduces platelet adhesion, and platelet thrombus formation [22]. Increasing volume of evidence points to the fact that pathogenesis in cardiovascular disorders including, hypertension, hypotension accompanying shock, atherosclerosis, and diabetes result from perturbations in NO metabolism [23].

NO is synthesized from l-Arg by NO synthases (NOS, EC 1.14.23). To date, three isoforms of NOS have been identified. They are all homodimeric (subunit size 125–160 kDa) and contain one FMN, one FAD, one non-heme iron, and one tetrahydrobiopterin molecule per subunit. Brain and endothelial NOS isoforms are constitutive (cNOS) and are regulated by Ca2+/calmodulin [24]. Brain (bcNOS) is a cytosolic enzyme whereas the endothelial isoform (ecNOS) membrane associated myristoylated [25]. The other isoform is the inducible NOS (iNOS) originally isolated from macrophages. Its expression is induced by proinflamatory cytokines such as tumor necrosis factor-α, lippopolysaccaride and interferon-γ. This isozyme is fully activated once synthesized, Ca2+-independent and has a tightly bound CaM 26, 27.

NOS displays multiple catalytic functions. Its principal function is the conversion of l-Arg to NO plus l-citrulline. However, under conditions of limiting l-Arg, NOS can produce superoxide anion (O2·−) from the NADPH-dependent reduction of O2 [28]. This can have potentially deleterious consequences as superoxide and NO react to form peroxynitrite (ONOO) a very potent oxidant.

Intraplatelet NO production has been directly determined [29]. We have isolated and identified a cNOS isoform from the human platelet [30]. More recently, Chen and Mehta 31, 32have shown that human platelets posses both ecNOS and iNOS with molecular weight and characteristics distinct from ecNOS [31]. Megacaryocytes of patients with coronary atherosclerosis were found to express more iNOS than cNOS (ratio 4.4:1). The ratio is reversed in normal patients (3.04) [33]. These results suggest a link between NOS expression and atherosclerosis.

l-Arg transporter has been identified in the human platelet [34]. We have evidence that l-Arg transport in the human platelet is affected by S-nitrosothiols [35]. NO also appears to regulate arachidonic acid metabolism in platelet as recent reports indicate that 12-lipoxygenase but not cyclooxygenase activity is inhibited by nitroprusside, an unstable compound that releases NO upon decomposition or by l-Arg (substrate of NOS) [36].

It is known that native LDL (a major risk factor in the pathogenesis of atherosclerosis) undergoes oxidative modification (Ox-LDL) within the blood vessels [37]. Previously, it has been shown that ox-LDL decrease the release of NO from endothelial cells [38]. Recently, Chen and Metha indicated that the l-Arg-NO pathway is involved in the effects of ox-LDL on platelet function. Interestingly, Zhao et al. [39]demonstrated that oxidized LDL inhibits the Ca2+-ATPase activity in purified platelet plasma membrane fractions. Further evidence for this was obtained with intact platelets where ox-LDL was shown to increase cytoplasmic Ca2+-levels, presumably via the inhibition of Ca2+-ATPase.

Glutathione (l-γ-glutamylcysteinylglycine) (GSH) is present in mM levels in most mammalian tissues including platelets [40]. GSH metabolic enzymes have been identified in the human platelet [41]. In the platelet, GSH is involved in the regulation of arachidonic acid metabolism. Liberation and metabolism arachidonic acid (AA) occurs concomitantly with the release reaction. Activation of the phospholipase A2 by the G-protein system results in the production of AA. AA is then converted to prostaglandin G2 (PGG2) or to 12-hydroperoxy-eiccosatetraenoic acid (12-HPETE) by the actions of cyclooxygenase or 12-lipooxygenase, respectively [42]. Both cyclooxygenase and 12-lipooxygenase are activated by hydroperoxides [43]. GSH indirectly regulates these enzymes through the action of GSH-peroxidase which destroys hydroperoxides in a GSH-dependent manner. In addition, 12-HPETE, an unstable metabolite is converted to a stable form, 12-hydroxy-eicosatetraenoic acid (12-HETE) by a GSH-dependent peroxidase [44]. PGG2 is then converted to PGD2 a weak platelet agonist and to thromboxane A2 (TXA2) a very potent agonist. Therefore, any metabolite like GSH that affects the production of platelet agonists like TXA2 is an important participant in platelet function.

Free thiols like that of GSH rapidly react with NO (NO+) giving rise S-nitrosothiols. NO-derivatives of BSA and GSH are thought to serve as stable NO-carriers in the serum and in the cytosol, respectively [45]. Approximately 60% of serum NO circulates as BSA-NO. BSA-NO lowers blood pressure and increases coronary blood flow by 165%. BSA-NO was shown to bind preferentially to denuded rabbit femoral vessels. Bound BSA-NO prevented platelet deposition and platelet aggregation [46]. Interestingly, once glycated BSA is no longer able to act as an NO-carrier [47]. In the platelet, the enzyme glyceradehyde-3-phosphate dehydrogenase has been implicated in the transport of NO via S-NO formation through its Cys-149 thiol [48].

Therefore, S-nitrosothiols represent an intimate link between the GSH and the l-Arg/NO pathways.

NO and superoxide anion (O2·−) react to produce peroxynitrite (ONOO) at diffusion controlled rates. ONOO is a relatively long lived strong oxidant as it has been shown to initiate lipid peroxidation [49]and the oxidation of thiols and the indole ring of Trp [50]. At pH 7.4, this anion will be ∼50% protonated (pKa = 7.49), forming peroxynitrous acid, which rapidly decomposes to H+ and NO3. In doing so it passes through an intermediate complex possessing both nitrogen dioxide (NO2 ·) character and hydroxyl radical (·OH) character [51]. NO2 · is oxidized to the nitronium ion (NO2+) by trace amounts of transition metal ions or their chelates [52]. NO2+ is a powerful nitrating agent for aromatic rings. In proteins, peroxynitrite decomposition most commonly leads to the nitration of Tyr and Phe side chains as well as the oxidation of thiols and Trp [50]. The specific nitration of Tyr catalyzed by active site residues have been reported in the case of Cu, Zn-superoxide dismutase [52], glutathione reductase [53], and metalloproteinase-1 [54].

In washed platelets ONOO induces aggregation. However, in platelet-rich plasma ONOO acted as an inhibitor of platelet aggregation. The antiaggregatory properties of OONO were duplicated by the addition of BSA or GSH to washed platelets, suggesting thiol status can regulate the action of this strong oxidant [54].

Section snippets

GSH Metabolism

Several reports indicate that platelet GSH metabolism is impaired in diabetics. In general intraplatelet GSH levels are lower in diabetics than in controls 55, 56. During platelet activation GSH levels have been shown to transiently decrease. In normal platelets GSH levels return to preactivation levels in the presence of subthreshold agonist concentrations. In contrast, in the diabetic platelet GSH levels do not return to preactivation levels. We have examined the kinetic properties of some

Concluding Remarks

As the work outlined in this review indicates several aspects of platelet metabolism are altered in diabetics. These metabolic events give rise to a platelet that has less antioxidants (GSH), and higher levels of peroxides, owing to impaired GSH peroxidase and lower GSH levels. The direct consequence of GSH-related alterations is the overproduction of arachidonic acid metabolites, which are strong platelet agonists.

Additional contributions to platelet hyperreactivity result from abnormal Ca2+

Acknowledgements

Laura Mazzanti wishes to thank Prof. Giovanna Curatola and Drs. Rosa Anna Rabini, Roberto Staffolani, Ferrero Foundation, and CNR for contributions to her work. Bulent Mutus wishes to thank the Canadian Diabetes Association and NSERC for research funding that made some of the work discussed in this review possible.

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