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- Materials and methods
- Note added in proof
Summary. Diabetes mellitus (DM) is associated with enhanced lipid oxidation and persistent platelet activation. We investigated whether oxidant stress (OS) also affects circulating proteins and is associated with an abnormal coagulative pattern. In 72 type 2 DM (T2DM) patients, urinary 8-iso-prostaglandin (PG) F2α and 11-dehydro-thromboxane B2 (TXM) were measured as markers of lipid peroxidation and platelet activation, respectively. The carbonyl content of plasma proteins (PCARB) was measured as global index of protein oxidation. 8-Iso-PGF2α and PCARB levels were higher in DM patients than in controls (P < 0.05). Likewise, both TXM and prothrombin F1+2 levels were higher in diabetics (P < 0.05). By contrast, anticoagulant markers, such as activated protein C, protein C activation peptide, and soluble thrombomodulin (TM) were depressed in T2DM (P < 0.05). In conclusion, OS in T2DM involves circulating proteins and is associated with an unbalanced promotion of procoagulant reactions. These effects in concert with platelet activation may contribute to atherothrombotic complications in T2DM.
Oxidant stress (OS) has been reported to be increased in diabetes mellitus (DM) and hypothesized to be involved in the pathogenesis of vascular damage and thromboembolic complications [1,2]. Diabetic patients are characterized by increased urinary excretion of F2-isoprostanes , which are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation, catalyzed by oxygen-free radicals on cell membranes and low density lipoprotein (LDL) particles [3,4]. Enhanced lipid peroxidation and the generation of bioactive isoprostanes may represent an important biochemical link between impaired glycemic control and persistent platelet activation [2–4]. In addition, the formation of advanced glycation end (AGE) products, such as pentosidine, acrolein, carboxymethyl-lysine, may lead to oxidant damage of proteins, which in turn may accelerate AGE formation [1,5,6].
Moreover, oxidant damage may promote coagulation and alter the structure and the function of coagulative proteins, as previously shown both in vitro and in vivo[7–13]. In fact, oxidized lipid micelles accelerate the activity of the prothrombinase complex in vitro, thus enhancing thrombin production [7,8]. In addition, upon exposure to oxidizing stimuli, fibrinogen, thrombin, and physiological anticoagulants, such as protein S and antithrombin III, undergo major structural and functional changes [9–12]. Furthermore, OS may affect thrombomodulin (TM), an endothelial protein which enables thrombin to activate the protein C (PC) pathway . For instance, oxidation of TM methionine 388 by either H2O2 or activated PMN reduces its cofactor capacity by 90% . Finally, oxidant agents such as homocysteine and oxidized LDL inhibit both the synthesis and surface expression of TM in cultured endothelial cells [16,17]. Therefore, OS may not only favor coagulative activation [7,8], but also impair anticoagulant pathway.
We have previously reported biochemical evidence of enhanced generation of F2-isoprostanes  and persistent platelet activation, as reflected by enhanced excretion of 11-dehydro-thromboxane B2, a major TXA2 enzymatic metabolite excretion, in patients with T2DM . We speculated that increased OS associated with DM could also induce oxidative changes in proteins, such as those involved in coagulation, thus altering their function. Therefore, in the present study we investigated whether the carbonyl content of plasma proteins is altered in T2DM patients when compared with age-matched non-diabetic subjects and whether it correlates with the rate of F2-isoprostane formation. We also measured both the activation peptide released from PC by the thrombin–TM complex, and the activation peptides released from procoagulant enzymes/substrates (prothrombin F1+2 fragment and fibrinopeptide A) along with plasma level of soluble TM. The association analysis of both pro- and anticoagulant markers with oxidation indexes provided a multidimensional picture of how oxidant stress affects the hemostatic balance in DM.
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- Materials and methods
- Note added in proof
Enhanced lipid peroxidation is associated with persistent platelet activation in diabetic patients . This study confirms and extends our previous findings. In fact, both lipid and protein oxidation were found significantly elevated in patients with T2DM. Plasma protein carbonyls provide a global index of protein oxidation involving the side chains of several amino acid residues. They represent the stable end-product generated upon formation of transient radical species, such as chloramines and nitrogen/carbon radicals, which are induced by oxidant stimuli [28,29]. However, direct oxidation of amino acid side chain is not the only way through which carbonyl groups can be formed in proteins. In fact, glycation may induce formation of protein carbonyls, such as ketoamine derivatives, thus generating reactive radicals and perpetuating a vicious cycle . Thus, protein oxidation may be generated in part by nonenzymatic glycation processes, consistently with the significant association between HbA1c and PCARB found in the present study (Table 2). Moreover, a potential mechanism linking lipid and protein oxidation has been recently reported . Thus, oxidized polyunsaturated fatty acids may form α,β-unsaturated aldehydes, that can attack lysyl side chains, contributing to the generation of stable protein carbonyl groups . Such a mechanism is consistent with our finding of a significant relationship between protein and lipid oxidation in vivo in the setting of T2DM. Although both urinary 8-iso-PGF2α and PCARB were associated with F1+2 levels in univariate analysis, only the former was significantly associated with F1+2 in a multiple regression analysis. The strong association of F2-isoprostane formation with both 11-dehydro-TXB2 and F1+2 levels suggests that lipid peroxidation can affect platelet as well as coagulative activation, thus contributing to a pro-thrombotic state in this setting. This observation is consistent with the in vitro demonstration that oxidized lipid micelles enhance the prothrombinase activity, thus enhancing thrombin generation [7,8]. The present results confirm and extend these in vitro findings to the clinical setting of T2DM.
Diabetic patients also showed an impairment of anticoagulant mechanisms. Plasma soluble TM and APC/PCP levels were in fact decreased in diabetic subjects. It has to be remarked that the finding of reduced soluble TM levels was in apparent disagreement with previous reports concerning DM . However, the mechanism(s) generating soluble TM in blood is unclear, and different levels of soluble TM have been reported in different clinical settings [33,34]. Generally, soluble TM level is thought to reflect the endothelial damage in peripheral atherosclerotic disorders . However, in primary pulmonary hypertension, despite of a severe damage of pulmonary endothelium, a net decrease of soluble TM levels has been reported [35,36]. Infusion of prostacyclin was reported indeed to enhance TM levels in these patients . Thus, TM levels may be considered the net resultant of multiple and complex phenomena, whose evolution may generate very different patterns of soluble TM levels. In our opinion, when the PC anticoagulant pathway has to be investigated, measurement of both soluble TM and APC/PCP levels should be performed at the same time to provide a coherent scenario. Moreover, the set of DM patients investigated in this study did not present signs of severe endothelial destruction, as revealed by normal values of VWF and fibrinogen levels as well. This may contribute to explain the apparent disagreement with previous results.
It has to be remarked that oxidizing agents, such as H2O2, oxidized LDL or homocysteine can down-regulate the activity and/or expression of TM on the surface of endothelial cells . Furthermore oxidation of methionine 388 in the TM molecule, inactivates almost completely the anticoagulant function of the endothelial protein . Thus, increased oxidation of circulating proteins may involve and impair TM function as well. Based on these considerations, oxidant stimuli might inhibit APC production by reducing TM concentrations on the endothelial surface, where PC activation takes place, and/or by chemical inactivation of the TM molecule. It may be of interest that two recent studies showed that regression of atherosclerosis in monkeys reduces also the vascular superoxide production, increases pulmonary TM activity and increases the anticoagulant response to thrombin in vivo[37,38]. The results obtained in the present study, showing a significant decrease of APC/PCP levels in T2DM patients under oxidative stress, may just represent the reversal of the medal, hinging on similar mechanisms.
An antioxidant pharmacological intervention aimed at investigating its effects on the anticoagulant PC pathway in this setting might be theoretically of clinical interest. However, recent studies raised many doubts on vitamin E supplementation, alone or in combination with other antioxidant agents like vitamin C and β-carotene, as protective agents against thrombotic cardiovascular diseases . Accordingly, a recent pilot study on the effects of vitamin E supplementation on both protein oxidation and coagulation parameters in normal subjects showed a modest effect on F1+2 production only, while markers of the TM and APC/PCP pathway and protein oxidation remained unchanged . The chemistry of biological oxidations is very complex and likely specific for a particular kind of chemical species. A molecule with antioxidant properties for lipids not necessarily shows the same degree of activity for protein oxidation. For instance, it is known that vitamin E is an inappropriate antioxidant against the superoxide anion, which can react directly and rapidly with proteins or nitric oxide, while it needs metal ions to peroxidize lipids . Further studies are thus needed before designing potential intervention studies with antioxidant agents in the T2DM setting.
In conclusion, the present study demonstrates increased oxidation of both lipids and proteins in T2DM. The capacity of oxidized lipids to activate platelets may constitute a thrombotic trigger per se. Furthermore, oxidized lipids present on activated platelets can provide a better surface for the assembly and activation of prothrombinase complex. This may trigger a vicious cycle that, together with the oxidation-linked depression of the anticoagulant pathway, may predispose to a pro-thrombotic state in this setting. This complex scenario may explain the limited efficacy of conventional antiplatelet therapy with low-dose aspirin in diabetics , providing a rationale for trials of more aggressive antithrombotic therapy in this setting.