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Platelet resistance to the antiaggregatory cyclic nucleotides in central obesity involves reduced phosphorylation of vasodilator-stimulated phosphoprotein.

Central obesity is a major risk factor for early cardiovascular events and other atherothrombotic diseases (1, 2). It is associated with insulin resistance, lipid disorders, oxidative stress, and low-grade chronic inflammation as well as with a prothrombotic state (1, 2). For this reason, central obesity has been considered in the recent classification of the International Diabetes Federation as a necessary component of the metabolic syndrome (3), a cluster of cardiovascular risk factors.

One possible mechanism contributing to enhanced thrombotic risk in obese patients is platelet hyperactivation, which is involved in the different steps of the atherosclerotic process (4, 5).

Our previous studies of platelets of obese persons identified multiple defects in the sensitivity to the antiaggregatory mediators. In particular, we observed reduction of the antiaggregatory effects of insulin and organic nitrates, which activate the cGMP pathway (6-9), and of adenosine and prostacyclin, which activate the cAMP pathway (10, 11). We also observed reduced sensitivity to the antiaggregatory effects of cGMP and cAMP themselves, which are the main intracellular messengers responsible for inhibition of platelet responses elicited by the large majority of platelet agonists (11).

Platelet response to cyclic nucleotides is complex and depends on nucleotide concentrations and the timecourse of platelet exposure. Most authors agree that the main effect of cyclic nucleotides is inhibitory and is exerted through activation of the corresponding cyclic nucleotide-dependent protein kinases, i.e., protein kinase G (PKG) [1] for cGMP and protein kinase A (PKA) for CAMP (12-16), which are involved in the regulation of basic mechanisms of platelet activation, such as agonist-induced increases of cytosolic calcium (12,15-17), fibrinogen binding (18), and cytoskeleton protein contraction (19). Recent evidence indicates, however, that increased concentrations of platelet cGMP are associated with enhanced platelet function (20, 21).

A relevant target of both cyclic nucleotide-regulated protein kinases is the focal adhesion protein vasodilator-stimulated phosphoprotein (VASP) (22, 23), which is strategically involved in platelet inhibitory pathways. VASP phosphorylation closely correlates with inhibition of fibrinogen binding to glycoprotein IIb/IIIa (GP IIb/IIIa) (18, 24), and it affects initial sequences of platelet adhesion and activation by modulating interactions of platelet actin filaments (4,19). VASP is considered a reliable mediator of cyclic nucleotide action (12, 24, 25).

The reduced platelet antiaggregatory activity exerted by both cyclic nucleotides (11) does not necessarily lead to impaired activation of cyclic nucleotide /specific kinase/ VASP pathways. Platelets from individuals with insulin resistance have higher free cytoplasmic calcium concentrations than platelets from controls (26); thus, calcium fluxes may present a primitive resistance that is also inhibited by efficient cyclic nucleotide /specific kinase/ VASP pathways. The aim of these studies was to clarify whether cyclic nucleotides activate downstream pathways in individuals with central obesity, as they do in lean controls.

Patients and Methods


The study participants were 12 healthy volunteers [6 men and 6 women, mean (SE) age 34.7 (1.9) years] and 12 individuals with central obesity [6 men and 6 women, age 35.4 (2.1) years]. The criterion for central obesity was a waist circumference, measured at its smallest point with the abdomen relaxed, >88 cm in women or >102 cm in men (27). All study participants gave informed consent before investigation and the Ethics Committee of our Department approved the study design. None of the study participants had smoked or taken medications that could influence platelet function during the previous 4 weeks. Obese participants were otherwise healthy on the basis of medical history, physical examination, and standard diagnostic procedures; had no family history of diabetes mellitus; were normotensive (i.e., arterial blood pressure value <140/90 mm Hg); and had fasting and postchallenge plasma glucose concentrations within reference intervals [fasting plasma glucose [less than or equal to]6.105 mmol/L (110 mg/dL) and plasma glucose <7.77 mmol/L (140 mg/dL) 2 h after a 75-g oral glucose load]. Biochemical variables were measured as described below.


In previous investigations we used 8-bromo analogs of cyclic nucleotides, which because of their hydrophilicity are poorly permeable through cell membranes (11). In this study we used the more lipophilic molecules 8-(4-Chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP), which are highly effective in PKA and PKG activation (28) and do not interfere with cyclic nucleotide phosphodiesterases (28); because these analogs have not been previously used in studies of platelets from obese individuals, we also evaluated whether their antiaggregatory effect is decreased in central obesity.

For platelets from both lean and obese study participants, we investigated (a) sensitivity to the antiaggregatory effects of the cyclic nucleotide analogs 8-pCPT-cAMP and 8-pCPT-cGMP on ADP-induced platelet aggregation; (b) PKA and PKG concentrations; and (c) concentrations of total VASP and of VASP phosphorylation in response to 8-pCPT-cAMP and 8-pCPT-cGMP.


Fasting venous plasma glucose, serum cholesterol, HDL cholesterol, and triglycerides were measured by automated chemical analyses in the central laboratory of our hospital. Fasting plasma insulin was measured by RIA with a reagent set from Biochem Immuno System S.p.A.; the cross-reactivity was 100% for human insulin, 14% for human proinsulin, and 0.0002% for C-peptide and glucagon. Fasting C-peptide was measured by RIA with a reagent set from Biochem Immuno System S.p.A.; the cross-reactivity was 100% for human C peptide, 3.2% for human proinsulin, and absent for glucagon. Insulin sensitivity in the fasting state was estimated with the Homeostasis Model Assessment Index of Insulin Resistance (HOMA IR) according to the following formula: fasting plasma glucose (mmol/L) x fasting serum insulin ([micro]U/ mL) divided by 22.5 (29). HOMA IR is commonly used in clinical studies as a marker of insulin resistance (30, 31); high HOMA IR scores denote low insulin sensitivity.


Blood samples were collected after study participants had fasted overnight. A venous blood sample was collected without stasis and anticoagulated with 1 volume of sodium citrate, 38 g/L, pH 7.4, to 9 volumes of blood. Platelet-rich plasma (PRP) was obtained from citrated whole blood by 20-min centrifugation at 1008 at room temperature; platelet-poor plasma (PPP) was prepared by further centrifugation at 2000g for 10 min. Platelet counts were determined on an S-plus Coulter Counter (Coulter Electronics). Mean (SE) platelet counts in PRP ([10.sup.9]/L) were 268 (25) in controls and 248 (18) in obese individuals (not significant). Because the study design consisted of measurement of platelet responses in samples from the same PRP after addition of buffer solutions or different substances for each study participant, platelet numbers were not adjusted. Platelet aggregation was carried out by following light-scattering changes as originally described by Born (32), using a model 500 Chrono Log aggregometer at a constant stirring speed of 900 rpm.

Platelet aggregation in response to ADP was reported as maximal aggregation, calculated as: 100 x [(initial absorbance--absorbance after addition of ADP)/(initial absorbance)], with ADP added at a final concentration of 4 [micro]mol/L. Half-maximal inhibitory concentration ([IC.sub.50]) values of the 2 cyclic nucleotide analogs were determined at the different incubation times, when possible.


PKA and PKG concentrations were determined by Western blotting as previously described by Dey et al. (33). Experiments were carried out in 50-mL blood samples antiaggregated with acid citrate-dextrose solution (vol/ vo1:1/6). ACD-anticoagulated PRP, obtained by centrifugation at 1008 for 20 min, underwent further centrifugation at 2000g for 10 min. The pellet was washed 2 times at 37 [degrees]C in HEPES-Na buffer (10 mmol/L HEPES Na, 140 mmol/L NaCl, 2.1 mmol/L MgS[O.sub.4], 10 mmol/L D-glucose, pH 7.4); 500 [micro]L of washed platelets (2.5 x [10.sup.9] platelets/mL) were sedimented by centrifugation at 2000g for 10 min and solubilized by lysis buffer (1% SDS, 0.1% Triton X-100, 10 mmol/L Tris-HCI, pH 7.4), supplemented with protease inhibitors (Sigma). After centrifugation at 30 000g for 60 min, 30 [micro]g protein from platelet lysates was subjected to 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred to polyvinylidenedifluoride membrane (Millipore). Membranes were incubated at room temperature for 1 h with rabbit polyclonal antibodies against PKA 1a/1[beta] (Santa Cruz Biotechnology; 1:3000), or rabbit polyclonal antibodies against PKG 1a/1[beta] (Calbiochem; 1:300). Then, membranes were washed 3 times for 10 min each with PBS (136 mmol/L NaCl, 2.7 mmol/L KC1,10 mmol/L NaHP[O.sub.4],1.8 mmol/L K[H.sub.2]P[O.sub.4])/0.1% Tween-20 (PBS-Tween) and incubated with antirabbit horseradish peroxidase-conjugated secondary antibody (1:10 000) for 45 min. After 3 final washes (10 min each) in PBS-Tween, membranes were subjected to chemiluminescence (Amersham Life Sciences) for detection of the specific antigen. Density of bands in Western blots was analyzed with Kodak 1D Image Analysis software.


PKA preferentially induces VASP phophorylation at Ser157 causing an upward shift in the apparent molecular weight from 46 kDa to 50 kDa in sodium dodecyl sulfatepolyacrylamide gel electrophoresis, whereas PKG preferentially induces VASP phosphorylation at Ser239 without any change in molecular mass (22,23).

Experiments were carried out in blood samples anticoagulated with ACD and washed platelets prepared as previously described; 500 [micro]L of washed platelets containing 2.5 x [10.sup.9] platelets/mL were preincubated in the presence of 8-pCPT-cAMP (10-500 [micro]mol/L), 8-pCPT-cGMP (10-500 [micro]mol/L), or buffer solution for 10 min. Platelets were then sedimented by centrifugation at 2000g for 10 min, solubilized, and subjected to Western blot analysis as described previously.

The incubation of membranes was carried out with the following antibodies (all obtained from Calbiochem): rabbit antihuman VASP protein (1:15 000); monoclonal antibody recognizing VASP phosphorylated at Ser157 (1:1000); and monoclonal antibody recognizing VASP phosphorylated at Ser239 (1:1000).

After 3 washes in PBS-Tween, membranes were incubated for 45 min with monoclonal antirabbit horseradish peroxidase-conjugated secondary antibody (1:10 000) for VASP protein detection or with horseradish peroxidaseconjugated rabbit antimouse IgG (1:50 000) for phosphorylated VASP detection. After additional washes, protein expression was visualized as described previously.


ADP, 8-pCPT-CAMP, 8-pCPT-cGMP, PBS, and Tween-20 were obtained from Sigma. The source of the specific antibodies for Western blotting has been previously indicated.


Data in the text and in the Figs. are expressed as mean (SE). Statistical analyses were performed with AMOVA for repeated measurements, and, when appropriate, by the Student t-test for unpaired data. [IC.sub.50], values were determined by probit analysis. Furthermore, simple and multiple regression analyses were carried out by use of the Stat View Software for Macintosh.


Characteristics of the study participants are summarized in Table 1. The 2 groups differed significantly in body mass index (P <0.0001), waist circumference (P <0.0001), fasting insulin (P <0.002), fasting C-peptide (P <0.0001), HOMA IR index (P <0.0001), systolic blood pressure (P <0.05), triglycerides (P <0.03), and HDL cholesterol (P <0.005). Although all of the obese study participants presented with central obesity, none fulfilled the criteria for the diagnosis of the metabolic syndrome according to the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III) [i.e., the simultaneous presence of 3 of the following variables: waist circumference >88 cm in women, >102 cm in men; HDL-C <1.036 mmol/L (40 mg/dL) in men, <1.295 mmol/L (50 mg/dL) in women; fasting triglycerides >1.695 mmol/L (150 mg/dL); blood pressure >130/85 mmHg; fasting glucose >6.105 mmol/L (110 mg/dL)] (27).


Platelet numbers were the same for obese and lean individuals, and platelet response to ADP was similar. In particular, mean (SE) maximal aggregation values in response to 4 [micro]mol/L ADP in obese and lean individuals were 53% (3%) and 51% (4%), respectively (not significant).


Platelet aggregation in response to ADP was reduced by 8-pCPT-CAMP (3-20 min incubation; 10-500 [micro]mol/L) in a concentration-dependent manner both in lean controls and in obese individuals (Fig. 1A; AMOVA for repeated measurements: P <0.0001 for both groups). The antiaggregatory effects of 8-pCPT-CAMP, however, were greater in controls for each concentration of the cyclic nucleotide analog at all experimental times (P <0.05-0.0001). In particular, the mean (SE) [IC.sub.50], values were lower in controls than in obese-individuals at 10 min [24 (7) vs 252 (41) [micro]mol/L; P <0.0001] and at 20 min [5 (1) vs 123 (33) [micro]mol/L; P <0.01] (Fig. 113). In individuals with central obesity, no 8-pCPT-CAMP concentration inhibited platelet aggregation to ADP by 50%, so the 8-pCPT-cAMP [IC.sub.50], for a 3 min platelet exposure could not be calculated. With simple regression analysis, the [IC.sub.50], value of 8-pCPTcAMP at 20 min was positively correlated with waist circumference, HOMA IR, HDL cholesterol, triglycerides, and systolic and diastolic blood pressure (Table 2). With multiple regression analysis, however, only HOMA IR was significantly correlated with 8-pCPT-CAMP [IC.sub.50].

In both lean controls and in obese individuals, 8-pCPTcGMP (3-20 min incubation; 10-500 [micro]mol/L) decreased ADP-induced platelet aggregation in a concentrationdependent manner (AMOVA: P <0.0001) (Fig. 2A). The 8-pCPT-cGMP antiaggregatory effects, however, were greater in controls for each concentration of the cyclic nucleotide analog at all investigated times (P <0.05-0.0001). Furthermore, mean (SE) 8-pCPT-cGMP [IC.sub.50], values were lower in the control participants than in obese individuals with exposure for 20 min [17 (8) [micro]mol/L vs 172 (43) [micro]mol/L; P <0.01] (Fig. 213). In individuals with central obesity, it was impossible to calculate the [IC.sub.50] value with shorter 8-pCPT-cGMP exposure, no concentration inhibited ADP-induced aggregation by 50%. When simple regression analysis was used, the [IC.sub.50], value of 8-pCPT-cGMP at 20 min was positively correlated with waist circumference, HOMA IR, HDL cholesterol, triglycerides, and systolic and diastolic blood pressure (Table 2). With multiple regression analysis, however, only HOMA IR was significantly correlated with 8-pCPT-cGMP [IC.sub.50].


Protein content of PKA and PKG was similar in platelets from obese individuals and from lean controls (Fig. 3A).


The total VASP protein content of resting platelets was similar in lean and obese individuals (Fig. 3A). In both lean and obese individuals 8-pCPT-CAMP increased VASP phosphorylation at Ser157 (AMOVA for repeated measurements: P <0.0001 for both groups) (Fig. 313); the increase was smaller in obese than in lean individuals (significance vs lean individuals: P <0.0001 with 10 [micro]mol/L, P <0.01 with 100 [micro]mol/L, and P <0.05 with 500 [micro]mol/L).

Platelet exposure to 8-pCPT-cGMP increased VASP phosporylation at Ser239 in both lean and obese individuals (AMOVA for repeated measurements: P <0.001 for both groups; Fig. 4); the increase was smaller in obese than in lean individuals (significance vs lean individuals: P <0.0001 with 10 [micro]mol/L, P <0.001 with 100 [micro]mol/L, and P <0.01 with 500 [micro]mol/L).


This study showed that decreased antiaggregatory action of the cyclic nucleotides cAMP and cGMP in platelets of individuals with central obesity was associated with decreased phosphorylation of VASP at specific sites, reflecting impaired activation of PKA and PKG.

This result strengthens our previous observation that the antiaggregatory action of CAMP and cGMP is reduced in patients with central obesity (11). In fact, in the present study we used highly lipophilic, permeable cyclic nucleotide analogs (i.e., 8-pCPT-CAMP and 8-pCPT-cGMP), which, unlike those previously used (11), do not interfere with phosphodiesterases and are very effective in the activation of specific protein kinases (28).

In obese individuals the inhibitory effect of these analogs on platelet aggregation induced by ADP was much reduced, as shown by the fact that their [IC.sub.50], values at 20 min were ~10-fold higher than in controls.


Univariate regression analyses showed that [IC.sub.50], values of both cyclic nucleotide analogs were correlated with HONIA IR and with other variables such as HDL cholesterol, triglycerides, and systolic and diastolic blood pressure, whose alterations are considered for the diagnosis of metabolic syndrome (27). It should be emphasized, however, that none of the obese individuals met criteria for the diagnosis of metabolic syndrome, because each of them presented at the most only 1 diagnostic feature in addition to central obesity.


When all parameters were pooled together, multiple regression analysis showed that only [IC.sub.50], values of both cyclic nucleotide analogs remained significantly correlated with HONIA IR, thus suggesting that the molecular defect involved in the resistance to cyclic nucleotides is directly related to insulin resistance.

Our observations may be of interest in the current debate on the causal relationship between insulin resistance and metabolic syndrome (34, 35), because they suggest that insulin resistance per se is directly involved in the pathogenesis of some platelet abnormalities occurring in central obesity, which is a classical component of the metabolic syndrome (27).


Mechanistically, the present study clearly showed impaired activation of the cyclic nucleotide /specific kinase/ VASP pathways in individuals with central obesity. Platelets from obese individuals showed a significant decrease in VASP phosphorylation both at Ser157 after exposure to 8-pCPT-CAMP and at Ser239 after exposure to 8-pCPT-cGMP, despite a similar platelet content of PKA, PKG, and VASP proteins. To the best of our knowledge, our study provides the 1st demonstration of an impaired activation of both cyclic nucleotide-dependent protein kinases in obese patients. Abnormalities of these kinases have been previously observed only in patients with neuro-psychiatric disorders (36).

This study, therefore, identified a novel feature of platelet dysfunction occurring in central obesity. Because the molecular defects observed in obese individuals are involved in crucial steps of the control of platelet function (12-16), the present results may be relevant in explaining resistance not only to the antiaggregatory effects of the cyclic nucleotide analogs but also to other antiaggregatory mediators, which we investigated in previous studies (6, 9,11). The reduced antiaggregatory effects of insulin, organic nitrates, and prostacyclin, which act through activation of cyclic nucleotide /protein kinase pathways (10, 11), may be explained, at least in part, by an impaired action of the cyclic nucleotides on their specific kinases.

VASP phosphorylation induced by PKA and PKG is known to be relevant to the inhibition of aggregation through modulation of actin polymerization and inhibition of fibrinogen binding to the platelet integrin GP IIb/IIIa (18, 24). The phosphorylation state of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases (37); therefore further studies are needed to evaluate whether increased activity of these phosphatases may play a role in the reduced content of phosphorylated VASP in platelets from obese individuals in response to cyclic nucleotide analogs.

In conclusion, the findings of this study further elucidate the complex picture of platelet alterations in obese individuals with insulin resistance. Our previous studies showed resistance to antiaggregatory action of agents due to impaired ability to increase cyclic nucleotide synthesis (6-11). The present results also show impaired ability of cyclic nucleotides themselves to activate downstream steps of antiaggregation, such as those related to VASP phosphorylation.

These defects, which are closely linked to insulin resistance, could contribute to the pathogenesis of the prothrombotic state described in the insulin resistance syndrome and justify, at least in part, the increased cardiovascular risk attributed to this syndrome (1,38-40).

Grant/funding support: This study was supported by a research grant from Italian Ministero dell'Istruzione, Universita e Ricerca (MIUR) COFIN 2004 within the project "The molecular basis of insulin resistance and their importance in the pathogenesis of the alterations of the vessel wall", Local

Coordinator: Prof. Giovanni Anfossi, National Coordinator: Prof. Amalia Bosia and by a research grant from Regione Piemonte (to G.A.).

Financial disclosures: None declared.

Acknowledgements: We thank Mrs. Anna Baker for her linguistic assistance.

Received July 22, 2006; accepted March 16, 2007. Previously published online at DOI: 10.1373/clinchem.2006.076208


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[1] Nonstandard abbreviations: PKG, protein kinase G; PKA, protein kinase A; VASP, vasodilator-stimulated phosphoprotein; GP IIb/IIIa, glycoprotein Ilb/IIIa; 8-pCPT-cAMP, 8-(4-Chlorophenylthio)-cAMP; 8-pCPT-cGMP, 8-(4Chlorophenylthio)-cGMP; HOMA IR, Homeostasis Model Assessment Index of Insulin Resistance; PRP, platelet-rich plasma; PPP, platelet-poor plasma; [IC.sub.50], half-maximal inhibitory concentration.


Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.

* Address correspondence to this author at: Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (Turin), Italy. Fax 39-011-9038639; e-mail
Table 1. Clinical characteristics of the study
participants. (a)

 Controls Obese Significance
Characteristics individuals
No. 12 12
Men/women (n/n) 6/6 6/6
Age, years 34.7 (1.9) 35.4 (2.1) NS
BMI, (b) kg/[m.sup.2] 21.5 (0.4) 32.0 (0.7) P <0.0001
Waist circumference, cm 78.5 (3.0) 106.5 (2.1) P <0.0001
Glucose, mmol/L 4.83 (0.14) 5.18 (0.37) NS
Insulin, pmol/L 33.7 (3.3) 217.8 (40.8) P <0.002
HOMA IR index 1.7 (0.2) 4.8 (0.6) P <0.0001
Fasting C-peptide, ng/mL 2.17 (0.18) 3.69 (0.21) P <0.0001
Systolic blood 118 (2.4) 125 (2.0) P <0.05
 pressure, mmHg
Diastolic blood 78 (1.3) 82 (2.1) NS
 pressure, mmHg
Triglycerides, mmol/L 1.1 (0.1) 1.7 (0.2) P <0.03
Total cholesterol, mmol/L 4.8 (0.3) 5.3 (0.3) NS
HDL cholesterol, mmol/L 1.6 (0.1) 1.1 (0.1) P <0.005
Platelet count in 268 (25) 248 (18) NS
 PRP, [10.sup.9]/L

(a) Values are expressed as mean (SE).

(b) BMI, Body mass index; NS, not significant.

Table 2. Simple and multiple regression analysis
concerning the relation between cyclic nucleotide
IC50 values and different variables measured in
controls and obese individuals.

 IC50 value of 8-pCPT-cAMP

Regression analysis Simple Multiple

 r P [beta] t P

Waist circumference 0.796 0.001 -0.035 -0.108 0.917
BMIa 0.858 0.0001 -0.197 -0.525 0.619
HOMA IR 0.944 0.0001 1.157 4.079 0.007
HDL cholesterol -0.531 0.051 0.258 1.789 0.124
Triglycerides 0.747 0.002 0.149 0.584 0.581
Systolic blood pressure 0.611 0.02 -0.014 -0.059 0.955
Diastolic blood pressure 0.667 0.009 0.128 0.626 0.554
[R.sup.2] 0.947

 [IC.sub.50] value of 8-pCPT-cGMP

Regression analysis Simple Multiple

 r P [beta] t P

Waist circumference 0.786 0.001 -0.109 -0.269 0.797
BMIa 0.839 0.0001 -0.362 -0.779 0.466
HOMA IR 0.929 0.0001 1.203 3.416 0.014
HDL cholesterol -0.534 0.049 0.215 1.203 0.274
Triglycerides 0.744 0.002 0.341 1.077 0.323
Systolic blood pressure 0.605 0.022 0.141 0.472 0.654
Diastolic blood pressure 0.628 0.016 -0.057 -0.224 0.83
[R.sup.2] 0.918

(a) BMI, Body mass index.
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Title Annotation:Hemostasis and Thrombosis
Author:Russo, Isabella; Del Mese, Paola; Doronzo, Gabriella; De Salve, Alessandro; Secchi, Mariantonietta;
Publication:Clinical Chemistry
Date:Jun 1, 2007
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