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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:417.)
© 2006 American Heart Association, Inc.

Thrombosis

Platelet Inhibition by Insulin Is Absent in Type 2 Diabetes Mellitus

Irlando Andrade Ferreira; Astrid I.M. Mocking; Marion A.H. Feijge; Gertie Gorter; Timon W. van Haeften; Johan W.M. Heemskerk; Jan-Willem N. Akkerman

From the Laboratory for Thrombosis and Haemostasis (I.A.F., A.I.M.M., G.G., J.-W.N.A.), Department of Hematology, University Medical Center Utrecht and the Institute for Biomembranes (I.A.F., A.I.M.M., G.G., J.-W.N.A.), Utrecht University, the Netherlands; Department of Biochemistry and Human Biology (M.A.H.F., J.W.M.H.), University of Maastricht, the Netherlands; Department of Internal Medicine (T.W.v.H.), University Medical Center Utrecht, the Netherlands.

Correspondence to Dr J.W.N. Akkerman, Thrombosis and Haemostasis, Department of Hematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. E-mail j.w.n.akkerman{at}azu.nl

	Abstract

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Objective— ADP-induced P2y₁₂ signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca²⁺ increases induced by ADP-P2y₁ contact through blockade of the G-protein G_i, and thereby with P2y₁₂-mediated suppression of cAMP.

Methods and Results— Here we show in patients with type 2 diabetes mellitus (DM2) that platelets have lost responsiveness to insulin leading to increased adhesion, aggregation, and procoagulant activity on contact with collagen. Using Ser⁴⁷³ phosphorylation of protein kinase B as output for insulin signaling, a 2-fold increase is found in insulin-stimulated normal platelets, but in DM platelets there is no significant response. In addition, DM2 platelets show increased P2y₁₂-mediated suppression of cAMP and decreased P2y₁₂ inhibition by the receptor antagonist AR-C69931MX.

Conclusion— The loss of responsiveness to insulin together with increased signaling through P2y₁₂ might explain the hyperactivity of platelets in patients with DM2.

Insulin inhibits Ca²⁺ mobilization in platelets by interfering with P2y₁₂-mediated cAMP suppression. Here we show that DM2 platelets have lost responsiveness to insulin and also show increased P2y₁₂ signaling. This might explain the increased platelet deposition and procoagulant activity under flow of DM2 platelets on contact with collagen.

Key Words: P2y₁₂ receptor • Ca²⁺ regulation • clopidogrel • protein kinase B/Akt • IRS-1

	Introduction

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Platelet activation leads to release of components that initiate formation of a thrombus and start inflammatory responses that contribute to atherosclerosis.¹ Signaling through the P2y₁₂ receptor is crucial for formation and stabilization of a thrombus.^2,3 Inhibition of the P2y₁₂ receptor reduces collagen-induced adhesion, aggregation and thrombin generation.^3,4 Subjects with a P2y₁₂ deficiency have a bleeding tendency^3,5 and individuals with an increased P2y₁₂ receptor copy number have platelets with an increased responsiveness to agonists, and these subjects experience peripheral arterial thrombosis.⁶ The CAPRIE trial shows that long-term administration of the P2y₁₂ antagonist clopidogrel is more effective than aspirin in reducing the combined risk of ischemic stroke, myocardial infarction, or vascular death in subjects with a prothrombotic condition such as diabetes mellitus type 2 (DM2).⁷ These findings illustrate the crucial role of P2y₁₂ signaling in platelet activation in vitro and in vivo.

The importance of P2y₁₂ signaling is explained by its capacity to initiate 2 pathways that directly interfere with platelet activating or inhibiting mechanisms. First, there is the activation of the G-protein subunit G_i, which inhibits adenylyl cyclase and thereby formation of the platelet inhibitor cAMP.⁸ This property is particularly evident after treatment with prostacyclin,⁹ and also in the absence of cAMP elevating agents, P2y₁₂ signaling controls cAMP production through adenylyl cyclase.^10,11 cAMP inhibits platelets through cAMP-dependent protein kinase (protein kinase A [PKA]),¹² which inhibits almost all platelet functions through blockade of multiple steps in platelet activation cascades including receptor activation, signaling through the mitogen-activated protein kinases pathway, formation of thromboxane A₂ (TxA₂), and the activation of key enzymes such as phospholipase C_ß and protein kinase C (PKC).¹³ Second, there is the release of the G_iß dimer leading to the activation of protein kinase B (PKB/Akt), and integrin _IIbß₃ via type 1B phosphatidylinositol 3-kinase (PI3-K).¹⁴ In animal models, type 1B PI3-K is crucial for platelet activation and its absence protects against thromboembolic vascular occlusion.¹⁵ In human platelets, the role of type 1B PI3-K is less well understood, because although being activated by ADP-P2y₁₂ contact, it appears under negative control by ADP-P2y₁ binding and activation of Src and PKC.^10,16

A prime example of abnormal platelet responsiveness is observed in patients with DM2, who are characterized by an impaired responsiveness to insulin or even complete insulin resistance. DM2 subjects have a 2- to 4-fold increased risk for cardiovascular disease and have both microvascular (nephropathy, retinopathy, neuropathy) and macrovascular (peripheral arterial disease) complications. DM2 subjects have platelets that show increased adhesion, aggregation, TxA₂ production, and P-selectin expression.¹⁷ In general, DM2 subjects have marked insulin resistance, mostly explained by their obesity.¹⁸ There is indirect evidence that the hyperactivity of their platelets may be caused by insulin resistance. In healthy individuals, platelets are inhibited by insulin leading to reduced Ca²⁺ mobilization and aggregate formation.^9,17 Interestingly, a euglycemic hyperinsulinemic clamp fails to trigger platelet inhibition in obese insulin-resistant subjects even in the absence of DM.¹⁹ The hyperactivity is likely to have pathological consequences, because the increased adhesion and aggregation will accelerate the formation of a thrombus and enhance the procoagulant activity that helps to stabilize the thrombus.²⁰

In the present study, we investigated whether platelet hyperactivity observed in DM2²¹ is associated with a defect in P2y₁₂ signaling. We demonstrated recently in healthy subjects that insulin interferes with ADP- and thrombin-induced platelet functions through interference with the P2y₁₂-mediated regulation of G_i⁹. After receptor binding, insulin activates the insulin receptor substrate-1 (IRS-1) through tyrosine phosphorylation, which initiates association with G_i-subunit. The result is inhibition of G_i activity and impaired suppression of adenylyl cyclase through P2y₁₂, introducing a phenotype that resembles platelets with a congenital P2y₁₂ defect or platelets from normal individuals who have been treated with the P2y₁₂ antagonist clopidogrel.⁹ Here we demonstrate that platelet hyperactivity in DM2 is likely to be caused by a defect in the mechanisms through which insulin interferes with signaling by the P2y₁₂ receptor.

	Materials and Methods

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The full methods can be found in the online data supplement at http://atvb.ahajournals.org.

	Results

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Disturbed Ca²⁺ Homeostasis in DM2
In healthy subjects, the increase in cytosolic Ca²⁺, [Ca²⁺]_i, by release from internal stores that accompanies platelet activation is easily disturbed by factors that interfere with P2y₁₂ signaling such as insulin.⁹ To detect possible abnormalities in this mechanism in DM2 subjects, Ca²⁺ regulation was measured in platelets from healthy subjects and from DM2 subjects (normal and DM2 platelets, respectively; please see http://atvb.ahajournals.org for physical and biochemical characteristics of study subjects in Table I) in the absence of extracellular Ca²⁺. The mean [Ca²⁺]_i in unstimulated DM2 platelets (64.4±21.8 nmol/L) was 2-fold higher than in normal controls (27.2±5.9 nmol/L; P<0.01). After stimulation with collagen, peak [Ca²⁺]_i levels did not differ between the 2 groups (378.2±139.0 and 305.4±208.2 nmol/L, P>0.05), respectively, but the time to peak was shorter in DM2 platelets than normal platelets (80.7±18.4 and 103.6±13.5 seconds, P<0.03). In normal platelets, addition of insulin failed to change the basal [Ca²⁺]_i but led to a dose-dependent reduction of collagen-induced Ca²⁺ mobilization (expressed as 100%) to 66±11% at 100 nmol/L (P<0.001). This agrees with earlier findings in platelets stimulated with ADP and thrombin.⁹ In contrast, inhibition by insulin was absent in DM2 platelets (Figure I, available online at http://atvb.ahajournals.org). These results indicate that DM2 platelets have a disturbed Ca²⁺ homeostasis that is unresponsive to inhibition by insulin.

Increased Responsiveness of DM2 Platelets to Aggregating Agents
Aggregation studies were performed to assess whether the disturbed Ca²⁺ homeostasis in DM2 platelets affected the responsiveness to collagen or ADP. Aggregation was initiated with 0.1 to 2.5 µg/mL collagen and 1 to 10 µmol/L ADP (Figure 1). Curves were fitted by nonlinear regression, which resulted in EC₅₀ values for collagen-induced aggregation of 0.44 and 0.35 µg/mL for normal and DM2 platelets, respectively. For ADP-induced aggregation, these data were 1.52 and 0.79 µmol/L, respectively. The aggregation response to collagen and ADP was higher in DM2 platelets (P<0.05). These results indicate that the responsiveness of DM2 platelets is increased especially for ADP.

View larger version (13K): [in this window] [in a new window]   Figure 1. Increased responsiveness of DM2 platelets to aggregating agents. Aggregation of normal (H) and DM2 (D) PRP was initiated with 0.1 to 2.5 µg/mL collagen and 1 to 10 µmol/L ADP. The maximal aggregation was measured and data were fitted by nonlinear regression (H: R2=0.91 and 0.79; DM2: R2=0.92 and 0.86 for collagen and ADP, respectively). Means±SD; n=7 healthy and 7 DM2 subjects; (please see http://atvb.ahajournals.org for the detailed Materials and Methods).

Absent Inhibition of Collagen- and ADP-Induced Platelet Aggregation by Insulin in DM2
Aggregation studies were performed to investigate whether the unresponsiveness to insulin observed in the regulation of Ca²⁺ had an effect on the role of insulin on platelet functions.^9,22 Platelets were treated with 1 nmol/L insulin and aggregation was initiated with collagen and ADP. In healthy subjects, 1 nmol/L insulin inhibited collagen- and ADP-induced aggregation to 76±11% and 75±8%, respectively (P<0.05). The inhibition by insulin was completely absent in platelets from DM2 subjects (Figure II, available online at http://atvb.ahajournals.org). Thus, DM2 platelets are unresponsive to insulin.

Inhibition of Collagen-Induced Platelet Deposition Under Flow by Insulin
Apart from the formation of aggregates, adhesion and generation of a pro-coagulant surface by exposure of phosphatidylserine (PS) are important steps in platelet deposition under flow. Earlier studies revealed an important role of P2y₁₂ signaling in these processes.³ We determined platelet deposition and binding of annexin V-fluorescein isothiocyanate (FITC) to PS-exposing platelets after perfusion over a collagen-coated surface at a shear rate of 1000 s^–1. Normal platelets rapidly adhered to collagen and formed aggregates (Figure 2A). The basal surface coverage by DM2 platelets (18.8±1.3%) was higher than by normal platelets (14.5±1.3%, P<0.001), which is in agreement with the hyperactivity of DM2 platelets observed in stirred suspensions (Figure 1; Figure I).²³ Insulin reduced surface coverage by normal platelets in a dose-dependent manner (10.7±1.4% at 100 nmol/L; P<0.001), whereas inhibition by insulin was absent in DM2 platelets (Figure 2A and 2B). Also, binding of annexin V-FITC in perfusates with DM2 platelets (20.5±3.4%) was higher than in controls (12.9±2.0%, P<0.001), probably as a result of the increased adhesion by DM2 platelets. In normal platelets, insulin reduced the binding of annexin V-FITC to 6.2±2.1% at 100 nmol/L (P<0.001), but in DM2 platelets no effect of insulin could be detected (Figure 2C). Together, these results indicate that DM2 platelets have an increased responsiveness to a collagen-coated surface under flow and that this property is insensitive to the presence of insulin.

View larger version (52K): [in this window] [in a new window]   Figure 2. Inhibition of collagen-induced platelet deposition under flow by insulin. Blood was perfused at an intermediate shear rate (1000 s–1) over a collagen-covered surface. A, Shown are 2 typical phase contrast images of normal blood perfused over collagen in the absence (upper panel) and presence (lower panel) of 100 nmol/L insulin. The arrow depicts the flow direction in the perfusion chamber. B, Surface coverage measured in phase contrast images. C, Surface coverage with annexin V-FITC staining platelets. Further details as in Figure 1.

Downstream Signaling of the Insulin Receptor/IRS-1 Complex
In adipocytes, ineffective insulin signaling or insulin resistance has been attributed to abnormalities in IRS-1 activation. To investigate whether the loss of insulin signaling to Ca²⁺ regulating mechanisms in DM platelets was accompanied by abnormal signaling initiated by the insulin receptor/IRS-1 complex, the phosphorylation of Ser⁴⁷³ on PKB was measured. Platelets were incubated with insulin for 15 minutes and samples were subjected to SDS-PAGE. In normal platelets, insulin increased the phosphorylation of PKB-Ser⁴⁷³ to 192.0±70.3% (P<0.03). DM2 platelets showed a complete lack of PKB phosphorylation. (Figure 3). Together with the absent interference with Ca²⁺, probably reflecting impaired signaling to G_i2, the absent activation of PKB suggests that a common step in the insulin signaling machinery is affected.

View larger version (17K): [in this window] [in a new window]   Figure 3. Downstream signaling of the insulin receptor/IRS-1 complex. Platelets were incubated with insulin for 15 minutes and subjected to SDS-PAGE. A, Blots were probed with P-PKB-Ser473. B, The basal phosphorylation of individuals was expressed as 100%. Further details as in Figure 1.

Insulin Inhibits Platelet Activation by Collagen by Interfering With P2y₁₂ Signaling to cAMP-Dependent PKA
In platelets from healthy subjects, insulin interferes with the P2y₁₂-mediated suppression of cAMP formation, thereby attenuating Ca²⁺ increases and reducing aggregation induced by ADP and thrombin.⁹ As shown in the present study, a similar inhibition is observed on stimulation by collagen. H89 is a specific inhibitor of PKA.²⁴ Pretreatment with H89 fully abolished the inhibition by insulin, confirming that insulin reduced platelet aggregation via interference with the cAMP-dependent activation of PKA. To address the question whether insulin alone changed the level of cAMP in the absence of activators or inhibitors of adenylyl cyclase, resting platelets were incubated with different concentrations of insulin followed by analysis of the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-dependent PKA.¹³ No phosphorylated VASP could be detected in platelets treated with insulin. In contrast, addition of PGI₂ that through the IP receptor and G_s activates adenylyl cyclase induced a rapid increase in phosphorylated VASP (Figure III, available online at http://atvb.ahajournals.org). Thus, although it interferes with the regulation of cAMP and PKA, insulin is unable to change these signaling molecules in the absence of platelet agonists and antagonists.

Increased P2y₁₂ Signaling in DM2 Platelets
Because in normal platelets insulin interferes with platelet functions through inhibition of P2y₁₂ signaling,⁹ the unresponsiveness to insulin in DM2 platelets might be caused by disturbances in signal transduction from P2y₁₂ to cAMP. To address this issue, platelets were incubated with the stable PGI₂ analog iloprost to raise cAMP and thereafter treated with increasing concentrations ADP to induce different extents of P2y₁₂ signaling. Basal cAMP levels did not differ between normal and DM2 platelets. In normal platelets, ADP dose-dependently reduced iloprost-induced cAMP (expressed as 100%) to 68.1±8.5% (P<0.001) at 10 µmol/L ADP. Addition of insulin partially reversed the effect of ADP in normal platelets leading to a reduction of cAMP to 85.1±12.7% (P<0.02). The decline of iloprost-induced cAMP was significantly steeper in DM2 platelets showing reduction to 48.8±11.1% (P<0.001) at the same ADP concentration (Figure 4A), which was unresponsive to insulin (Figure 4B). These results indicate that P2y₁₂ signaling is increased in DM2 platelets and not affected by the presence of insulin.

View larger version (30K): [in this window] [in a new window]   Figure 4. Increased P2y12-signaling in DM2. Platelets were stimulated with 1 to 10 µmol/L (A) or 10 µmol/L (B) ADP in the presence or absence of iloprost. Iloprost-induced cAMP accumulation was expressed as 100%. C, Platelets were incubated with 0.1 to 500 nmol/L AR-C69931MX, and Ca2+ mobilization was induced by 10 µmol/L ADP. D, Platelets were incubated with 1 to 50 nmol/L AR-C69931MX, and stimulated with 10 µmol/L ADP. Further details as in Figure 1.

To investigate whether this abnormality affected the sensitivity to the P2y₁₂ receptor antagonist AR-C69931MX, dose-inhibition studies were performed in normal platelets. To this end, Ca²⁺ mobilization was induced by ADP-P2y₁ contact and different concentrations AR-C69931MX were added to interfere with the P2y₁₂-mediated support of [Ca²⁺]_i increases (Figure 4C). Optimal inhibition was observed at 100 nmol/L AR-C69931MX, at which point the inhibition amounted to 70%. Using 2 suboptimal concentrations of AR-C69931MX that induced 50% and 25% inhibition, studies were repeated in DM2 platelets. At 50 nmol/L AR-C69931MX normal and DM2 platelets showed a similar inhibition of ADP-P2y₁ Ca²⁺ mobilization, but at 10 nmol/L AR-C69931MX the inhibition in DM2 platelets was significantly lower than in normal controls (23.6±4.3% and 39.3±11.9% respectively; P<0.05; Figure 4D). These findings suggest that in DM2 platelets the ADP-P2y₁₂-mediated support of ADP-P2y₁ induced Ca²⁺ increases has a decreased sensitivity to the P2y₁₂ receptor antagonist AR-C69931MX.

	Discussion

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The present data show that platelets from DM2 subjects have lost their responsiveness to insulin and show increased responsiveness to ADP, which supports platelet activation by many agonists.⁸ In the presence of insulin, DM2 platelets show increased collagen-induced Ca²⁺ mobilization. Collagen- and ADP-induced aggregation are slightly higher than controls and DM2 platelets show increased adhesion to surface-coated collagen under flow with a concomitant increase in PS exposure. In normal platelets, inhibition of ADP-induced aggregation by insulin is abolished by the PKA inhibitor H89. This is in line with the concept that insulin interferes with P2y₁₂-induced suppression of adenylyl cyclase and cAMP formation through tyrosine phosphorylation of G_i2, resulting in its inhibition. In DM2 platelets, P2y₁₂ signaling is present and functional, but the pathway appears to be upregulated and less sensitive to P2y₁₂ inhibition. These findings indicate that insulin interferes with platelet activation by collagen through the same mechanism as in platelets stimulated with ADP and thrombin.⁹ They also indicate that the cause for the loss of insulin sensitivity in platelets from DM2 subjects must be sought in a defect in a pathway that triggers the inhibition of G_i2.

A human platelet contains 570 insulin receptors.²⁵ After receptor activation, IRS-1 is recruited and tyrosine phosphorylated leading to activation of pathways involving PKB and p38 mitogen-activated protein kinases in addition to inhibition of G_i2^9,18,26. Possibilities for abnormal insulin signaling in DM2 platelets are defects in the insulin receptor ß-subunit, IRS-1, and the tyrosine phosphorylation of G_i2 together with the different tyrosine kinases and phosphatases that control these processes.^9,18,27,28 Defects in the insulin receptor are associated with severe abnormalities such as growth disorders, lipodystrophy, and acanthosis nigrans, which were absent in the DM2 study group. A more likely explanation for the loss of insulin sensitivity in DM2 platelets is a defect in IRS-1. In transfected cells, the IRS-1 gene G972R variant causes impaired activation of PI3-K and PKB.²⁹ The same mutation is found in obese patients with DM2,³⁰ where it is associated with an increased risk of cardiovascular disease.³¹ However, the gene variant is also found in a healthy individuals, indicating that it is not the decisive factor that makes individuals diabetic.³² Analysis of IRS-1 and G_i2 in the DM2 platelets stimulated with 100 nmol/L insulin revealed reduced and often absent tyrosine phosphorylation of these intermediates. Unfortunately, the normal controls also showed varying levels of tyrosine phosphorylation, which made it difficult to identify a precise block in insulin signaling. Instead, analysis of PKB activation consistently showed strongly impaired Ser⁴⁷³ phosphorylation in DM2 platelet but not in the controls. Thus, loss of insulin signaling to G_i2 and Ca²⁺ is accompanied with loss of signaling to PKB. A likely cause is a defect in IRS-1, which is an upstream regulator of both pathways. The nature of the defect in IRS-1 regulation remains to be elucidated but apparently it is has a great effect on G_i. Gain- and loss-of- function defects of G_i2 have been described in bipolar affective and bleeding disorders,^3,33 whereas defects in tyrosine protein phosphorylation have been described in congenital thrombocytopenia, Wiscott-Aldrich syndrome, and the Scott syndrome.^34–36

In agreement with earlier studies in DM2 subjects,^23,37,38 we found an increased basal [Ca²⁺]_i in DM2 platelets before stimulation with collagen. Studies with thapsigargin, an inhibitor of sarco/endoplasmatic Ca²⁺-ATPase (SERCA), made clear that the Ca²⁺ level is determined by a constant release from and re-uptake of Ca²⁺ by the endoplasmatic reticulum.³⁹ Apparently, in DM2 platelets these processes have reached a new steady-state with a 2-fold higher [Ca²⁺]_i than in normal platelets. This did not lead to a higher Ca²⁺ response after collagen stimulation, but the response was faster and accompanied with increased aggregation. In addition, adhesion to collagen under flow was higher with DM2 platelets than with normal platelets suggesting that abnormalities in Ca²⁺ regulation made platelets more reactive toward a collagen-coated surface. At high shear platelet adhesion to collagen through interaction of glycoprotein (platelet glycoprotein [GP]) Ib with von Willebrand factor bound to collagen is followed by integrin ₂ß₁ mediated firm adhesion, which halts platelet rolling and allows collagen to interact with GPVI.^40,41 Signaling mediated via GPVI increases the binding affinity of ₂ß₁ to collagen and induces release of ADP.^42,43 Adhesion to collagen of platelets deficient of the P2y₁₂ receptor is >20% lower than with normal platelets, illustrating the potent enhancement by P2y₁₂ signaling through released ADP.³ The binding of GPVI to collagen is independent of intracellular control indicating that changes in adhesion to collagen reflect modulation of ₂ß₁⁴².

Another abnormality found in DM2 platelets is the increased signaling capacity of the P2y₁₂ pathway. DM2 platelets induced a steeper fall in cAMP in iloprost-treated platelets than their normal counterparts. A similar enhancement has been found in carriers of the P2y₁₂-H2 haplotype, which is characterized by an increased P2y₁₂ receptor number at the plasma membrane.⁶ Carriers of the P2y₁₂-H2 haplotype are characterized by peripheral arterial disease due to the presence of hyper-responsive platelets that are less sensitive to pharmacological strategies that inhibit P2y₁₂ signaling, such as clopidogrel.⁶ DM2 platelets showed a decreased responsiveness to a suboptimal concentration of the P2y₁₂ antagonist AR-C69931MX but at an optimal concentration the difference disappeared. Clopidogrel resistance has been described in a patient population with a general high risk for recurrent vascular events, although it was not specific for DM.⁴⁴ Our present findings might indicate that clopidogrel resistance can be overcome by applying higher doses of the drug.

Platelet hyperactivity in DM2 correlates with an increased risk of atherothrombotic complications.^45–48 In addition to hyperactive platelets, the coagulation mechanism shows abnormalities in DM2 subjects with elevated levels of coagulation factors factor VII, VIII, XI, and XII.⁴⁹ The enhanced adhesion of platelets in DM2 with a concomitant increase in PS exposure that facilitates the coagulation cascade might contribute to the hypercoagulable state observed in this disease.

	Acknowledgments

 
The support of the Dutch Diabetes Research Foundation (1999.046; Amersfoort, the Netherlands) and the "Fonds voor het Hart" (The Hague, the Netherlands) is gratefully acknowledged. J.W.N.A. is supported by the Netherlands Thrombosis Foundation. We thank Dr S.J. Korporaal, M. van Lier, Dr E.J.P. de Koning, and J.P. van Wijk (UMC Utrecht, the Netherlands) for their assistance, and the volunteers for their help.

Received August 4, 2005; accepted November 17, 2005.

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