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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0137
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Right arrow Diabetes and Insulin
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 920-927
Copyright © 2005 by The Endocrine Society

P2Y12 Receptors Play a Significant Role in the Development of Platelet Microaggregation in Patients with Diabetes

Hiroyuki Matsuno, Haruhiko Tokuda, Akira Ishisaki, Yuan Zhou, Yasuo Kitajima and Osamu Kozawa

Department of Clinical and Pathological Biochemistry (H.M.), Doshisha—Women’s College of Liberal Arts, Kyotanabe 610-0395, Japan; Departments of Pharmacology (A.I., O.K.) and Dermatology (Y.Z., Y.K.), Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan; and Department of Internal Medicine, Chubu National Hospital: National Institute for Longevity Sciences (H.T.), Obu 474-8511, Aichi, Japan

Address all correspondence and requests for reprints to: Dr. Hiroyuki Matsuno, Department of Pharmacology, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan. E-mail: leuven{at}cc.gifu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Ninety-eight diabetic patients (type 2) were studied together with 24 healthy normotensive controls. Microaggregates (particle scale, <25 µm) of platelets were detected by a laser scattering system. Microaggregates in the control group showed a time-dependent reversible change; however, they existed continuously in 82 of 98 diabetic patients. When platelets of diabetics were stimulated by a shear stress alone without ADP, 74 also showed spontaneous and irreversible microaggregates even though they were not observed in all control subjects. In control subjects, microaggregates were inhibited by MRS2279 (a P2Y1 antagonist), but not AR-C69931MX (a P2Y12 antagonist). However, AR-C69931MX prevented irreversible microaggregates in diabetic patients. When either aspirin or ticlopidine was administered to diabetic patients with irreversible microaggregates, both drugs significantly decreased microaggregates induced by a low dose of ADP. Ticlopidine additionally reduced the microaggregates induced by shear stress alone. In conclusion, microaggregates of platelets via P2Y12 receptors could play a key role in the hypersensitivity of platelets in diabetic patients, and the measurement of microaggregation could be a useful marker to estimate of thrombogenesis. These findings present a possible new means for patients with diabetes to prevent ischemic events.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PLATELET AGGREGATION PLAYS a central role in the development of thrombus formation. Currently available antiplatelet agents, such as aspirin, show clinical efficacy in the treatment of arterial thrombotic disorders by inhibiting platelet aggregation (1, 2). However, because microthrombi produced in the early phase of platelet activation may be a trigger for additional development of larger thrombi, the inhibitory effect of these agents on the formation of microthrombi should be evaluated. Patients with diabetes have a few fold increases in the risk of dying of cardiovascular diseases (3, 4). Vascular stenosis due to thrombus formation is a major contributor, and it is generally accepted that platelets play an important role. Diabetic microvascular disease is a leading cause of coronary artery disease and renal failure, and these phenomena are major causes of blindness (5, 6, 7). Altered platelet function, such as hypersensitivity of platelets to collagen, is prevalent in diabetes and may participate in the pathogenesis of diabetic vascular complications by promoting microthrombus formation (8, 9). However, the excess risk of thrombosis in patients with diabetes cannot be fully abrogated by antidiabetic therapy.

Platelet aggregation is usually measured using either the OD method (6) or the impedance method (7), both of which are indispensable for clinical evaluation of platelet function. However, these methods provide no information about subtle, but significant, changes in the number of platelet microaggregates in response to some small increment in aggregating stimuli or in the proaggregatory status of the platelets. The light-scattering (LS) method, which has primarily been used for experimental research, provides a tool with a greater sensitivity for detecting microaggregates of platelets than the conventional light transmittance method (7). Recently, a particle-counting method that employs LS has been developed (9), allowing identification of particle size in terms of light intensity and minimizing the interference with neighboring platelets, which may attenuate high intensity light scattered by larger particles (10). More recently, an important role of platelet microaggregation in the circulation and a significant alteration of platelet microaggregation in pathogenesis were reported by the creation of a laser LS system for microaggregates (12, 13, 14).

ADP is considered to be a weak agonist by itself compared, for example, with thrombin or collagen (15); however, ADP is a necessary cofactor for normal activation of platelet by other agonists. Low concentrations of ADP potentiate or amplify the effects of agonist for platelet activation (16). Moreover, recent investigation revealed that von Willebrand factor (vWF) and its interaction with platelet receptor protein glycoprotein Ib/V/IX (GPIb/V/IX) and GPIIb/IIIa played important roles in the onset of platelet thrombosis at sites exposed to shear stress (17, 18). We previously reported that the measurement of microaggregated forms is a useful tool for the evaluation of antithrombotic compounds (19, 20). In the present study we applied an LS system for the detection of microaggregates of platelets in patients with type 2 diabetes, because a variety of platelet abnormalities have been suggested to play a role in the thrombogenesis of diabetes (21, 22, 23). Moreover, we investigated the mechanism of development of microaggregates via ADP receptors or GP receptors and compared diabetic patients with normal subjects.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Inclusion criteria for study entry were the presence of type 2 diabetes with hemoglobin A1c (HbA1c) greater than 6.5, no insulin therapy, and the absence of any clinically manifest micro- or macroangiopathy. Of 122 total patients screened, 98 type 2 diabetic patients fulfilled the criteria and were enrolled in the present study. Sixty-seven of the patients were treated with oral sulfonylurea therapy. Fourteen of 67 patients were also treated with an oral {alpha}-glucosidase inhibitor. The others were treated with diabetic diet alone. Twenty-four healthy nondiabetic subjects were examined following the same protocol, and all of them qualified to form a control group. For 2 wk before testing we carefully counseled each subject about the effects of medication on platelet activation. We strictly checked the condition of medication before the start of the experiments, and we excluded patients with medication in this study. All subjects were advised to avoid sleep deprivation and blood donation. All participants signed an informed consent agreement as approved by the local institutional review board after a detailed explanation of the study.

Reagents

AR-C 69931MX and GR144053 were gifts from AstraZeneca Co. Ltd. (Wilmington, DE) and GlaxoSmithKline Co. Ltd. (London, UK), respectively. Low molecular weight aurino-tricarboxytic acid (ATA) was purified from commercial ATA (nonfractionated ATA was obtained from Sigma-Aldrich Corp., St. Louis, MO). This compound prevents a vWF-GPIb/V/IX axis on platelets (24, 25, 26). The other chemical substances were obtained from Sigma-Aldrich Corp.

Blood drawing

To preserve steady state conditions, blood (total of 18 ml from each patient) was drawn while the subject was in the supine position between 0800–0900 h after 15 min of rest. Blood was drawn with minimal use of a tourniquet. Either EDTA (1 µg/ml) or sodium citrate (14 µM) was used as an anticoagulant.

Preparation of platelets

Platelet-rich plasma (PRP) was obtained from blood samples including sodium citrate by centrifugation at 155 x g for 12 min at room temperature. Platelet-free plasma was prepared from residual blood by centrifugation at 2500 x g for 15 min. In the experiments using mice, PRP was obtained by centrifugation at 240 x g for 12 min at room temperature. To obtain washed platelets, PRP was centrifuged at 1750 x g for 10 min at room temperature in the presence of prostaglandin I2 (PGI2) (20 µM), and the platelet pellet was washed with HEPES-Tyrode’s buffer. Platelet count was adjusted to 4 x 108 cells/ml for each experiment.

Measurement of platelet aggregation induced by ADP

Platelet aggregation using citrated PRP was followed in an aggregometer (AG 10 apparatus, Kowa Co. Ltd., Tokyo, Japan) at 37 C with a stirring speed of 800 rpm. Platelets were preincubated for 2 min, then platelet aggregation was monitored for 5 min after the addition of various dose of ADP (0.3–30 µM). The percentage of transmittance of the isolated platelets was recorded as 0%, and that of the appropriate platelet-free plasma (blank) was recorded as 100%.

Detection of microaggregated from

Aggregation was simultaneously determined by evaluating the maximum percent decrease in the OD and by assessing the LS intensity using an AG 10 apparatus (Kowa Co. Ltd.). The principles of the LS methods have been described previously (20). Briefly, a laser beam measuring 40 µm in diameter was generated using a 20-mW diode laser (675 nm; Toshiba, Tokyo, Japan) and was passed through PRP (200 µl) stirred in a cylindrical glass cuvette with a 5-mm internal diameter. The light scattering in the volume studied (48 x 140 x 20 µm) was detected by a photocell array. Light intensity corresponds to particle size. The signal frequency was recorded at 10-sec intervals. Data were expressed as the change over time in the number of aggregates (counts per second) of individual sizes (determined by light intensity, expressed in volts). Data were recorded as a two-dimensional graph showing the change over time in total light intensity expressed as a cumulative summation at 10-sec intervals of scattered light intensity (Ii) and the number of particles corresponding to that intensity (Ni) in terms of particle size (intensity): ({sum}IiNi)(volt x counts/sec). The total light intensities of small, medium, and large aggregates were determined. Particles with an intensity of 25–400 mV represented small aggregates (9–25 µm), those with an intensity of 400-1000 mV represented medium aggregates (25–50 µm), and those with an intensity of 1000–2048 mV represented large aggregates (50–70 µm) (9, 10). The degree of microaggregates was described by measurement of the area under the curve (AUC) of each detection line. Platelets were continuously stimulated by a shear stress with a stirring speed of 1000 rpm during the observation period.

Electron microscopic analysis

To identify the aggregated form of platelets induced by shear stress, we performed histological analysis using SEM. Various aggregated forms were detected by the LS system, then each sample was taken 8 min after the observation period. Activated platelets in each cuvette were removed and transferred into 2% glutaraldehyde for 2 h. Fixed platelets were spread on a dish to allow visual inspection for SEM as previously described (14, 27).

Biochemical analysis

HbA1c was measured using the Hemoglobin A1c Micro Column Test (Bio-Rad Laboratories, Hercules, CA). Total cholesterol and triglyceride levels were measured in whole plasma and lipoprotein fractions using standardized automated methods.

Treatment with ADP receptor antagonists or platelet GP inhibitors on microaggregations

Either AR-C69931MX (0.1 µM), a P2Y12 receptor antagonist (28), or MRS2279 (1.0 µM), a P2Y1 receptor antagonist (29), was added to PRP of either diabetic patients with microaggregates or normal subjects, then PRP was stimulated by a low dose of ADP (1.0 µM). In separate experiments, either GR144053 (30), a platelet GPIIb/IIIa antagonist (1 µM) or low molecular weight ATA (24, 26), a platelet GPIb/V/IX-vWF axis inhibitor (30 µM), was added to PRP of diabetic patients with microaggregates before the start of stimulation with low shear stress alone without any agonists. Samples (n = 20 in each group) were randomly picked for this experiments.

Study of reduction of microaggregates in diabetic patients

Diabetic patients with microaggregates were randomly divided into two groups, a group with aspirin (100 mg/d; n = 22) and a group with ticlopidine (200 mg/d; n = 19). Blood samples were taken from each patient 4–6 wk after the start of treatment with each drug.

Statistical analysis

All data are expressed as the mean ± SEM. Significance was determined by ANOVA, followed by the Student-Newman-Keuls test. Data were analyzed using statistical software Stat Mate (Nazan-do, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Microaggregated form of platelets

Figure 1Go shows a typical pattern of detection of platelet aggregation using an LS system. In control subjects, the microaggregated form (particle diameter, 9–25 µm) showed a reversible change (Fig. 1AGo) when PRP was stimulated by a low dose of ADP (1.0 µM). The aggregated form changed to a large form after stimulation with a high dose of ADP (10 µM) until the end of the observation period (Fig. 1DGo). In contrast, 82 of 98 diabetic patients (group I) showed an irreversible change in microaggregates of platelets (Fig. 1BGo). The other patients with diabetes did not show this irreversible change of microaggregates (Fig. 1CGo). However, aggregated patterns were almost the same as the control pattern when platelets in groups I (Fig. 1EGo) and II (Fig. 1FGo) were stimulated by a high dose of ADP (10 µM). Typical aggregated forms of platelets were analyzed by a scanning electron microscope (Fig. 2Go). In control subjects, platelets dissociated 8 min after stimulation with a low dose of ADP at a dose of 1.0 µM (Fig. 2AGo). In contrast, irreversible microaggregates were observed in diabetic patients (Fig. 2BGo). When platelets were stimulated with a high dose of ADP at a dose of 10.0 µM, large aggregates were found (Fig. 2CGo).



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  		FIG. 1. Typical patterns of the platelet-aggregated form determined by a LS system. The platelet-aggregated form induced by a low dose of 1.0 µM ADP (A–C) or 10.0 µM ADP (D–F) was assessed by the LS intensity (LSI) on samples obtained from diabetic patients (B, C, E, and F) and control subjects (A and D). Red line, Large aggregates; green line, mid-sized aggregates; blue line, small aggregates (microaggregates). Reversible microaggregates were observed in control when platelets were stimulated by a low dose of ADP (A). This phenomenon was also observed in diabetic patients of group II (C). In contrast, irreversible and marked microaggregates of platelets were clearly found in diabetic patients of group I (B). However, when platelets were stimulated with a high dose of ADP (10.0 µM), platelets in all groups completely aggregated, as shown in Fig. 2CGo.

 


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  		FIG. 2. Electron microphotographs of typical platelet aggregation. Scanning electron microphotographs showed platelets of a diabetic patient in control (A) and diabetic (B and C) subjects with the stimulation of ADP at doses of 1.0 µM (A and B) and 10.0 µM (C). Each sample was taken from PRP in a cuvette after the end of the observation period of platelet aggregation by an LS system. Reversible microaggregates were observed in a control subject (A). Irreversible microaggregates were exited in a diabetic patient (B). Large aggregates were normally observed in all subjects when platelets were stimulated by ADP at a dose of 10.0 µM. C, Large aggregates of diabetic patients. Scale bar, 10 µm (A and B) and 100 µm (C).

 
Characterizations of study groups

Diabetic patients were divided into two groups according to the detection of microaggregates. Eighty-two of 98 diabetic patients showed irreversible microaggregates under a low dose of 1.0 µM ADP (group I). The AUC of the microaggregated form in group I was significantly higher than that in the control group. The other diabetic patients (group II) and control subjects show reversible microaggregates stimulated by a low dose of ADP. The clinical and biochemical characteristics of the subjects are presented in Table 1Go. Compared with control subjects, the groups of patients with type II diabetes differed significantly in plasma glucose levels and HbA1c levels. Diabetic obesity was not observed in every patient. Anthropometric indexes and metabolic variables were within the normal limits, and there was no statistical difference between the two groups of diabetic patients. Antiplatelet drugs were not administered during the last 2 wk. Therapeutic drugs for diabetes, such as sulfonylurea derivatives or {alpha}-glucosidase inhibitors, were used in both groups. Therapeutic doses of these compounds do not directly affect platelet aggregation in vivo. In group I, nine patients and three patients were treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A inhibitor (pravastatin) and an angiotensin-converting enzyme inhibitor (captopril), respectively. In group II, one patient was treated with an angiotensin-converting enzyme inhibitor. Treatment with a 3-hydroxy-3-methyl-glutaryl-coenzyme A inhibitor could affect platelet aggregation (31); however, significant reduction of platelet aggregation was not observed in nine patients.


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  		TABLE 1. Characterization of the study subjects

 
Platelet aggregation induced by ADP

The 50% maximum aggregation induced by ADP in diabetic patients or control subjects was calculated. PRP from each group were stimulated with various doses of ADP. The values in group I, group II, and control subjects were 2.2 ± 0.8, 14.8 ± 2.3, and 16.3 ± 4.5 µM, respectively. The value in diabetic patients with microaggregates (group I) was significantly lower than that in control subjects.

Correlation between microaggregated form and biological parameters

A possible correlation was found between the AUC of the microaggregated form induced by a low dose of ADP and HbA1c levels (Fig. 3Go) in all diabetic patients. No significant correlation was found between the microaggregated form and the other parameters, such as age, body mass index, systolic blood pressure, total cholesterol, and low density lipoprotein (data not shown).



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  		FIG. 3. Correlation of microaggregated form with HbA1c levels. The degree of microaggregates was shown by the AUC calculated by an automatic computer-analyzed system. The AUC of microaggregates induced by a low dose of ADP (1.0 µM) was well correlated with HbA1c. Correlation coefficients by Spearman rank-order correlation were significant in both groups (P = 0.032). {circ}, Group I; •, group II.

 
Effects of ADP receptor antagonist on microaggregates

When PRP was stimulated by a low dose of 1.0 µM ADP, microaggregates of platelets were observed in all subjects. In control subjects, MRS2279 inhibited the development of microaggregates; however, it did not reduce the amount of microaggregates in diabetic patients. In contrast, AR-C69931MX markedly reduced microaggregates of platelets in diabetic patients, whereas it had no effect on those in control subjects (Fig. 4Go).



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  		FIG. 4. Effects of ADP receptor antagonists on microaggregates of platelets. PRP from control subjects (A) or diabetic patients with irreversible microaggregates (B) was preincubated for 1 min with either AR-C69931MX (AR) at a dose of 0.1 µM or MRS2279 (MR) at a dose of 1.0 µM before the addition of ADP (1.0 µM). Data are presented as the mean ± SEM.

 
Detection of microaggregates without stimulation by ADP

Microaggregates were not measured in all subjects in the control group when ADP was not added to PRP (Fig. 5AGo). However, 74 of 82 diabetic patients with microaggregates induced by a low dose of ADP also showed spontaneous platelet microaggregation without stimulation by ADP. These microaggregates immediately increased after the start of measurement, and the microaggregated form was present continuously during the observation period (Fig. 5BGo).



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  		FIG. 5. Effects of ADP receptor antagonist and GP receptor inhibitors on spontaneous microaggregates of platelets. The typical spontaneous microaggregated form without the stimulation of agonists was assessed by LS intensity (LSI) on samples obtained from diabetic patients with irreversible microaggregates (B–F) and a control subject (A). PRP from diabetic patient was preincubated with MR2279 (MR) at a dose of 1.0 µM (C), AR-C69931MX (AR) at a dose of 1.0 µM (D), GR144053 (GR) at a dose of 1.0 µM (E), or ATA at a dose of 30 µg/ml (F) for 1 min before the start of stimulation by shear stress. A and B, Alteration of microaggregates stimulated by shear stress after preincubation for 1 min in a control subject or a diabetic patient without antagonists (NA), respectively. G, Average amount of microaggregates induced by low shear stress alone. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01 (vs. microaggregates without antagonists).

 
Inhibition of spontaneous microaggregates in diabetic patients

The effects of ADP receptor antagonists and GP receptor inhibitors were investigated in diabetic patients with spontaneous microaggregates when there was no addition of ADP. Typical changes in microaggregates are shown in Fig. 5Go, C–F. Treatment with AR-C69931MX, but not MRS2279, significantly reduced the development of microaggregates (Fig. 4Go, C and D, respectively). Next, prevention of platelet GPIIb/IIIa partially reduced microaggregates (Fig. 5EGo). Inhibition of the platelet GPIb/V/IX-vWF axis almost completely prevented the formation of microaggregates (Fig. 5FGo). Figure 5GGo shows the inhibitory effects of these compounds.

Effects of aspirin or ticlopidine on spontaneous microaggregates in diabetic patients

Diabetic patients with spontaneous microaggregation with no stimulation by ADP were treated with either aspirin or ticlopidine. Figure 6Go shows alterations in the amount of platelet microaggregation before or after treatment with each drug. Treatment with either aspirin or ticlopdine significantly reduced microaggregation induced by a low dose of ADP (Fig. 6AGo). The inhibitory effect of ticlopdine was much greater than that of aspirin. Moreover, ticlopidine significantly reduced microaggregates without stimulation by ADP (Fig. 6BGo). A tendency for bleeding was not observed in any subject, and hemorrhage events were not seen in any subject during the observation period.



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  		FIG. 6. Effects of aspirin and ticlopdine in patients with diabetes. Alterations of the microaggregated form were measured before or after treatment with aspirin ({square}) or ticlopidine ({cjs2112}) in diabetic patients. ADP (1.0 µM)-induced microaggregates (A) in diabetic patients (Before) were significantly reduced by treatment with either aspirin or ticlopidine (After). Spontaneous microaggregates were significantly diminished by treatment with ticlopidine (B). Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01 (vs. microaggregated form before treatment with each drug).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study we focused on microaggregates of platelets in type 2 diabetic patients and compared the roles of ADP receptors and GP receptors on platelet aggregation in a group of 98 diabetic patients with those in a group of 24 nondiabetic control subjects. Additionally, to define the effects of antiplatelet drugs in patients with diabetes, we treated them diabetic patients with irreversible microaggregates with aspirin or ticlopidine.

Microthrombi in the circulation play a crucial role in vascular stenosis after ischemic events. Recently, a detection system for platelet microaggregation was established (10), and the role of platelet microaggregation was investigated in basic research (20, 32) and clinical experiments (11, 12, 13). Detection of platelet microaggregate formation was much more sensitive using the LS system than with the light transmission system. Platelet microaggregation was certainly affected by several kinds of diseases, and microaggregates were normally induced by a very low dose of platelet agonist, such as ADP (1.0 µM), collagen (0.1 µg/ml), thrombin (0.1 U/ml), and other such compounds (11, 13). In the present study microaggregates of platelets were detected in all subjects when platelets were stimulated with a low dose of ADP (1.0 µM). However, 81.6% of type 2 diabetic patients showed irreversible microaggregates of platelets even if microaggregates in all control subjects were gradually dissociated at the end of the observation period. Microaggregates produced in the early phase of platelet activation are considered to potentially aggravate thrombus formation, leading to vascular occlusions by the formation of a platelet-rich thrombus (33). Indeed, the 50% maximum aggregation induced by ADP in diabetic patients with irreversible microaggregates was significantly lower than that in either normal subjects or the other diabetic patients. Surprisingly, these microaggregates were also clearly detected without stimulation by ADP in 91.3% of diabetic patients with irreversible microaggregates induced by a low dose of ADP. The other diabetic patients (24.5%) and all normal subjects did not show microaggregates of platelets without stimulation by agonists. During the measurement of platelet aggregation, shear stress could be produced by a rotating bar with a stirring speed of 1000 rpm in a cuvette. Indeed, high shear stress (108 dyn/cm2) induced irreversible platelet aggregation (34, 35), and low shear stress (6–12 dyn/cm2) induced transient aggregation of platelets, but these clots were gradually dissociated in normal subjects (35). Therefore, we speculate that platelets from these diabetic patients were easily activated by some kind of shear stress. These findings indicated that microthrombi formed mainly from platelets could continuously exist in the circulation of patients with type 2 diabetes.

ADP is considered to be a weak platelet agonist by itself compared with thrombin or collagen (1, 15). Indeed, addition of exogenous ADP to human platelets results initially only in shape change and reversible aggregation in the presence of physiological concentrations of ionized calcium in the external medium. ADP receptors are divided into two main groups: the G protein-coupled or metabotropic superfamily termed P2Y, and the ligand-gated ion channel or ionotropic superfamily termed P2X (36). Molecular identification of the platelet P2 receptors has recently made it possible to separately assign the known effects of ADP and ATP to the three cloned P2 receptors found on platelets, namely, P2Y1, P2Y12, and P2X1. In our experiments, inhibition of P2Y1 receptors by treatment with MRS2279 was successful in preventing microaggregates of platelets in control subjects, which demonstrated that the P2Y1 receptor must be absolutely necessary for ADP-induced microaggregates of platelets. This is an agreement with a previous finding that adenosine does not restore aggregation in the presence of a P2Y1-selective antagonist (37). This has been confirmed in P2Y1-deficient mice (17), and the refractory state is, in fact, found to be entirely due to desensitization of the P2Y1 receptor with resultant loss of shape change (38). However, in diabetic patients, MRS2279 slightly reduced development of microaggregates induced by a low dose of ADP, but did not completely inhibit it. In contrast, irreversible platelet microaggregation in diabetic patients was significantly reduced by treatment with AR-C69931MX, a P2Y12 antagonist. The P2Y12 receptor is normally responsible for complication and amplification of the aggregation induced by ADP (36). These findings showed that the P2Y12 receptor could play a key role in the development of microaggregates in diabetic patients.

The platelet-aggregated form consists of two types of platelet membrane receptors, GPIIb/IIIa and GPIb/V/IX. GPIIb/IIIa not only binds fibrinogen and vWF to mediate platelet aggregation and adhesion, but also serves as a signaling receptor. ADP induces the signaling, which activates the receptor function of GPIIb/IIIa for soluble fibrinogen, and ADP-induced platelet aggregation involves binding of fibrinogen to GPIIb/IIIa (39). The P2Y12 receptor is involved in the constitution of stable macroaggregates (irreversible change) through full activation of the GPIIb/IIIa, whereas P2Y1 is involved in the centralization of platelet granules and the formation of filopodia in platelets (18, 40). Our results showed that reversible microaggregates in control subjects were prevented by treatment with a GPIIb/IIIa antagonist. However, irreversible microaggregates of platelets in diabetic patients were not completely inhibited by a GPIIb/IIIa antagonist. In contrast, inhibition of the GPIb/V/IX-vWF complex by ATA markedly reduced microaggregates of diabetic patients. Binding of vWF with the GPIb/V/IX complex is also important in formation of platelet activation. Shear-dependent platelet aggregation occurs by a mechanism initiated by binding of vWF to the GPIb/V/IX complex (41). Indeed, microaggregates produced by shear stress alone in diabetic patients were also significantly reduced by adding vWF to the GPIb/V/IX complex inhibitor. These findings were supported by the results of clinical evidence using aspirin and ticlopidine. Aspirin has been used as a primary and a secondary strategy to prevent cardiovascular events in nondiabetic and diabetic individuals. Substantial evidence from meta-analysis of large scale collaborative trials suggested that a low dose of aspirin should be used as a primary prevention strategy with diabetes that is at high risk for cardiovascular diseases (42). In our clinical study, treatment with aspirin reduced the development of irreversible microaggregates induced by a low dose of ADP, but not that induced by shear stress alone. In contrast, inhibition of P2Y12 receptors by treatment with ticlopidine significantly reduced irreversible microaggregates induced by either a low dose of ADP or shear stress alone. Recent investigations reported that the P2Y12 receptor blockade prevents shear-induced platelet aggregation (43) and blocks the function of P2Y12 in platelet activation initiated by the vWF-GPIb complex (44). These findings indicated that the hypersensitivity of P2Y12 receptors in diabetic patients could induce abnormal microaggregates of platelets via platelet GPIb/V/IX.

Finally, glucose and insulin have been shown to affect platelet function by different mechanisms (45, 46, 47). Different intracellular pathways may be activated in response to platelet activation. This study was not designed to determine which pathway is pivotal in the development of irreversible microaggregation of platelets. However, in the present study we found that inhibition of P2Y12 receptor or inhibiting the GPIb/V/IX-vWF axis significantly prevented the development of microaggregates of platelets in diabetic patients. These findings indicated that microaggregates of platelets were mainly stimulated by the P2Y12 receptor of ADP and were mostly constructed via GPIb/V/IX on the platelet membrane. Additionally, body index and biological values without HbA1c of Japanese diabetic patients were not significantly different from those of normal subjects. Therefore, we could discuss the relationship between microaggregates of platelets and diabetes without obesity, hypercholesterolemia, and any other fundamental change with diabetes. In conclusion, irreversible microaggregates occurred frequently in patients with type 2 diabetes, and the development of microaggregates is a trigger of platelet activation. This phenomenon could be caused by an increase in the sensitivity of P2Y12 receptors and the GPIb/V/IX-vWF axis on platelets, which enhance platelet aggregation. This suggests that a biological role of microaggregates is to provide a local thrombogenic mechanism that hinders clots from growing into occlusive intravascular thrombi. Therefore, detection of the microaggregated form by the LS system could be a useful tool for the prevention of ischemic events in diabetic patients. Moreover, inhibition of P2Y12 receptors and GPIb/V/IX might have a key role in preventing the development of microthrombi in patients with type II diabetes. Our findings could open a new avenue for the prevention of cardiovascular diseases in diabetes.


   Footnotes
 
First Published Online October 13, 2004

Abbreviations: ATA, Aurino-tricarboxytic acid; AUC, area under the curve; GP, glycoprotein; HbA1c, hemoglobin A1c; LS, light scattering; PRP, platelet-rich plasma; vWF, von Willebrand factor.

Received January 26, 2004.

Accepted September 27, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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