Shosaku Nomura MD, First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi Osaka 570, Japan. E-mail: email@example.com
Levels of platelet-derived microparticles (PMPs), platelet activation markers (P-selectin expressed on, or annexin V binding to, platelets (plt:P-selectin or plt:annexin V, respectively)), chemokines (IL-8, monocyte chemotactic peptide-1 (MCP-1), and regulated on activation normally T-cell expressed and secreted (RANTES)), and soluble P- and E-selectins were compared in peripheral blood from diabetic and control patients in order to develop a better understanding of their potential contribution to diabetic vascular complications. Significant increases were found for PMPs, plt:P-selectin, MCP-1, RANTES and soluble P- and E-selectins in diabetic individuals, whereas IL-8 levels were similar. Furthermore, after ticlopidine treatment, most of these factors receded to baseline levels observed in non-diabetic patients. Our findings indicate that ticlopidine might be able to prevent or reduce vascular complications in diabetic patients.
More than 75% of diabetic patients die of vascular complications associated with the disease [1,2]. Although diabetes itself is a risk factor for these complications , diabetes is often also associated with a hypercoagulable state [4,5]; increased platelet adhesion and aggregation have been reported in patients with diabetes . Abrams et al.  reported the detection of platelet activation-dependent antigens, such as P-selectin, by using MoAbs and flow cytometry and, using this technique, it has been directly demonstrated that the number of P-selectin-positive platelets (plt:P-selectin) is increased in diabetic patients .
Platelet-derived microparticles (PMPs) are produced by either platelet activation or physical stimulation under various conditions [9–12]. PMPs contain inner granules, membranous microvesicles released from activated platelets and membranous fragments produced by mechanical destruction , and have coagulative activity . A few studies on the potential role of PMPs in diabetic complications can be found in the literature [7,8,11,12].
Levels of cell adhesion molecules have been demonstrated to be increased in both type 1 and type 2 diabetes  and have been implicated in the development of microvascular complications. In particular, E-selectin, which is expressed on endothelial cells and contributes to monocyte adhesion to the endothelium , is up-regulated at the site of atherosclerotic lesions , and serum levels of soluble E-selectin (sE-selectin) are elevated in patients with diabetes [13,16].
Monocytes and macrophages may play an important role in glomerular disease  and glomerular infiltration of macrophages is associated with diffuse glomerulosclerosis in patients with diabetic nephropathy . Monocyte chemotactic peptide 1 (MCP-1) is a C-C chemokine for monocytes [19,20] and it has recently been reported that MCP-1 may contribute to glomerular monocyte infiltration in diabetic nephropathy . Another member of the C-C chemokine family, regulated on activation normally T-cell expressed and secreted (RANTES) , is a potent chemoattractant for memory T lymphocytes, monocytes, eosinophils, and basophils. RANTES may also activate specific effector cell populations of these cells [22,23]. To our knowledge however, no in vivo findings exist concerning RANTES levels in diabetic patients. We therefore measured and compared the levels of platelet activation markers (plasma PMPs, annexin V binding to platelets (plt:annexin V) and plt:P-selectin), chemokines (IL-8, MCP-1, and RANTES), and soluble P- and E-selectins, in order to develop a better understanding of their potential contribution to diabetic vascular complications. Furthermore, we investigated the effects of the anti-platelet drug ticlopidine on these markers in diabetic patients.
Materials and methods
All 20 healthy volunteers and 37 diabetic patients gave their informed consent to participate in this study, according to the Declaration of Helsinki. All of the diabetic patients had type 2 diabetes. Table 1 shows the characteristics of both patients and controls.
Table 1. Clinical characteristics of patients and healthy controls
Control (n = 20)
Type 2 diabetes (n = 37)
Data are shown as the means ± s.e.m.
NS, Not significant; BMI, body mass index; PLT: platelet count; HbA1c, haemoglobin A1c; T-CHOL, total cholesterol; TG, triglycerides.
48 ± 6
56 ± 5
23·3 ± 1·0
25·4 ± 1·2
4970 ± 752
5650 ± 824
PLT ( × 104/μl)
23·1 ± 4·7
23·5 ± 5·2
4·8 ± 0·3
7·8 ± 0·5
198 ± 35
214 ± 29
143 ± 37
162 ± 44
Flow cytometry of activated platelets and microparticles
Blood was collected into tubes containing 3·8% sodium citrate (9:1 v/v). Platelet-rich plasma (PRP) was prepared by centrifugation at 200 g for 10 min at room temperature, and the supernatant (the PRP) was then centrifuged at 1400 g for 10 min at room temperature. The resultant platelet pellet was washed twice with washing buffer (9 mmol/l Na2EDTA, 140 mmol/l NaCl, and 26 mmol/l Na2HPO4, pH 7·2) and resuspended in HEPES-Tyrode's buffer (129 mmol/l NaCl, 8·9 mmol/l NaHCO3, 0·8 mmol/l KH2PO4, 0·8 mmol/l MgCl2, 5·6 mmol/l glucose, and 10 mmol/l HEPES, pH 7·4). A equal volume of 2% paraformaldehyde was added to the washed platelets, and the mixture incubated for 15 min at room temperature. The platelets were washed twice, resuspended in stock solution (9 mmol/l Na2EDTA, 26·4 mmol/l Na2HPO4 2H2O, 140 mmol/l NaCl, 0·1% NaN3, and 2% fetal bovine serum (FBS), pH 7·2), and stored at 4°C until analysis. Analysis always took place within 48 h of platelet collection.
PMPs were detected using a modified version of a previously reported method [11,12,24]. Ten microlitres of the platelet suspension (3 × 108/ml) were added to 100 μl of HEPES-Tyrode's buffer containing 5 nmol/l EGTA, and both intact and aggregated platelets were removed by centrifugation at 1000 g for 15 min to yield a supernatant containing microparticles only. Next, 10 μl of washed intact platelets (3 × 108/ml) were added to the supernatant and incubated with 5 μg/ml KMP-9 (the FITC-labelled anti-platelet GPIX MoAb)  for 30 min in the dark at room temperature. After incubation, samples were diluted 1:10 with HEPES-Tyrode's buffer containing 5 mmol/l EGTA and analysed using an Ortho Cytoron Absolute analyser (Ortho Diagnostic Systems, Tokyo, Japan). Only the cells and particles positive for GPIX were gated to distinguish platelets and PMPs from electronic noise. To differentiate between platelets and PMPs, the lower limit of the platelet gate was set at the left-hand border of the forward-scatter profile of resting platelets. Ten thousand FITC-positive particles in the PMP gate were then counted to determine the number of PMPs released per 10 000 platelets.
As an index of platelet activation, plt:P-selectin expression was quantified by staining with anti-P-selectin (CLB-thromb/6; Immunotech, Marseille, France) MoAbs. Fixed platelets were washed twice with washing buffer, and incubated with FITC–P-selectin antibody for 30 min in the dark at room temperature. After incubation, samples were diluted with HEPES-Tyrode's buffer containing 2 m m EDTA and analysed using the Ortho Cytoron Absolute analyser. This instrument was calibrated for fluorescence and side scatter using 2·0 μm beads. In forward and side scatter levels, the gating area was set for the platelet population only, excluding aggregating platelets, debris, and machine noise. The setting of the negative line was for unstained platelets and platelets incubated with fluorescence-labelled mouse IgG. Assessment of platelet levels of glycoprotein (GP)IIb/IIIa and GPIb was performed using anti-GPIIb/IIIa (NNKY1-32) [24,26] and anti-GPIb (NNKY5-5)  MoAbs.
Assessment of PMPs and plt:P-selectin in whole blood
In order to eliminate the problems associated with platelet washing, a whole blood flow cytometric assay was also designed. The saturating concentration of FITC–P-selectin was predetermined on the basis of platelet activation by 2 μm A23187 (Sigma Chemical Co., St Louis, MO). Blood was collected into tubes containing 3·8% sodium citrate (9:1 v/v). Immediately after exposure to A23187 for 5 min, 10 μl of the collected whole blood were added to 100 μl of HEPES-Tyrode's buffer containing 5 mmol/l EGTA. Next, an equal volume of 2% paraformaldehyde was added and the sample incubated for 15 min at room temperature. Fixed samples were incubated with FITC–P-selectin antibody for 30 min. Flow cytometric analysis was determined as described in the previous section.
Assessment of annexin V binding to platelets
Suspensions of platelets were incubated with FITC-labelled annexin V (150 nmol/l) for 10 min at room temperature, diluted with 500 μl HEPES-Tyrode's buffer, and analysed using an Ortho Cytoron Absolute analyser as described earlier.
Measurement of chemokines and soluble factors
Blood samples were collected from patients and healthy controls into plastic tubes. Aliquots were distributed to tubes containing either sodium citrate or without any anticoagulant. Blood was allowed to clot at room temperature for a minimum of 1 h, and serum or citrated plasma was collected after centrifugation for 20 min at 1000 g (4°C), and was then stored at −30°C until assayed. The serum levels of chemokines and soluble factors were measured using commercial kits according to the manufacturers' instructions. Human MCP-1 and RANTES ELISA kits were from Toyobo Diagnostic, Inc. (Tokyo, Japan). IL-8 was measured using IMMULIZER (Diagnostic Products Corp., Los Angeles, CA). Levels of soluble (s)P-selectin and sE-selectin were measured using MoAb-based ELISA kits from R&D Systems (Toray Fuji Bionics Inc., Tokyo, Japan). Soluble thrombomodulin (sTM) levels were determined by enzyme immunoassay (Teijin Diagnotics, Tokyo, Japan). As positive controls, recombinant chemokines, as well as the standard solutions provided with the commercial kits, were used in each assay.
Anti-thrombosis with ticlopidine
Twenty-four of the diabetic patients had complications. We administered ticlopidine 200 mg/day for 8 weeks to 15 of these 24 patients, who gave their informed consent for the use of the drug. We did not change the other therapeutic drugs used for diabetes management during ticlopidine treatment. The patients included seven with neuropathy, four with retinopathy, four with nephropathy (without renal dysfunction), and one with retinopathy and nephropathy. In addition, we assessed seven non-diabetic patients (three with cerebral infarction, two with unstable angina, one with myocardial infarction, and one with Raynaud's disease). We compared the levels of the measured variables before and after 8 weeks administration of ticlopidine.
Statistical analysis was performed using Student's t-test, and P < 0·05 was considered significant. Comparison between patients with type 2 diabetes and healthy controls was performed using an unpaired t-test. In contrast, the paired t-test was used for comparison of data in the same patient before and after ticlopidine administration. A correction factor was applied for multiple comparisons.
We first examined the levels of PMPs and plt:P-selectin in washed platelets and whole blood ( Table 2). Both the extent of plt:P-selectin staining and the number of PMPs in washed platelets were higher than those in whole blood, but the differences were not statistically significant. In fact, the levels were significantly correlated (plt:P-selectin: r = 0·9815, P < 0·001; PMPs: r = 0·8646, P < 0·05).
Table 2. Platelet-derived microparticle (PMP) and P-selectin-positive platelet (plt:P-selectin) levels measured by A23187 in whole blood and washed platelet suspensions
PMP (/104 platelets)
Data are shown as the mean ± s.d. (n = 5). P values compare the levels of plt:P-selectin or PMPs between whole blood and washed platelet suspensions.
Washed platelet suspension
35·8 ± 7·3 (%)
1320 ± 215
32·4 ± 6·6 (%)
1195 ± 227
P < 0·001
P < 0·05
Table 3 shows the levels of PMPs and platelet activation markers in the control and diabetic groups. There were no differences in the binding of the anti-GPIIb/IIIa and anti-GPIb MoAbs between the two groups; however, there were significant differences in the levels of PMPs and platelet activation markers (PMPs: 576 ± 71 versus 338 ± 45, P < 0·05; plt:P-selectin: 9·4 ± 1·3% versus 4·5 ± 0·8%, P < 0·01 for diabetic versus control groups, respectively).
Table 3. Relationship between platelet-derived microparticle (PMP)/platelet activation markers in controls and diabetic patients
Control (n = 20)
Diabetic patients (n = 37)
Values are the means ± s.e.m. NS, Not significant.
94·3 ± 3·4
95·6 ± 3·5
95·7 ± 4·3
93·4 ± 4·0
4·5 ± 0·8
9·4 ± 1·3
plt:annexin V (%)
5·2 ± 0·8
10·9 ± 1·9
PMP (/104 plt)
338 ± 45
576 ± 71
Table 4 shows the levels of chemokines and soluble selectins. There were no differences in IL-8 levels between the control and diabetic groups; however, the levels of MCP-1, RANTES, and soluble factors were higher in the diabetic patients compared with the controls (MCP-1: 345 ± 19 versus 234 ± 17, P < 0·05; RANTES: 112 ± 25 versus 41 ± 5, P < 0·01).
Table 4. Relationship between chemokines/soluble selectins in controls and diabetic patients
Control (n = 20)
Diabetic patients (n = 37)
Values are the means ± s.e.m. NS, Not significant.
7·2 ± 0·9
10·1 ± 2·5
234 ± 17
345 ± 19
41 ± 5
112 ± 25
124 ± 15
196 ± 28
43·2 ± 3·9
68·4 ± 5·5
Figure 1 shows the relationship between the levels of plt:P-selectin and chemokines in diabetic patients. The IL-8 level did not significantly correlate with that of plt:P-selectin, but the MCP-1 and RANTES levels were positively correlated (P < 0·05 and P < 0·001, respectively).
Table 5 shows the clinical and biochemical characteristics of the diabetic patients who received ticlopidine. PMPs and platelet activation markers before and after ticlopidine treatment are shown in Fig. 2. Levels of plt:P-selectin, plt:annexin V and PMPs (per 104 platelets) were reduced from 9·3 ± 2·7% to 4·9 ± 1·8% (P < 0·01), from 11·4 ± 3·1% to 8·6 ± 2·8% (P < 0·05), and from 534 ± 98 to 394 ± 54/104 platelets (P < 0·05), respectively.
Table 5. Clinical profile of the ticlopidine-treated patients
Figure 3 shows the values of chemokines and soluble selectins before and after treatment. IL-8 levels did not change significantly; however, the levels of MCP-1, RANTES, sP-selectin, and sE-selectin were significantly decreased after treatment. On the other hand, the levels of FBS and HbA1 did not differ significantly after ticlopidine treatment (data not shown). The levels of chemokines in non-diabetic patients receiving ticlopidine did not change significantly, although platelet activation markers did decrease significantly following treatment ( Table 6).
Table 6. Levels of chemokines and selectins before and after ticlopidine treatment in seven non-diabetic patients
Values are the means ± s.e.m. (n = 7). NS, Not significant.
13·5 ± 2·9
8·5 ± 1·7
Plt:Annexin V (%)
15·4 ± 1·7
12·8 ± 2·6
PMP (/104 plt)
588 ± 101
439 ± 96
9·43 ± 1·8
9·7 ± 2·2
246 ± 25
226 ± 21
107 ± 12
88 ± 14
133 ± 27
121 ± 33
49·6 ± 3·6
45·5 ± 2·9
Table 7 shows the changes in diabetic patients with elevated (> mean + 2 s.d. of healthy controls) sTM levels. A total of eight patients had high-sTM levels (sTM > 8·5 Teijin units (TU) /ml) and these patients included all four with nephropathy. All chemokine, selectin, and platelet activation marker levels were higher in those patients with elevated sTM levels compared with the other diabetic patients. The sTM, chemokine, soluble selectin, and platelet activation marker levels were significantly decreased after ticlopidine treatment, but the IL-8 level was not changed.
Table 7. Levels of soluble thrombomodulin (sTM), chemokines, selectins, and platelet activation markers before and after ticlopidine treatment in diabetic patients with elevated sTM levels
Values are the means ± s.e.m. (n = 8).
NS, Not significant; MCP-1, monocyte chemotactic peptide 1; RANTES, regulated on activation normally T-cell expressed and secreted; plt:P-selectin, P-selectin-positive platelets; plt:Annexin V, annexin V binding to platelets; PMP, platelet-derived microparticles. TU, Teijin unit.
sTM (TU /ml)
13·1 ± 1·9
9·5 ± 2·3
10·3 ± 1·5
10·1 ± 1·7
398 ± 22
275 ± 31
124 ± 11
86 ± 9
245 ± 29
144 ± 23
70·3 ± 6·1
45·9 ± 5·3
11·2 ± 1·5
4·5 ± 2·1
plt:Annexin V (%)
11·3 ± 1·7
8·6 ± 1·8
PMP (/104 plt)
693 ± 75
420 ± 63
The present study shows that the levels of two C-C chemokines, two soluble selectins, PMPs, and two platelet activation markers were elevated in patients with diabetes, and that these elevations were significantly decreased by ticlopidine treatment.
Platelets in diabetic patients often have increased sensitivity to secondary aggregation in response to agonists . Hyperactive platelets may lead to capillary microembolization via the formation of microaggregates . In the present study, the level of plasma PMPs was significantly higher in diabetic patients than in healthy subjects. PMPs play an important role in coagulation mechanisms . Therefore, these increased levels may be a cause of hypercoagulability . In addition, an increase in PMP levels has been observed in disease states where activated platelets have been detected in vivo[11,12].
The levels of plt:P-selectin were also higher in diabetic patients than in healthy subjects. An increase in P-selectin expression on platelets is known to be a marker of platelet activation  and flow cytometric analysis of plt:P-selectin levels has been standardized [7,30]. Using this technique, it has been directly demonstrated that elevated levels of P-selectin-positive platelets circulate in patients with coronary artery disease or those with diabetes . Our findings agree with these previous reports and, in conjunction with the PMP findings, also suggest that enhanced platelet activity and related procoagulant activity in diabetes may induce hypercoagulability.
We evaluated the plasma concentrations of chemokines and soluble selectins in diabetic patients, although it is unclear whether the serum levels of P- and E-selectin reflect the tissue levels of these selectins. In the present study, the levels of the two C-C chemokines MCP-1 and RANTES were increased in diabetic patients compared with healthy subjects. Enhanced levels of C-C chemokines have been found in inflammatory and immune-mediated disease [32–34]. Thus, we believe that our findings support the notion that immunological and inflammatory processes are important features of diabetic complications. MCP-1 and RANTES are produced by a variety of leucocytes; RANTES is also produced by platelets, whereas MCP-1 is also produced by endothelial cells and fibroblasts [23,35]. In the present study, not only RANTES but also MCP-1 levels were positively correlated with the plt:P-selectin level. Activated platelets have been found to stimulate MCP-1 production in monocytes through enhanced RANTES secretion and direct platelet–monocyte contact mediated by P-selectin expression on the platelet surface . This mechanism of enhancement of MCP-1 expression in leucocytes has recently been shown to be operative in patients with acute myocardial infarction .
We next studied the effects of the anti-platelet drug ticlopidine on the levels of PMPs, platelet activation markers, chemokines, and soluble selectins. Ticlopidine is a well-known inhibitor of platelet aggregation and it has undergone extensive clinical trials to test its effectiveness at reducing the risk of ischaemic stroke, myocardial infarction, and death from vascular disease . The levels of plt:P-selectin decreased significantly after ticlopidine administration, demonstrating the anti-platelet effect of this drug. In addition, the level of PMPs, which are produced in association with platelet activation, was decreased significantly by ticlopidine administration. These results of ticlopidine on platelet activation markers were similar in non-diabetic patients.
In diabetic patients, arteriosclerosis and microangiopathy are both major complications  and diabetes is characterized by the premature development of micro- and macrovascular disease . PMPs have procoagulant activity that is capable of generating thrombin  and thrombin, in turn, induces platelet adhesion to endothelial cells, resulting in vascular injury . Thus, anti-platelet therapy with ticlopidine is also thought to be useful for anti-thrombin therapy, including anticoagulation therapy, since it suppresses the production of intrinsic coagulants released following platelet activation.
The levels of chemokines and soluble selectins in diabetic patients were also decreased significantly by ticlopidine. By contrast, these levels did not exhibit any significant changes in non-diabetic patients, presumably because the chemokine levels in non-diabetic patients were not greatly elevated prior to drug administration. RANTES and MCP-1 have chemotactic activity for both monocytes and lymphocytes and, in particular, MCP-1 has been postulated to be a major signal for the accumulation of mononuclear leucocytes in disease states . These monocytes and lymphocytes interact and activate endothelial cells. The increased expression of endothelial adhesion molecules following activation may have a potential role in accelerating diabetic macroangiopathy. These adhesion molecules include E-selectin, which is a product of both endothelial cells, as well as P-selectin, a product of endothelial cells and platelets [43,44]. In the present study, both soluble E- and P-selectin levels were slightly increased in diabetic patients. Thus, the finding of a significant decrease in chemokine and soluble selectin levels following ticlopidine administration suggests concomitant inhibition of diabetic macroangiopathy development.
Finally, we examined sTM levels, as this might predict early nephropathy or be a marker for microvascular complications in diabetic patients . In accordance with this, our patients with high sTM levels included all four patients with nephropathy. We investigated the effects of ticlopidine in all the diabetic patients with high sTM levels. Levels of chemokines (MCP-1 and RANTES), soluble selectins (P-selectin and E-selectin), and platelet activation markers (plt:P-selectin, plt:P-annexin V and PMP) all decreased significantly after ticlopidine treatment, as did sTM levels. Ticlopidine has been reported to have a direct effect on the immune system [46,47]. In particular, May et al.  reported that it reduced monocyte–platelet interactions. Our results appear to reflect the effect of ticlopidine on the immune system, in addition to its anti-platelet effect. The present findings thus suggest that ticlopidine may be useful for inhibiting the development of diabetic complications.
In conclusion, levels of plt:P-selectin, plasma PMPs, chemokines, and soluble selectins were significantly higher in patients with diabetes than in normal controls, and administration of ticlopidine significantly decreased these levels. These effects were more pronounced in patients with high sTM levels. These findings suggest that ticlopidine may prevent the development of complications, in which chemokines, selectins, and activated platelets participate, in diabetic patients.
We thank Mr Tatsunori Matsuzaki, Mr Mabanu Yamaoka, and Miss Misao Abe of the Department of Blood Transfusion, Kansai Medical University, for providing technical support. This study was supported in part by a Research Grant for Advanced Medical Care from the Ministry of Health and Welfare of Japan.