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Association between mean platelet volume in the pathogenesis of type 2 diabetes mellitus and diabetic macrovascular complications in Japanese patients

Abstract Aims/Introduction Mean platelet volume (MPV) is a widely used biological marker of platelet function and activity. Increased MPV is associated with accelerated thrombopoiesis and an elevated risk of cardiovascular disease. However, it is not known whether higher MPV is related to the pathogenesis of type 2 diabetes and diabetic macrovascular complications in Japanese patients. Therefore, we analyzed MPV and its correlation with atherosclerosis in Japanese patients with type 2 diabetes and those who had prediabetes. Materials and Methods We divided the patients into three groups: normoglycemic patients (n = 56), prediabetes patients (n = 44) and type 2 diabetes patients group, (n = 115). We measured platelet parameters and evaluated arterial stiffness in the three groups. Results Significantly higher MPV was found in the type 2 diabetes mellitus and prediabetes patients compared with normoglycemic patients. MPV was significantly correlated with fasting blood glucose and glycated hemoglobin levels. Multiple linear regression analysis showed that MPV was positively correlated with HbA1c, even after adjustment for confounding factors. In the evaluation of arterial stiffness by measuring the cardio‐ankle vascular index and maximum intima‐media thickness, MPV showed a positive correlation with these parameters. Conclusions These findings suggest that MPV was significantly increased in the early stage of type 2 diabetes. We showed positive correlations between MPV and HbA1c levels, and between MPV and arterial stiffness in Japanese patients with type 2 diabetes.


INTRODUCTION
Platelets have central roles in hemostasis and thrombosis 1 . When platelets are activated by vascular injury, they secrete various substances essential for mediating coagulation, thrombosis, inflammation and atherosclerosis 2 . Therefore, evaluation of platelet hyperactivity is important. However, the optimal method for platelet testing is complicated and requires specialized equipment, making direct measurement of platelet activity impractical in routine practice. Generally, large platelets show greater metabolic and enzymatic activity than small platelets 3,4 . Mean platelet volume (MPV) is widely used as a biological marker of platelet function and activity 5 . MPV can be measured easily and cheaply by using an automated blood cell counter, which is routinely available in most hospitals. Increased MPV has been linked to the development of myocardial infarction 6 and atherosclerosis 7 , as well as to increased risk of cardiovascular disease 7,8 .
The risk of cardiovascular disease is high in patients with an underlying disease, such as hypertension, dyslipidemia and type 2 diabetes mellitus. Type 2 diabetes mellitus is a metabolic disorder and a critical factor in cardiovascular disease onset. Its pathogenesis is characterized mainly by insulin resistance and pancreatic b-cell failure. Microvascular complications (retinopathy, nephropathy and neuropathy) and macrovascular complications (ischemic heart disease, peripheral vascular disease and cerebrovascular disease) are features of type 2 diabetes mellitus associated with hyperglycemia. Higher MPV is observed in patients with metabolic disorders, such as obesity 9 and type 2 diabetes mellitus [10][11][12] ; however, these studies only reported increased MPV in patients with type 2 diabetes mellitus, and it is still unclear whether higher MPV is present in the early stage of type 2 diabetes mellitus. To our knowledge, no studies have examined whether increased MPV is associated with progression of atherosclerosis in patients with type 2 diabetes mellitus.
In the present study, we hypothesized that MPV is associated with metabolic disorder in the early stage of type 2 diabetes mellitus in Japanese patients, and that increased MPV results in the progression of diabetic macrovascular complications.

Participants
The required number of patients in each group was determined statistically by R software (R Foundation for Statistical Computing, Vienna, Austria), and at least 41 patients were required in each group to carry out the analysis. On the basis of this calculation, we analyzed the data of 215 patients (normoglycemic patients [Control group], n = 56; prediabetes patients [Pre-DM group], n = 44; and type 2 diabetes mellitus group, n = 115) from 2014 to 2015. They were classified into three groups based on criteria outlined by the American Diabetes Association 13 : Control (fasting blood glucose levels <110 mg/dL), Pre-DM (fasting blood glucose levels 100-125 mg/dL and glycated hemoglobin [HbA1c] 5.7-6.4%) and type 2 diabetes mellitus (fasting blood glucose levels >126 mg/dL and HbA1c ≥ 6.5%). We could not divide the Pre-DM group into impaired fasting glucose and impaired glucose tolerance groups, because an oral glucose tolerance test was not carried out. Patients with type 1 diabetes or an abnormal platelet count (<100 or >450 9 10 9 platelets/L) were excluded.

Ethics approval
The protocol for this research project was approved by a suitably constituted ethics committee of the institution and it conformed to the provisions of the Declaration of Helsinki. The institutional review board of Nara Prefecture General Medical Center, Approval No. 398.

Measurement of platelet parameters
Venous blood was sampled using K3-ethylenediaminetetraacetic acid tubes. The samples were tested within 1 h of collection to ensure variations due to sample aging were minimized. We measured platelet count and MPV using an ADVIA â 2120i Hematology System (Siemens, Munich, Germany), which uses two-angle laser flow cytometry with sodium dodecyl sulfate treatment for isovolumetric sphering measurement of erythrocytes and platelets. As a result, platelets and other blood cells can be discriminated accurately.

Biochemical measurements
Biochemical analyses of fasting blood glucose levels, C-reactive protein, triglycerides, and high-and low-density lipoprotein cholesterol were determined by standard methods. HbA1c was analyzed using high-performance liquid chromatography (HLC723-G9; Tosoh Bioscience, Tokyo, Japan). The clinical laboratory of Nara Prefecture General Medical Center carried out all laboratory measurements.

Assessment of arterial stiffness
The cardio-ankle vascular index (CAVI) is a measure of the arterial stiffness of the arterial tree between the origin of the aorta and the ankle. This assessment is independent of blood pressure during measurement. CAVI assessment was carried out using a VaSera VS-1500A system (Fukuda Denshi, Tokyo, Japan) 14 .
Ultrasonography of carotid intima-media thickness B-mode ultrasonographic imaging of the carotid artery was performed using an ultrasound imaging system (LOGIQ S8; GE Healthcare Japan, Tokyo, Japan) 15 . Maximum intima-media thickness (IMT) was used because it has been reported to be more useful than mean IMT for evaluating the progression of carotid artery disease [16][17][18][19] .

Statistical analysis
One-way analysis of variance (ANOVA) was used for comparisons among the three groups. If ANOVA showed a significant difference, we used the Tukey-Kramer test for multiple comparisons to identify which group differed. Student's t-test was used for comparisons between two groups. Pearson's correlation model was used to calculate correlations between MPV and several parameters (fasting blood glucose levels, HbA1c levels, CAVI and maximum IMT). To examine the relationships between MPV and diabetic parameters after adjustment for clinical characteristics (age, sex, body mass index, blood pressure, smoking status and type of antidiabetic medication), multiple linear regression analysis was carried out. Data are presented as the meanstandard deviation. P < 0.05 was considered to show statistical significance.

RESULTS
Platelet size is positively correlated with glucose intolerance Table 1 summarizes patients' characteristics, whereas Table 2 shows the types of antidiabetic medication taken in the type 2 diabetes mellitus group. The clinical conditions of the Control group are shown in Table 3. No significant difference was evident between all groups for age, body mass index, C-reactive protein, low-and high-density lipoprotein cholesterol or diastolic blood pressure. The only significant differences observed in the type 2 diabetes mellitus group compared with the Control group were significantly increased triglycerides and systolic blood pressure.
There was a significant increase in MPV in the type 2 diabetes mellitus and Pre-DM groups compared with the Control group ( Figure 1a). No significant difference was seen in platelet counts among the three groups ( Figure 1b). MPV was positively correlated with fasting blood glucose and HbA1c levels ( Figure 1c,d). To examine whether MPV was affected by the type of antidiabetic medication, we carried out multiple linear regression analysis. However, there was no positive relationship between MPV and the type of antidiabetic medication in the type 2 diabetes mellitus group after adjusting for medication type (Table 4).
In addition to MPV, the ADVIA 2120i Hematology System can measure another marker of platelet size, namely, the large platelet (LPLT) morphology flag. When ≥10% of platelets in the blood are >20 fL, the LPLT flag becomes positive. In LPLT-positive patients, fasting blood glucose and HbA1c levels were significantly increased compared with LPLT-negative patients (Figure 2a,b). Furthermore, we carried out multiple linear regression analysis to determine the independent relationships between MPV and HbA1c among the confounding factors. MPV was positively correlated with HbA1c and blood pressure, even after adjustment for the confounding factors (Table 5).
Next, we examined the possibility that MPV could change according to variations in glucose levels. We defined an improvement of hyperglycemia when fasting blood glucose levels were decreased by <160 mg/dL or HbA1c levels were decreased by >0.5% in 1 year, regardless of the type of treatment used. Then, 38 patients in the type 2 diabetes mellitus group were observed to have achieved an improvement of   fasting blood glucose ( Figure 3a) and HbA1c (Figure 3b) levels after treatment for 1 year. We found that MPV after treatment was significantly reduced compared with that before treatment (Figure 3c).
Progression of atherosclerosis in patients with type 2 diabetes is positively correlated with increased MPV To evaluate how MPV is related to arterial stiffness, we measured CAVI and maximum IMT, which are widely used markers of arterial stiffness. A significant increase in CAVI and maximum IMT was found in the type 2 diabetes mellitus group compared with the Control group (Figure 4a,b). Furthermore, there was a significant increase in CAVI in the Pre-DM group compared with the Control group ( Figure 4a). Atherosclerosis has been found in the early stage of type 2 diabetes mellitus 20,21 . Indeed, we found that MPV and CAVI were positively correlated (Figure 4c). However, the relationship between increased MPV and atherosclerotic condition in the type 2 diabetes mellitus group was not completely clear. We included type 2 diabetes mellitus patients with mild atherosclerosis (8.0 < CAVI < 9.0) in the group of patients without atherosclerosis 14 and analyzed MPV in these patients. MPV was observed to be significantly increased in the type 2 diabetes mellitus group without atherosclerosis compared with the Control group (Figure 4d).

DISCUSSION
Our findings showed that increased MPV and diabetic conditions are positively correlated in Japanese patients with type 2  diabetes mellitus. Furthermore, we observed a significant increase in MPV in the early stage of type 2 diabetes mellitus according to the findings in the Pre-DM group. In this study, we examined the association between platelet size and hyperglycemia by analyzing LPLT, which showed that platelet size might be influenced by chronic hyperglycemia, because LPLT was positively associated with HbA1c levels. Multiple linear regression analysis showed that there was no positive relationship between MPV and the type of antidiabetic medication used in the type 2 diabetes mellitus group after making adjustments for medication type. Furthermore, we found that MPV could be changed by improving hyperglycemia in Japanese patients with type 2 diabetes mellitus. A significant decrease in MPV due to the improvement of hyperglycemia by 6 months of metformin treatment has been shown in patients with type 2 diabetes mellitus 22 . These results suggested that platelet size was influenced in the early stage of type 2 diabetes mellitus, and baseline treatment did not have a positive association with MPV. The chronic change in blood glucose levels might be associated with MPV. Factors reported to affect MPV include the length of time from venous blood sampling to testing 5 , seasonal changes 23 and hypertension 24 . However, we tested all blood samples within 1 h from sampling and observed no seasonal differences in the recruited patients. Therefore, these factors did not affect the present results. In the type 2 diabetes mellitus group, we observed significant elevation in only systolic blood pressure compared with the Control group. In addition, multiple linear regression analysis showed that MPV was positively correlated with HbA1c levels and blood pressure after adjustment for confounding factors. From these results, we could not completely discount the influence of blood pressure, but at the very least, blood pressure did not affect MPV and arterial stiffness in the Pre-DM group. Nevertheless, the mechanism by which MPV was increased in patients with type 2 diabetes mellitus remains unclear. Platelet size is determined during megakaryopoiesis, and there is a positive association between platelet morphology and levels of thrombopoietin and interleukin-6, which regulate megakaryocyte ploidy 25,26 . Hyperglycemia increases the production of thrombopoietin by neutrophils in the liver through the receptor for advanced glycation end-products 27 . Interleukin-6 is well known for linking inflammation and insulin resistance in type 2 diabetes mellitus patients 28,29 . Thrombopoietin and interleukin-6 production might be regulated by  We examined MPV as a biomarker for predicting macrovascular complications in Japanese patients with type 2 diabetes mellitus. In patients with coronary artery disease, MPV is reported to be a potentially useful prognostic biomarker 32 . The population-based Gutenberg Health Study showed a strong association between MPV and arterial stiffness. Furthermore, higher MPV was considered a potential marker of platelet activation and increased stiffness of the arterial vessel wall 31 . The present results showed a positive association between higher MPV and progression of atherosclerosis in the type 2 diabetes mellitus and Pre-DM groups. In addition, we showed the possibility that progression of atherosclerosis in type 2 diabetes mellitus was due to increased MPV. In light of this possible relationship, we suggest that measuring MPV might be important in terms of evaluating atherosclerosis and cardiovascular outcomes in Japanese patients with type 2 diabetes mellitus. This study had several limitations. First, due to its retrospective nature, we could not examine how increased MPV is related to future clinical events. Second, we were unable to divide the Pre-DM group into impaired fasting glucose and impaired glucose tolerance groups, because we did not have oral glucose tolerance test data. Third, we could not analyze platelet activity or functions, because we did not have any suitable methods or specialized equipment. Thus, the correlation between platelet functions and MPV is still unclear.
In conclusion, these findings suggest that increased MPV is positively associated with HbA1c levels in Japanese type 2 diabetes mellitus patients, and is positively related to the progression of atherosclerosis. Measuring MPV could be a beneficial marker for determining diabetes-induced macrovascular complications in patients with type 2 diabetes mellitus.