Relationship between Mean Platelet Volume and Critical Limb Ischemia in Diabetes Mellitus Patients

Abstract

The prevalence of diabetes mellitus continues to increase from year to year. Critical limb ischemia (CLI) is one of the complications of diabetes mellitus with a high mortality rate and requires amputation if not treated properly. Mean platelet volume (MPV) is an indicator of platelet activation and is expected to be a predictor of CLI in diabetes mellitus patients.

This article investigates the relationship between MPV and the incidence of CLI in diabetes mellitus patients.

This case–control study was conducted using the vascular registry of Dr. Sardjito Hospital, Yogyakarta, Indonesia, from January 2016 to December 2016. The relationship between MPV and the incidence of CLI was analyzed using bivariate and multivariate analysis.

There was a significant association between MPV and incidence of CLI in diabetes mellitus patient both on bivariate analysis ( p  = 0.035) and multivariate analysis ( p  = 0.029). Diabetes mellitus patients with MPV values of ≥ 9.8 fl had a protective effect to prevent the incidence of CLI (bivariate analysis: odds ratio [OR] = 0.366, 95% confidence interval [CI] = 0.142–0.943; multivariate analysis: adjusted OR = 0.288, 95% CI = 0.09–0.88). Confounding factors such as sex, age, obesity, and use of antiplatelet agents were not associated with the incidence of CLI ( p  > 0.05). Meanwhile, history of dyslipidemia as a confounding factor was significantly associated with the incidence of CLI ( p  < 0.05).

Low MPV was found to be significantly associated with the incidence of CLI in diabetes mellitus patients.

Introduction

The prevalence of diabetes mellitus (DM) is continually increasing from year to year. In 2015 there were 415 million people with DM around the world with the mortality rate of diabetes reaching nearly 5 million people annually. It is estimated that by 2040, there will be 642 million people with diabetes globally. Indonesia is ranked as the 7th highest country/region with an estimated number of 10 million patients with DM. Globally, the financial cost of treating DM has reached 673 million USD. ¹

Diabetes is associated with various chronic complications, both macrovascular and microvascular complications. One of the macrovascular complications of DM is peripheral artery disease.

Peripheral artery disease is the narrowing of the peripheral arteries to the legs, stomach, arms, and head—most commonly in the arteries of the legs. ² Critical limb ischemia (CLI) is considered as the end-stage of peripheral artery disease. ³ Diabetes increases the risk of peripheral artery disease including CLI in the lower extremities by two- to fourfold compared with non-DM patients. ^{4 5} The risk of amputation in DM patients with CLI is 7 to 15 times higher compared with non-DM patients. ⁶

Approximately 40% of patients with CLI will require amputation on the affected limb, and approximately 150,000 amputations are performed in the United States per year. ^{7 8} Diabetes complications such as CLI and amputation of a foot or leg will affect the quality of life of the patient. Amputation will also have many other psychological, social, and economic impacts (i.e., no continuity of work) so it is a source of fear for people with DM. ⁹

Besides these negative consequences, the mortality rate of DM patients with CLI increases 20% within 6 months after CLI diagnosis is established and exceeds 50% in 5 years. ¹⁰ Therefore, prevention efforts for the occurrence of CLI complications in people with DM become essential for their short- and long-term survival.

Mean platelet volume (MPV) is an indicator of platelet activation associated with cardiovascular risk factors, such as DM. MPV is used to measure platelet size and is a routine method used for both inpatients and outpatients because of the relatively low cost. ¹¹

Materials and Methods

An observational case–control study was conducted to investigate the relationship between MPV and the incidence of CLI in patients with DM. The study was conducted from May 2017 to July 2017. Data were taken from the vascular registry of Dr. Sardjito General Hospital between January 2016 and December 2016. The target population in this study was adult patients with DM. Accessible population was adult patients with DM who come to Dr. Sardjito General Hospital, Yogyakarta, Indonesia. The eligible subjects in this study were patients who met the following inclusion criteria: patients with DM over 18 years of age and who have undergone duplex ultrasonography. The independent variable in this study was MPV. The dependent variable in this study was the incidence of CLI. This study was approved by the Medical and Health Research Ethics Committee of the Faculty of Medicine Gadjah Mada University – Dr. Sardjito General Hospital (Ref. KE/FK/0653/EC/2017). During this study period, 74 patients were eligible to be analyzed.

Statistical Analysis

Statistical analysis used IBM SPSS software version 23.0. Baseline characteristics are presented using percentages in the study population. The relationships between MPV and CLI were analyzed using bivariate and multivariate analyses. Bivariate analysis was done using chi-squared tests to compare proportions between the two variables with p  < 0.05, which was considered significant. Multivariate logistic regression analysis was done to evaluate relationships between the independent and dependent variables with any confounding factors.

Results

A total of 74 subjects were involved in this study. The subjects were divided into DM patients with CLI/without CLI ( n  = 37/37). The baseline characteristics of subjects in this study are shown in Table 1 . The overall age range of the study subjects ranged from 19 to 89 years. In the case subjects of DM patients with CLI, 20 (54.1%) were men and the other 17 (45.9%) were women with mean age of 63.5 ± 10.6 years. One patient (2.7%) was obese and 3 patients (8.1%) had dyslipidemia. A total of 15 patients (40.5%) received dual antiplatelet therapy, 11 patients (29.7%) received single antiplatelet therapy, and 11 patients (29.7%) did not receive antiplatelet therapy.

Table 1. Baseline characteristics of study subjects.

Variable	DM with CLI ( N  = 37) # (%)	DM without CLI ( N  = 37) # (%)	p
Sex
Male	20 (54.1%)	22 (59.5%)	0.639
Female	17 (45.9%)	15 (40.5%)
Age
< 60	16 (44.4%)	20 (55.6%)	0.352
> 60	21 (55.3%)	17 (44.7%)
Obese
No	36 (97.3%)	31 (83.8%)	0.107 a
Yes	1 (2.7%)	6 (16.2%)
Antiplatelet therapy
No	11 (29.7%)	10 (27%)	0.311
Single therapy	11 (29.7%)	17 (45.9%)
Dual therapy	15 (40.5%)	10 (27%)
History of dyslipidemia
No	34 (91.9%)	26 (70.3%)	0.018
Yes	3 (8.1%)	11 (29.7%)

Abbreviations: CLI, critical limb ischemia; DM, diabetes mellitus.

^aFisher exact test.

In the control subjects of DM patients without CLI, 22 patients (59.5%) were males and 15 patients (40.5%) were females. The mean age in the control subjects was 59.9 ± 11.5 years. Six patients (16.2%) were obese and 10 (27%) had dyslipidemia. A total of 10 patients (27%) received dual antiplatelet therapy, 17 patients (45.9%) received single antiplatelet therapy, and 10 patients (27%) did not receive antiplatelet therapy. Patients receiving dual antiplatelet therapy got aspirin and clopidogrel for treatment, while patients receiving single antiplatelet therapy got aspirin or clopidogrel for treatment.

Determination of the cutoff point for MPV values used median value due to its nonnormal distribution. The descriptive statistical test was used to determine the median value and the result was 9.8 fl. This cutoff point was used to divide MPV values into two categories: high MPV values (≥ 9.8 fl) and low MPV values (< 9.8 fl). Chi-square test with odds ratio (OR) and 95% confidence interval (CI) was used to determine the association between MPV and CLI ( Table 2 ).

Table 2. Association between mean platelet volume and critical limb ischemia.

		Group		OR	95% CI	p
		Case	Control
MPV values	High (≥ 9.8)	16	25	0.366	0.142–0.943	0.035
	Low (< 9.8)	21	12

Abbreviations: CI, confidence interval; MPV, mean platelet volume; OR, odds ratio.

Results showed that MPV was significantly associated with CLI ( p  = 0.035). In addition, subjects with low MPV values (< 9.8 fl) had 2.73 times increased risk of developing CLI (OR = 0.366, 95% CI = 0.142–0.943) compared with those with high MPV values (≥ 9.8 fl).

Logistic regression was used to evaluate the relationship between independent and dependent variables ( Table 3 ).

Table 3. Multivariate analysis.

Variable	OR (ExpB)	95% CI	p
MPV values
High (≥ 9.8 fl)	0.288	0.09–0.88	0.029
Low (< 9.8 fl)	1
Age (y)
≥  60	1.898	0.61–5.91	0.269
< 60	1
Sex
Female	1.566	0.49–4.98	0.447
Male	1
Antiplatelet therapy
Dual therapy (aspirin and clopidogrel)	1.523	0.39–5.96	0.546
Single therapy (aspirin or clopidogrel)	0.35	0.08–1.45	0.148
No	1
Obese
Yes	0.227	0.02–2.53	0.228
No	1
History of dyslipidemia
Yes	0.124	0.03–0.61	0.01
No	1

Abbreviations: CI, confidence interval; fl, femtoliter; MPV, mean platelet volume; OR, odds ratio.

In the multivariate analysis, MPV was significantly associated with CLI ( p  = 0.029). In addition, subjects with low MPV values (< 9.8 fl) had 3.47 times increased risk of developing CLI (adjusted OR = 0.288, 95% CI = 0.09–0.88) compared with those with high MPV values (≥ 9.8 fl). Other variables such as age (adjusted OR = 1.898, p  = 0.269), sex (adjusted OR = 1.566, p  = 0.447), use of dual anti platelet therapy (adjusted OR = 1.523, p  = 0.546), use of single antiplatelet therapy (adjusted OR = 0.35, p  = 0.148), and obesity (adjusted OR = 0.227, p  = 0.228) were not independent factors of CLI in patients with DM. Meanwhile, the history of dyslipidemia as a confounding variable was an independent factor of CLI in patients with DM (adjusted OR = 0.124, p  = 0.01).

Discussion

Among the subjects who participated in this study, most were males. These results are consistent with studies conducted by the International Diabetes Federation in 2015 where it was mentioned that the prevalence of diabetes occurs more frequently in males than females. ¹

The mean age of DM patients with CLI or without CLI was over 50 years. This result is in accordance with other studies where age is a risk factor that affects the occurrence of DM disease. In addition, age is a major risk factor for atherosclerosis. ¹² Atherosclerosis is considered a major source of pathogenesis of CLI disease.

In this study, most subjects who participated both in the CLI and non-CLI groups are not categorized as patients with obesity. There was only one subject (2.7%) of the CLI group and six subjects (16.2%) of the non-CLI group categorized with obesity. Obesity is a factor that affects the occurrence of DM. Obesity is associated with the lifestyle of each person. The small number of subjects with obesity in this study may be because researchers did not know the duration of DM suffered by the subjects nor adherence to the therapy given. As a result, it could be that subjects were previously categorized with obesity with body mass index > 27.5, but after undergoing treatment they became categorized as a nonobesity subject.

The subjects in this study mostly received antiplatelet therapy either dual antiplatelet therapy (aspirin and clopidogrel) or single antiplatelet therapy (aspirin or clopidogrel). In the DM group with CLI, 11 subjects (29.7%) received single antiplatelet therapy and 15 subjects (40.5%) received dual antiplatelet therapy. Meanwhile, the number in the DM group without CLI who received single antiplatelet therapy included 17 subjects (45.9%) while 10 subjects (27%) received dual antiplatelet therapy. Administration of dual antiplatelet therapy in patients with CLI is expected to provide benefits in the recovery process. Aspirin and clopidogrel alone have an action mechanism by inhibiting platelet function. However, aspirin and clopidogrel have a negligible effect on MPV.

The subjects with a history of dyslipidemia in this study accounted for only 14 subjects with 3 subjects (8.1%) from the CLI group and 11 subjects (29.7%) from the non-CLI group. Meanwhile, 34 subjects (91.9%) of the CLI group and 26 subjects (70.3%) of the non-CLI group had no history of dyslipidemia. The condition of dyslipidemia affects the occurrence of atherosclerosis. In dyslipidemia an increase in fat levels occurs in the blood circulation. High levels of fat will accelerate and affect the accumulation of lipids in the arterial intima and also the formation of foam cells in atherosclerosis.

The results of this study indicate that the relationship between MPV and CLI events in patients with DM has a significance value of p  = 0.035. This result is statistically significant and shows that there is a relationship between MPV and CLI events in patients with DM. With the multivariate test, the result was p  = 0.029, which shows that MPV is an independent factor of CLI events. In this study, the significant results obtained in both bivariate and multivariate tests can explain the relationship between DM occurrence, atherosclerosis, and CLI so that MPV levels can be used as predictors of CLI events in patients with DM.

So far, researchers have only found one previous study that discussed the relationship between MPV with CLI events in patients with DM. Rief et al in 2018 found that low MPV was associated with a higher occurrence of CLI in a large number of peripheral arterial occlusive disease patients. ¹³ The findings of this study were similar to our study in which low MPV was associated with the incidence of CLI; however, this study differs in which we specifically focused on the incidence of CLI in DM patients.

One study conducted by Ranjith et al in 2016 found that increased MPV was associated with poor outcomes in patients with acute myocardial infarction. ¹⁴ Similarly, research conducted by Degerli et al in 2016 suggests that MPV may be used as an indicator of acute mesenteric ischemia only if the patient does not have an accompanying disease. ¹⁵ Both studies' findings are consistent with the results of this study in which an association of MPV with the disease was found to be a dependent variable. Another study conducted by Arican Ozluk et al in 2015 found there was no significant change in MPV levels in patients with peripheral angiographic artery disease. ¹⁶ The findings of that study were not in accordance with the results of this study which showed there is a relationship between MPV with CLI events in patients with DM.

In patients with DM, platelets present in the blood circulation are hyperreactive and are correlated with blood glucose, HbA1c, and metabolites from thromboxane A2. Increased insulin resistance experienced by patients with DM will increase platelet size which will cause high MPV. In addition, under CLI complications, the condition of prothrombosis also causes high MPV.

The OR results indicate that patients with CLI are more likely to have low MPV levels (< 9.8 fl) which may be caused by the process of atherosclerosis. Atherosclerosis occurs in the process of inflammation and formation of thrombus in areas that have an endothelial injury. Platelets form in the blood circulation that are hyperreactive and large because of DM and CLI. The process of attaching thrombocytes to the endothelial injury is massive and intense so that large platelets become attached to the endothelial lesions. This large platelet attachment will affect MPV measurements because MPV is a measure of the average volume of platelets. Accordingly, many large platelets may be used in the process of atherosclerosis in the endothelial injury so that the large platelets present in the circulation are reduced.

Another possible reason for association of low MPV and CLI in DM is the role of platelet-derived microparticles. Recent studies suggest that the platelet-derived microparticles enhance platelet adhesion and thrombus formation in vessel wall with vascular injury. ¹⁷ Low MPV could be considered as platelet-derived microparticles that play a major role in hemostasis and thrombosis. ¹³ In DM, microparticles are elevated and play a role in its pathogenesis. ¹⁸ This may support the finding that low MPV might act as a microparticle which is already elevated in DM, thereby accelerate the thrombus formation in CLI.

In addition, the patient's glycemic index status also affects MPV levels. DM patients with good and controlled glycemic status (HbA1c < 7%) may have decreased MPV levels. ¹⁹ In this study, subjects did not disclose whether their DM condition was well controlled or not. According to the OR results in both bivariate and multivariate analyses, the risk of CLI is greater in patients with low MPV levels. As a result, it is possible in this study the condition of patients with DM was well controlled.

In the results of this study, a history of dyslipidemia had a significant p -value. Dyslipidemia conditions increase the release of CD40 ligand, interleukin-1 β, platelet factor-4, and other chemokines from platelets. ²⁰ Increased production of platelet chemokines will support the occurrence of the thrombosis process. The use of drugs that lower lipids can lead to decreased levels of MPV. ²¹ Among the subjects in this study it was also not known whether patients with dyslipidemia underwent lipid-lowering therapy or not. According to the OR results in both bivariate and multivariate analyses, the risk of CLI is greater in patients with low MPV levels. As a result, it is possible that in subjects in this study with a history of dyslipidemia had received lipid-lowering therapy which led to a decrease in MPV levels.

This study has two limitations. First, this is a retrospective study which relied on the accuracy of registry data, medical records, and laboratory data. Second, the lack of a standardized MPV cutoff point was another limitation of this study.

Conclusion

Low MPV is significantly associated with the incidence of CLI in DM patients. The results of this study suggest that using the cutoff point of 9.8 fl, MPV is an independent factor of the incidence of CLI in patients with DM.

Funding Statement

Funding There is no financial relationship or competing interests regarding the content of this article.