The cosegregation of traditional risk factors, such as smoking, hypertension, dyslipidemia, and hyperglycemia, per se do not fully account for the excessive cardiovascular risk in diabetes (1,2), thus suggesting that there are other contributory factors. Diabetes is associated with several defects of coagulation and fibrinolysis that predispose to a thrombogenic tendency (3,4,5). Phospholipids are distributed asymmetrically between the inner and outer leaflets of the normal cell membrane lipid bilayer (6), with the cholinephospholipids (phosphatidylcholine and phosphatidylsphingomyelin) predominantly in the external leaflet and the aminophospholipids (phosphatidylserine [PS] and phosphatidylethanolamine) located internally. This equilibrium can be disrupted by activation of the calcium-dependent scramblase enzyme (6). PS is a potent activator of thrombin and other clotting factors (6), thus promoting a procoagulant environment that may increase the risk of cardiovascular disease. Therefore, we investigated whether there is an increase in PS in the outer leaflet of the lipid bilayer of erythrocyte and platelet cell membranes in type 1 diabetic patients and whether this is associated with changes in the appearance of scramblase.

Consent for the study was obtained from 13 healthy control (HC) subjects; 11 normoalbuminuric type 1 diabetic patients of ≥15 years diabetes duration without macrovascular disease (DM subjects); and 18 type 1 diabetic patients with microalbuminuria, defined as a urine albumin-to-creatinine ratio (ACR) >2.5 mg/mmol in males and >3.5 mg/mmol in females on at least three occasions, of which two were consecutive and at least 6 months apart, without macrovascular disease (DM-MA subjects). There were no significant differences in age, sex distribution, and smoking status between HC, DM, and DM-MA subjects. Furthermore, DM and DM-MA subjects were well matched for retinopathy, neuropathy, blood pressure (BP), BMI, and lipid and metabolic control. Venous blood was collected into acid-citrate-dextrose and then centrifuged at 120g for 15 min at 15°C to obtain platelet-rich plasma and erythrocytes. These were used to determine the median number of external PS molecules and their within-subject variance (intra-coefficient of variation) by using the binding of the cell membrane–impermeant annexin V-fluorescein isothiocyanate (FITC) conjugate (Beckman-Coulter, Paris) and quantified by a fluorescence-activated cell-scanner (Becton-Dickinson FACScan supporting Lysis II software) (7,8). The number of external scramblase molecules on platelets and erythrocytes were measured by fluorescence-activated cell scanning using a rabbit anti-scramblase antibody (directed to the extracellular COOH-terminal domain) and FITC–anti-rabbit IgG. Differences between groups were compared by the Mann-Whitney U test and corrected for multiple comparisons by a Bonferroni correction factor.

The results clearly show that the PS molecules per cell in the external leaflet of the cell membrane lipid bilayer of circulating platelets and erythrocytes were higher in DM subjects than in HC subjects (Table 1). Furthermore, the externalized PS was further increased in cells from DM-MA subjects (Table 1). Although increases in total cell membrane PS have previously been reported in type 1 diabetes (9), this is the first investigation of the appearance of PS in the outer leaflet of the lipid bilayer. The increased expression of PS on blood cells is likely to be an important contributor to the elevated cardiovascular risk in type 1 diabetic patients. This is supported by the even higher levels of PS on the exterior of blood cells from patients with microalbuminuria who have a higher risk of cardiovascular disease than patients without microalbuminuria (10).

The lipid bilayer enzyme scramblase, which can externalize PS when activated, was also increased in platelets and erythrocytes from DM subjects, with further increases in DM-MA subjects (Table 1). This suggests that scramblase may be involved in the increased externalized PS on cells from the diabetic subjects. This was further supported by the positive correlation between the number of external cell membrane PS and scramblase molecules in erythrocytes (rsp = 0.774; P < 0.001), and platelets (rsp = 0.715; P < 0.01) from all subjects. However, there were no significant correlations within each subject group. There were no significant relations among HbA1c, BP, ACR, BMI, or plasma lipids and outer-leaflet PS or scramblase, which suggested that the abnormality in the latter was not a simple reflection of glycemic control or renal function.

Although the median values for platelet and erythrocyte PS were increased in DM subjects, the percentage of variance in the cells within each subject was similar to that of HC subjects (59 [31–88] vs. 61 [51–87] and 62 [49–88] vs. 63 [55–91], respectively), suggesting that in DM subjects, the cells were able to control their external levels of PS but did so at a new increased steady state level. In contrast, the variance in externalized PS in the cell populations from DM-MA subjects was markedly increased (110 [76–178] and 111 [72–192], P < 0.01), suggesting that these cells not only had increased levels of external PS, but that their control mechanisms were impaired. Therefore, the results suggest that platelets and erythrocytes from DM-MA subjects not only had increased external PS with the accompanying thrombogenic tendency, but that the cells also had a loss of control of phospholipid asymmetry and would be at much greater risk of a catastrophic breakdown.

S.T.W. was supported by funding provided by the NHS Culyer Program to South Tees Acute Hospitals NHS Trust, and T.H.T. is supported by the Northern Counties Kidney Research Fund.

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Address correspondence to Dr. T.H. Thomas, Senior Lecturer, School of Clinical Medical Sciences, The Medical School, Framlington Place, 4th Floor William Leech Bldg., University of Newcastle, Newcastle upon Tyne, NE2 4HH, U.K. E-mail: t.h.thomas@newcastle.ac.uk.