The origin of circulating CD36 in type 2 diabetes.

OBJECTIVE: Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36+MPs in persons with T2DM. DESIGN: An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI)=39.9±6.4 kg m(-2); age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI=23.6±1.8 kg m(-2) and 33.5±5.9 kg m(-2), respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235a, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations. RESULTS: CD36+MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235a+=35.8±14.6%); although this did not correlate with haemoglobin A(1c). By contrast, the main source of CD36+MPs in non-T2DM individuals was endothelial cells (CD105+=40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 µg ml(-1) and was positively correlated with CD36+MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36+MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively). CONCLUSIONS: Both the levels and cellular profile of CD36+MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.

The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles.

BACKGROUND: CD36 is a widely expressed cell surface receptor that binds lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell-free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. OBJECTIVE: To determine the nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). METHODS: Lipoproteins were fractionated by density gradient centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight-488 anti-CD36 conjugate in combination with antibodies against different cellular markers. RESULTS: Cell-free plasma CD36 was not observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. CONCLUSIONS: sCD36 is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.

Bio-maleimide as a generic stain for detection and quantitation of microparticles.

Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy-maleimide (or bio-maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13,000 g and stained with Annexin V and bio-maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio-maleimide staining was comparable with that by Annexin V. The total mean MP level with bio-maleimide staining was 34 +/- 19.7/microl (range of 11.6-68.1/microl) and with Annexin V staining it was 38.9 +/- 29.8/microl (range of 10.6 to 112.9/microl). There was no significant difference using a paired t-test and methods were comparable using a Bland-Altman plot. Bio-maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.

Spontaneous major bleeding in acquired factor X deficiency secondary to AL-amyloidosis.

Abnormal bleeding may be seen in up to a third of patients with amyloidosis. Factor X deficiency is the commonest acquired coagulopathy in amyloidosis. While mild intracutaneous bleeding is common, spontaneous life-threatening haemorrhage is rare. We report a case of acquired FX deficiency due to amyloid light chain (AL)-amyloidosis presenting with spontaneous retroperitoneal bleeding. The diagnostic and management issues in this patient are discussed.