Development and Assessment of Enzyme Immunoassay for Platelet-derived Microparticles

Abstract

SummaryPlatelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, anti-phospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 m filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Extracellular Vesicles in Renal Diseases

“…17,21 It is very clear that absolute quantification of EVs substantially differs among the methods described above. A gold standard for quantification of such particles has not yet been established but is being addressed by 43,55 In addition, these techniques lack the capacity for visual differential confirmation between small cells, debris, and MPs or exosomes but can be used as confirmatory test, such as to show an exosome-specific marker in an EV sample preparation. 43 EM (especially cryo-EM) serves currently as the gold standard to characterize the morphology of EVs; however, it is not useful for high-throughput analysis because of costs and time ( Figure 4, A and B).…”

Plasma Level of Platelet-Derived Microparticles Is Associated with Coronary Heart Disease Risk Score in Healthy Men

“…Clinically, elevated levels of plasma PDMP are associated with thrombotic disorders 12,18,27,30) . PDMPs play an important role in atherothrombotic events; however, their role in the early phase of atherosclerosis in apparently healthy individuals remains to be clarified.…”

“…Storage of the samples at RT for 2 − 3 hr did not affect the PDMP level 35) . Immediately after centrifugation, 200 L of upper-layer supernatants from 2-ml samples were collected to avoid contamination of the platelets 11,12,25,30) and the samples were stored at 40 until analysis. The PDMP level was measured in duplicate and the mean values were recorded.…”

“…The PDMP level was measured in duplicate and the mean values were recorded. For the measurement of PDMPs we used an ELISA kit (JIMRO Co. Ltd., Japan) and monoclonal antibodies against glycoprotein CD42b and CD42a (glycoprotein Ib and IX) 11,12,25,30) . Uncoated vacutainers were used for hsCRP assay; these were stored at 40 until analysis.…”

“…Clarifying the relationship between PDMPs and the 10-yr coronary heart disease (CHD) risk score 29) in healthy individuals is of importance. We measured the plasma level of PDMP by enzyme-linked sorbent assays (ELISA) using monoclonal antibodies against CD42b (GPIb) and CD42a (GPIX) 11,12,30) , and analyzed the association of the PDMP level with the 10-yr CHD risk score.…”