Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction.

Secretory phospholipase A(2) (sPLA(2)) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA(2) will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA(2), at levels of sPLA(2) observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nM/10(8) RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 microM LPA, and the combination of PS-exposing red blood cells with PLA(2) caused a similar increase in permeability. In the presence of N-palmitoyl L-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 microM LPA in vivo were abolished. The present study demonstrates that increased sPLA(2) activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.

Measuring chromosome breaks in patients with thalassemia.

Iron-mediated oxidative stress plays an important role in the pathophysiology of thalassemia. Oxidative stress can cause lesions in DNA, including double-strand breaks. DNA damage, which is a cause of cancer (although not the only one), is recognized as deleterious. Unlike cancer, DNA damage can be assayed easily and relatively inexpensively in humans. In this study, a sensitive micronucleus assay was used to measure the frequency of chromosomal breaks in patients with alpha- and beta-thalassemia. The micronucleus test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. Micronuclei are readily apparent in red blood cells (RBCs), which otherwise lack DNA. We combined an immunomagnetic separation technique with single-laser flow cytometry to isolate and analyze reticulocytes in peripheral blood for the presence of micronuclei before these cells are removed by the spleen. Blood samples were obtained from patients with thalassemia and healthy volunteers. After immunomagnetic enrichment of CD71-positive reticulocytes, the cells were stained for micronuclei using the DNA dye 7-aminoactinomycin D (7-AAD) and evaluated by flow cytometry. Our findings indicate that higher levels of micronuclei frequencies are present in thalassemic RBCs.