Quantitation of serum prostacyclin-binding in thrombotic thrombocytopenic purpura.

We quantitated serum PGI2 binding of 8 normal subjects and two TTP (thrombotic thrombocytopenic purpura) patients by gel filtration and gel partition methods using a stable PGI2 analogue, iloprost. The dissociation constant (KD) and the binding capacity (or binding stoichiometry) determined for the normals were 94 +/- S.D. 19 microM and 1.8 +/- S.D. 0.5 mM (or 2.0 +/- .6, iloprost:HSA). Corresponding values for serum samples obtained from TTP patient I were KD 200 microM, and Bmax 2.3 mM in the acute phase, and 75 microM and 1.8 mM respectively in the remission phase. The serum samples from TTP patient II exhibited a higher KD. Values of 299 microM (acute phase) and 147 microM (remission phase) were obtained. The corresponding binding capacities were 2.1 mM and 1.5 mM. Binding affinity change appears to be the main factor which resulted in the PGI2 binding defect in TTP.

Selective internalization of arachidonic acid by endothelial cells.

The asymmetric distribution of phospholipids in bovine endothelial-cell membranes was probed with 2,4,6-trinitrobenzenesulphonate and purified phospholipase A2. The data suggest that phosphotidylethanolamine is primarily located in the inner lipid bilayer, as reported for other cell types. Stearic acid is taken up by the endothelial cells and is randomly distributed among the membrane phospholipids. In contrast, the polyunsaturated fatty acids (arachidonic, eicosatrienoic and eicosapentaenoic acids) have initial incorporation into the phosphatidylcholine fraction. These fatty acids then undergo a time-dependent transfer from phosphatidylcholine to phosphatidylethanolamine. Thus we propose that endothelial cells possess a mechanism for the selective internalization of polyunsaturated fatty acids.

Interaction between lymphocytes and platelets in the synthesis of prostacyclin.

To test the hypothesis that prostacyclin (PGI2) is formed via a biochemical interaction between platelets and lymphocytes, we measured eicosanoids by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). A distinct 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) peak was noted when [14C]arachidonic acid ([14C]AA) was added to the mixed cell preparations which was increased by pretreating platelets with 1-benzylimidazole (1-BI). Lymphocytes prelabeled with [14C]AA failed to form 6KPGF1 alpha when stimulated with phytohemagglutinin (PHA) or ionophore A23187. When the prelabeled platelets were suspended together with aspirin-treated lymphocytes and stimulated with ionophore, thrombin, or collagen, a 6KPGF1 alpha peak was detected and enhanced by 1-BI. These results were supported by quantifying the 6KPGF1 alpha content in the HPLC-purified fraction by RIA. Adding prostaglandin H2 (PGH2) directly to lymphocytes led to 6KPGF1 alpha production. Platelet aggregation and release were inhibited by lymphocytes in a dose-related manner. We conclude that lymphocytes possess PGI2 synthase activity which is capable of converting platelet-derived PGH2 into PGI2. PGI2 formed is sufficient to inhibit platelet function.

von Willebrand factor abnormalities and endothelial cell perturbation in a patient with acute thrombotic thrombocytopenic purpura.

The plasma of a 63-year-old patient with an initial acute, fatal episode of thrombotic thrombocytopenic purpura (TTP) contained agglutinated platelets and a factor VIII-related von Willebrand factor (vWF) antigen level that was elevated seven-fold above normal. Unusually large vWF multimers derived from endothelial cells were detected in her plasma at the onset of the TTP episode. This is the first patient in whom vWF abnormalities indicative of in vivo endothelial cell damage or perturbation have been found during an acute episode of TTP.

Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo.

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.