Involvement of Fc gamma receptor polymorphism in the therapeutic response of idiopathic thrombocytopenic purpura.

Clearance of autoantibody-sensitized platelets through Fc gamma receptors on phagocytic cells is one of the main mechanisms of thrombocytopenia in idiopathic thrombocytopenic purpura (ITP). We examined the Fc gamma RIIA-131R/H and Fc gamma RIIIA-158V/F polymorphisms in 104 adult chronic ITP patients, and in 59 healthy control subjects using polymerase chain reaction-based allele-specific restriction analysis. The frequency of Fc gamma RIIA genotypes (131H/H, H/R, R/R) was not significantly different between patients and controls, and did not correlate with the responsiveness to treatment. In contrast, among Fc gamma RIIIA genotypes, frequency of 158F/F homotype was smaller in ITP (P < 0.05). Furthermore, in Fc gamma RIIIA-158V/V homotype, the complete remission (CR) rate with medication (treatment with corticosteroid or other immunosuppressive agents) was significantly higher (60%) than that in 158V/F (10%) or 158V/F plus 158F/F, (P < 0.01, P < 0.05). Conversely, the CR rate after splenectomy in 158F/F and 158V/F types (64.3% and 54.6%) was higher than in 158V/V (25%). Our results indicate that the polymorphism of Fc gamma RIIIA, but not Fc gamma RIIA, influences the response to treatment in ITP.

A new approach to detect reticulated platelets stained with thiazole orange in thrombocytopenic patients.

Recent studies have shown that reticulated platelets stained with Thiazole Orange (T.O.) are useful markers for thrombopoiesis. The percentage of T.O. positive platelets tends to be inconsistent using the original method, especially when the peripheral blood platelet count is very low. We measured T.O. positive platelet levels in patients with severe thrombocytopenic disorders, using concentrated platelet-rich plasma and carrying out a two-color analysis involving T.O. and an anti-glycoprotein IIb/IIIa monoclonal antibody. This method allowed us to obtain consistent T.O. positive platelet rates in patients with thrombocytopenia whose platelet counts were below 20 approximately 30x10(9)/l. By this method, the T.O. positive rates of platelets from idiopathic thrombocytopenic purpura patients were found to be significantly higher than in the control group. The T.O. positive rates of other thrombocytopenic disorders were similar to those of the control group. These results are consistent with those previously reported. We conclude that our technique of measuring of T.O. positive platelets using platelet-rich plasma is useful for analyzing severe thrombocytopenic disorders.

Cloning and characterization of the gene encoding the murine glycoprotein V: the conserved thrombin-cleavable protein on platelet surface.

Glycoprotein V (GPV) is a platelet membrane protein present as a subunit of the GPIb/V/IX complex, a major receptor for von Willebrand factor, and is specifically cleaved by thrombin. In this study, we have cloned and characterized murine GPV gene. The entire coding sequence of murine GPV consisted of 1704 nucleotides and coded 567 amino acids, which were 70% identical with human GPV. Fifteen leucine-rich tandem repeats were present and the consensus sequence of the repeats was completely matched with that of human GPV. The thrombin-cleavage site was also conserved exactly at the same position. In Northern blot, murine GPV mRNA was specifically expressed in murine platelets, bone marrow cells and megakaryocytic cell lines. In the survey of other organs, GPV was not expressed at all. These results demonstrate that GPV is highly conserved, thrombin-cleavable protein beyond the species, and is a specific protein in the platelet-megakaryocyte lineage.

Expression and functional characterization of the P-selectin glycoprotein ligand-1 in various cells.

We have examined the expression and function of P-selectin glycoprotein ligand-1 (PSGL-1), which is a high affinity ligand for P-selectin. Northern blot and flow cytometric analysis demonstrated that a variety of hematopoietic cells and cell lines expressed PSGL-1. However, P-selectin binding ability was dependent on the additional expression of a carbohydrate structure, sialyl Lewis x (sLex). All the peripheral lymphocytes expressed PSGL-1 and subpopulation expressed sLex. Two color analysis showed that the majority of the cells that bound P-selectin were sLex-negative I lymphocytes, and most of the sLex-positive cells were B lymphocytes that did not blind P-selectin, indicating that the carbohydrate on T lymphocytes recognized by P-selectin is not sLex, and that the sLex on B lymphocytes is not readily presented for P-selectin recognition. Transfected 293 cells detectably bound P-selectin only when the cells expressed both PSGL-1 and sLex. When cysteine 310 of PSGL-1 was mutated to alanine, P-selectin binding was markedly reduced, suggesting the importance of dimerization of PSGL-1. These findings indicate that a preferable conformation of both carbohydrate and protein structure is necessary for a functional P-selectin ligand.

Synthesis and properties of a photoaffinity labeling reagent for protoporphyrinogen oxidases, the target enzymes of diphenyl ether herbicides.

A diazoketone 3 has been synthesized in two steps from acifluorfen 1, a diphenyl ether herbicide. Like the parent compound 1, the diazoketone 3 is toxic to plant cells and inhibits protoporphyrinogen oxidase, the molecular target of diphenyl ether herbicides. On photolysis of 3 in methanol, the generated carbene mainly undergoes the Wolff rearrangement to a ketene which further adds methanol, but many other products are observed. A tritiated derivative of 3 has been prepared which is suitable for photoaffinity labeling experiments.