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M. Failure of the Ipsogen MutaScreen kit to detect the JAK2 617VϾF mutation in samples with additional rare exon 14 mutations: implications for clinical testing and report of a novel 618CϾF mutation in addition to 617VϾF [letter]. Blood. 2010;115(15) To the editor: Beta 2 glycoprotein I is a substrate of thiol oxidoreductasesBeta 2 glycoprotein I (␤2GPI) is an abundant plasma protein recognized as the major autoantigen in the antiphospholipid syndrome. Although the crystal structure of ␤2GPI has been resolved, 1,2 its normal function remains unknown. We have been intrigued by the presence of a C-terminal cysteine (Cys326), which forms a loop-back disulfide link in the fifth domain of ␤2GPI. In the current study we examined ␤2GPI's potential to participate in thiol exchange reactions with the thiol oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). The incorporation of free thiols into ␤2GPI after reaction with TRX-1 or PDI was shown by labeling the products of this reaction with the selective sulfhydryl probe N a -(3-maleimidylpropionyl) biocytin (MPB). The biotinylated proteins were visualized by Western blotting with streptavidin-horseradish peroxidase. Because ␤2GPI does not contain unpaired cysteines, no labeling was observed after incubation with MPB ( Figure 1A). Free thiols could not be introduced into ␤2GPI by incubation with the reducing agent dithiothreitol (DTT) alone. However, free thiols could be introduced into ␤2GPI after incubation with the reduced forms of the thiol oxidoreductases TRX-1 and PDI, identifying ␤2GPI as a thiol oxidoreductase substrate ( Figure 1A-C). An interesting effect caused by the reduction of ␤2GPI by TRX-1 was a marked decrease in the affinity of anti-␤2GPI monoclonal and polyclonal antibodies as noted on the immunoblots.To determine the cysteine residue(s) in the ␤2GPI molecule involved in thiol exchange reactions, ␤2GPI treated with the TRX-1/TRX-1 reductase/NADPH system and labeled with MPB was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Figure 1B). Gel bands were excised, digested, and analyzed by liquid chromatography-tandem mass spectrometry. Mass spectral data were searched using Mascot (Version 2.2; Matrix Science) or converted to MzXML file format using ReAdW (Version 4.0.2) 3 and submitted to the database search program X!Tandem (Release 2008.12.01). 4 The analysis revealed Cys326 to be predominantly labeled with biotin (F.H.P., S.R., M.Q., M.J.R., J.W.H.W., K.T., Y.I., J.Y.Z., R.G., J.C.Q., B.G., W.E.H., P.J.H., S.A.K., manuscript submitted). The structural features of the disulfide bond containing Cys326 and all disulfide bonds of the 2 structures of ␤2GPI (PDB 1C1Z and 1QUB) were determined using the disulfide bond analysis tool available at www.cancerresearch.unsw.edu.au/CRCWeb. nsf/page/DisulfideϩBondϩAnalysis. 5 The analysis showed that the Cys288-Cys326 disulfide is a Ϫ/ϩ right-handed hook (Ϫ/ϩRHHook) configuration in both crystal structures of the protein. 1,2 Although there is no other structu...

Background:β2-Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods:β2GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe Na-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced β2GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin-1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin-1, in comparison with non-reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

Summary.Seventy-five patients with chronic myeloproliferative disorders were studied to investigate platelet function by simultaneous measurement of platelet aggregation by the impedance method and ATP dense granule release using a whole blood platelet lumi-aggregometer, in an attempt to identify patients at risk for thrombosis and bleeding.Thirty-nine patients had at least one abnormal result indicating platelet hyperactivity (i.e. impedance or release with one agonist being above the reference range); 16 patients had platelet hypoactivity (i.e. at least one result was below the reference range), whilst 14 had co-existence of hyper-and hypoactivity. Six patients had normal results. 20/ 53 patients with platelet hyperactivity (alone or mixed) had a positive history of venous and/or arterial thrombosis; in comparison, only two of the other 22 patients had a positive history. During a median follow-up of 33 months, nine patients with and one patient without platelet hyperactivity respectively developed new thrombotic events before the addition of specific therapy. A total of 50 patients with and eight patients without platelet hyperactivity respectively received specific treatment including aspirin and/or cytotoxic therapy. All but one elderly patient with platelet hyperactivity have remained free of new thrombotic events on specific therapy. Two of the 17 patients with platelet hypoactivity had major clinical bleeding.These observations highlight the need to test platelets for hyper-as well as hypo-function and suggest a useful role for routine whole blood platelet aggregation studies to identify the patients at risk for thrombosis or bleeding.