Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.
The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.
Heparin-induced thrombocytopenia (HIT), an adverse drug effect, has gained much attention. Affected patients have a high risk of new thrombotic complications. In addition, HIT is also a model to study mechanisms of immunemediated disorders. Platelet factor 4 (PF4) is the key protein involved. It is the basis for many diagnostic tests for HIT and is used for in vitro studies and in mouse models on the pathogenesis of HIT. Purified PF4 is known to easily form aggregates, which can cause artifacts in experiments. The impact of storage buffer, storage period, lyophilization, and temperature on the size of PF4 and PF4/heparin (H) complexes was assessed by dynamic light scattering (DLS), while enzyme immunoassay (EIA) was used to test the binding of anti-PF4/H antibodies (aPF4/H Abs) to PF4/H complexes. PF4 size was more stable in Hank's balanced salt solution (HBSS) compared to phosphate-buffered saline (PBS), especially during storage. Lyophilization further facilitated the formation of PF4 aggregates, while incubation of reconstituted lyophilized PF4 in PBS at 37 °C reduced PF4 aggregates. Complexes formed between lyophilized PF4 and heparin were larger, and they enhanced the binding of aPF4/H Abs in EIA compared to complexes between nonlyophilized PF4 and heparin, both in HBSS and PBS, possibly influencing in vitro test results. Our results may be helpful for mechanistic studies on the biological function of PF4 and for the improvement of assays for detecting aPF4/H Abs.
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