Yoji Shibayama

Mast cell derived heparin activates the contact system: a link to kinin generation in allergic reactions

Contact activation occurs when plasma comes in contact with negatively charged man‐made surfaces but no substance that initiates contact activation in vivo has been identified. We have isolated a mast cell heparin proteoglycan (MC‐HepPG) from a Furth mouse mastocytoma‐derived cell line that is analogous to human tissue‐type mast cell HepPG. This material and other glycosaminoglycans (GAGs) were tested for their ability to accelerate the reciprocal activation of factor XII and prekallikrein and the autoactivation of factor XII. Quantitative analysis showed the MC‐HepPG to be as active as dextran sulfate on a weight basis; hog intestine heparin, dermatan sulfate. keratan polysulfate and chondroitin sulfate C were less active, other sulfated polysaccharides were essentially inactive. Incubation of MC‐HepPG in 1:4 diluted plasma resulted in complete cleavage of high molecular weight kininogen in a factor Xll‐dependent reaction. All of the MC‐HepPG dependent reactions described above were inhibited by preincubation of MC‐HepPG with heparinase I and II but not by pretreatment with heparitinase. chondroitinase ABC or the serine protease inhibitor aPMSF thus indicating that heparin proteoglycan is indeed acting as an initiating ‘surface’. We analysed the proteoglycan preparation by HPLC gel filtration. Fractions spanning a molecular weight range of > 400000–8000 were active initiators. Comparison of the chromatograms obtained before and after cleavage of GAG side chains from the protein core suggested that dissociated GAGs in the MW range 69000–17000 are the most active species rather than the complete proteoglycan. MC‐HepPG GAGs therefore represent a physiologic macromolecule with activity comparable to non‐physiological surfaces in a purified system and with the capability to induce activation of the contact system in diluted plasma. Its ability to promote kinin generation links cellular and humoral inflammatory responses in the perivasculature and provides a possible explanation for the elevated kinin levels observed after allergen exposure.

Simple method for determination of A1c-type glycated hemoglobin(s) in rats using high performance liquid chromatography.

We have developed a new chromatographic method for the determination of rat glycated hemoglobins by cation exchange high-performance liquid chromatography. The hemoglobins were eluted by a two-step gradient, and the total assay time, including re-equilibration of the column, was 27 min. Two A1c type and one pre-A1c type rat glycated hemoglobins were separated and measured. The change in major HbA1c of rats, in which diabetes was induced by streptozotocin and which were subsequently treated with insulin, was monitored. In diabetic rats (n = 10, average blood glucose greater than 300 mg/dL), major HbA1c rose to 3.39 +/- 0.06% compared with controls (n = 10, 1.20 +/- 0.03%) during 5 wk. Insulin treatment decreased the HbA1c from 3.48 +/- 0.16% to 2.74 +/- 0.15% (p less than 0.01) in 6 wk. This method was also effective for the determination of mouse HbA1c.

Effect of Neurotropin® on the Binding of High Molecular Weight Kininogen and Hageman Factor to Human Umbilical Vein Endothelial Cells and the Autoactivation of Bound Hageman Factor

Bradykinin is generated by activation of the plasma kallikrein-kinin (K-K) cascade and contributes to the symptoms of allergic reactions and the perception of pain. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus, which is widely used clinically in Japan as an effective agent for these disorders. Factor XII (FXII) and high molecular weight kininogen (HK), two critical constituents of the plasma K-K cascade, bind to endothelial cells, and bound FXII is autoactivated in the presence of zinc ions. We have investigated the effects of Neurotropin on the interactions of FXII and HK with endothelial cells. Neurotropin inhibited the binding of both proteins to cultured human umbilical vein endothelial cells (HUVEC) and inhibited autoactivation of FXII upon HUVEC in a concentration-dependent manner. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and, consequently, the formation of bradykinin.

Activation of the Plasma-Kinin-Forming Cascade Along Cell Surfaces

SummaryProteins of the plasma-bradykinin-forming cascade can be assembled along the surface of endothelial cells and the cell receptors identified include gC1qR and cytokeratin 1, which interact with high-molecular-weight kininogen (HK) and Factor XII. Antisera to gC1qR and cytokeratin 1 inhibit not only binding of HK to the cell surface, but also prevent activation of normal plasma to produce bradykinin. Factor-XII-deficient or HK-deficient plasma, incubated with endothelial cells do not activate; therefore, the surface of cells may be a physiologic initiator of the plasma-bradykinin-forming cells and the process may be augmented when tissue injury occurs.ZusammenfassungPlasmaproteine der Bradykinin-Kaskade finden sich auf der Oberfläche endothelialer Zellen. Zu den identifizierten Zellrezeptoren gehören gC1qR und Zytokeratin 1, die mit dem hochmolekularen Kininogen (HK) und Faktor XII reagieren. Anti sera gegen gC1qR und Zytokeratin 1 hemmen nicht nur die Zellbindung von HK, sondern ver hindern auch die Aktivierung der Bradykininsynthese im Plasma. Plasma ohne Faktor XII oder HK aktiviert nach Inkubation mit Endothelzellen die Bradykininsynthese nicht. Somit scheint die Zelloberfläche ein physiologischer Initiator der Plasma-Bradykinin-bildenden Zellen zu sein und der Vorgang könnte durch Gewebsverletzungen verstärkt werden.