Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

Collagenase-induced oedema in the rat paw and the kinin system.

Collagenase (100 micrograms) induced a large plasma extravasation, during the first 15 min after its injection in rat paw, associated with the rapid development of oedema which subsided after 6 h. The extent of the oedema was similar in normal and kininogen-deficient rats. The swelling induced in normal rats was reduced by HOE 140 (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), a bradykinin B2 receptor antagonist, and by three serine protease inhibitors, soybean trypsin inhibitor (SBTI), Leucaena leucocephala trypsin inhibitor 1 (LLTI-1) and Leucaena leucocephala trypsin inhibitor 2 (LLTI-2). These agents had no effect on the oedema induced in kininogen-deficient rats. The swelling was also reduced by methysergide, indomethacin, ketoprofen and methylprednisolone. It was increased by heparin, but it was not modified by mepyramine, WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]-triazolo- [4,3-a][1,4]-diazepine-2-yl]-1-(4-morpholinyl)-1-propanone) and NG-nitro-L-arginine. In vitro, collagenase did not release kinins from rat plasma or from purified T-kininogen. LLTI-1 and LLTI-2 did not inhibit collagenase activity for one of its specific substrates. Kinins are thus involved in the development of collagenase oedema in normal rats. Their generation would be indirect following changes in matrix proteins in extravascular spaces. Nevertheless, kinins are not the decisive mediators of the swelling. Serotonin, possibly released from platelets, and prostanoids participate in the inflammatory process.

Measurement of red blood cell antibodies in autoimmune hemolytic anemia.

The enzyme-linked antiglobulin test (ELAT) was employed to measure the number of IgG molecules per red blood cell (IgG/RBC) in 11 patients with autoimmune hemolytic anemia (AIHA). All patients with AIHA had high levels of red cell-associated IgG (110-3, 650 IgG/RBC). The control group consisted of normal volunteers (N = 10) and patients with hereditary spherocytosis (N = 1), beta 0-thalassemia (N = 1), immunologic thrombocytopenic purpura (N = 3) and IgG multiple myeloma (N = 4). All control individuals presented low levels of red cell IgG (less than 38 IgG/RBC) with the exception of one of four patients with myeloma who had a mildly elevated value (50 IgG/RBC). Since the multiple myeloma patients had greater than 2 g/dl IgG, the possible nonspecific uptake of IgG onto the RBCs of patients with elevated serum IgG values did not interfere with the results of ELAT. ELAT proved to be a useful method for accurate quantification of the amount of IgG specifically bound on the surface of RBC of patients with AIHA.

Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies.

An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.

Acute turpentine inflammation and kinin release in rat-paw thermic oedema.

Livers from rats at 2-3 days after s.c. injection of turpentine, when perfused, synthesized prekallikrein nearly 3 times faster than did livers from normal rats. On the other hand paw oedema, produced by heating to 46 degrees, in rats injured in this way was less marked. Likewise in such rats the amount of bradykinin release by 50 min. of coaxial perfusion of the paw was only 13.6 +/- 4.6 compared with 63.1 +/- 13.4 ng in normal rats. A possible explanation for the observed reduction in production of bradykinin may be inhibition of kallikrein due to an increased concentration of alpha 2-macroglobulin.

Quantitation of red cell-bound IgG by an enzyme-linked antiglobulin test in human immunodeficiency virus-infected persons.

Anemia, thrombocytopenia, and neutropenia have been observed in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. To investigate whether red cells (RBCs) of patients with human immunodeficiency virus infection were coated with IgG and/or complement (C3), blood samples of 239 patients were tested. The prevalence of a positive direct antiglobulin test on RBCs was 16.7 percent. By use of an enzyme-linked antiglobulin test (ELAT) to measure more accurately the number of IgG molecules per RBC in a group of 67 patients, 30 of the 67 individuals were observed to have increased numbers (mean, 155) compared to normal controls and to patients with hypergammaglobulinemia due to multiple myeloma or chronic liver disease. Hemoglobin level was correlated with the number of IgG molecules per RBC (p = 0.008), but no correlation could be demonstrated between those numbers and serum immunoglobulin (p = 0.10) or circulating immune complexes (p = 0.38). Our results with ELAT suggest that some AIDS patients may have specific binding of IgG on the surface of their RBCs, rather than nonspecific uptake; further clinical correlations are necessary to confirm these findings.