Identification of tissue kallikrein messenger RNA in human neutrophils.

Expression of tissue kallikrein in human neutrophils has been suggested by previous studies using enzymatic and immunochemical techniques. Secretion of this potent biological factor by neutrophils would be of marked significance in the inflammatory process. The present study utilized the polymerase chain reaction following reverse transcriptase generation of total neutrophils cDNA to demonstrate the presence of tissue kallikrein mRNA in the human neutrophils. In addition, use of sequence-specific primers demonstrated the presence of mRNA for the hGK-1 gene, but not for the hPK gene product or the gene for prostate-specific antigen. These results confirm that tissue kallikrein is present in neutrophils and may be secreted as part of the inflammatory process.

Factors in biological fluids affect gel filtration behavior of epidermal growth factor.

There have been conflicting reports on the presence of multiple forms of epidermal growth factor (EGF) in human saliva. The present study was initiated to study the gel filtration behavior of EGF in saliva and other biological fluids. In addition, studies were performed to determine if there were factors in saliva or other biological fluids that would influence the gel filtration behavior of EGF. Gel filtration studies with saliva and urine demonstrated EGF migration consistent with a molecular weight of 35 kDa with no heterogeneity observed. The addition of radiolabeled EGF to either saliva or urine resulted in an altered migration on gel filtration. These results provide evidence for a protein-peptide interaction in saliva and other biological fluids that alters the apparent physical properties of mature EGF.

Molecular diversity of tissue kallikrein in human saliva.

The present study was conducted to explore the extent of heterogeneity of tissue kallikrein in saliva using immunological analysis and to demonstrate that such heterogeneity resulted from secretory phenomena and not degradation secondary to secretion. Human mixed saliva was collected by paraffin-stimulation and centrifuged at 10,000 rpm for 10 minutes. Special collectors were used to obtain parotid and submandibular/sublingual saliva using citric acid stimulation. Western blot analysis of human mixed saliva demonstrated major immunoreactive species with molecular masses of < 20 KD, 45 KD, 60 KD, 90 KD and > 200 KD. The polyclonal antibody used for these blotting studies was monofunctional with respect to reaction with purified salivary tissue kallikrein. Only the < 29 KD and 45 KD species were active using an enzyme overlay technique. While a similar distribution of molecular weight material was observed in both parotid and submandibular saliva, the amount of immunoreactive material was markedly less in parotid secretion. In addition, the < 20 KD material was essentially absent in parotid saliva. Treatment of the saliva with thermolysin eliminated the immunoreactive band at 60 KD while treatment with various glycosidases also eliminated some heterogeneity. These results demonstrate considerable variation in tissue kallikrein expression in salivary gland secretions.

Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin.

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.

The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein.

Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.

Influence of divalent and monovalent cations on some active site properties of human factor Xa.

The effect of divalent and monovalent cations on the hydrolysis of BzIleGlu(OR)GlyArgpNA(S-2222) was compared to the rate of inactivation of factor Xa by dansyl-GluGlyArg-chloromethylketone(DERG-CK). At substrate concentrations below Km, an approximate four-fold increase in amidase activity was observed in the presence of manganese ions while a three-fold increase was observed with calcium ions. The presence of magnesium ions resulted in a two-fold increase in amidase activity. Similar increases in the rate of inactivation of factor Xa by DERG-CK were observed. Na+ ions had a marked enhancing effect of both factor Xa amidase activity and inactivation by DERG-CK. Kinetic parameters for the hydrolysis of S-2222 by factor Xa were obtained in the presence and absence of Ca++ and Na+. Vmax values increased in the presence of either Ca++ or Na+. Km values increased in the presence of Ca++ while there was a modest decrease in Km in the presence of Na+. It is suggested that the enhanced activity of factor Xa is a reflection of changes in the reactivity of active site residues.

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Treatment of calcinosis universalis with low-dose warfarin.

Patients with calcinosis universalis secondary to dermatomyositis or systemic sclerosis have increased levels of the calcium-binding amino acid, gamma-carboxyglutamic acid. The enzyme that effects gamma carboxylation of glutamic acid is warfarin-sensitive. Four patients with calcinosis universalis were treated with 1 mg per day of warfarin for 18 months in a non-blind initial study. Two patients had both decreased gamma-carboxyglutamic acid urinary concentration and decreased extra-skeletal uptake on technetium 99m-diphosphonate whole-body nuclear scanning. In a subsequent double-blind placebo study, two thirds of the patients receiving 1 mg per day of warfarin had decreases in extra-skeletal nuclear tracer uptake after 18 months, compared with none of the four patients receiving placebo. No patient had a change in clinical assessment, bleeding complication, or baseline normal prothrombin time. This low-dose warfarin regimen appears to have no demonstrable adverse effects, and these results suggest a beneficial effect on the progression of calcinosis in these rheumatic diseases.

Comparison of five techniques for the determination of protein content in mixed human saliva.

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.