Activation of FXII during haemodialysis.

This study was designed to provide information on the kinetics of FXIIa activity and C3d release during haemodialysis. Dialysis was performed twice in 9 stable patient for 240 min with the same conditions except for a change in dialysate sodium profile (using a linear sodium programme starting at 138 and ending at 148 mmol/l or vice versa). Using paired statistics there was a significant (p < 0.02) increase in both C3d release and the FXIIa activity. The sodium profiles did not alter the outcome. The increase in FXIIa activity, which is maximal at the end of dialysis, is continuous, unlike the C3d release, which is maximal within 15 min of dialysis and then levels off. Both interactions are induced by the blood membrane contact. These results indicate that the FXIIa activation is not strictly coupled to the activation of the complement system.

Coacute heparin: a new simple monotest for monitoring heparin treatment.

Anti-Factor Xa methods have been generally accepted for the monitoring of heparin treatment, mainly due to their sensitivity to LMW heparin and excellent performance on automated equipment. When such equipment is not available, as in small laboratories or on the night shift, there is a need for a simple manual method. In the present method activated Factor X and a chromogenic peptide substrate are colyophilized in plastic cuvettes under conditions that avoid reaction between the two constituents. The assay is performed accordingly: Plasma (200 microL) is diluted in 3.0 mL predispensed Tris buffer containing dextran sulfate 8000 to avoid losses of heparin due to, for example, platelet factor 4. The diluted plasma (400 microL) is added to a cuvette. The reagents are rapidly dissolved and the reactions start. The rate of Factor Xa inhibition is proportional to the heparin activity. During the incubation, the substrate is also hydrolyzed by the remaining enzyme. This combination of reactions is insensitive to changes in temperature, making it possible to perform the assay at room temperature. After 10 minutes incubation, the reaction is stopped by adding 400 microL of 5% acetic acid. The absorbance is read in a well-calibrated photometer and the results plotted on a graph available in the kit. Eleven commercially available heparins have been tested with the assay and they all fell within a narrow range, showing the relevance of using a standard plot for all heparins. The LMW heparins behaved differently and it may also be that they demand different therapeutic strategies.

Coacute heparin: a new simple monotest for monitoring heparin treatment.

Anti-factor Xa methods have been generally accepted for the monitoring of heparin treatment mainly due to their sensitivity to LMWH and excellent performance on automated equipment. When such equipment is not available, as in small laboratories or on the night shift, there is a need for a simple manual method. In the present method, activated Factor X and a chromogenic peptide substrate are colyophilized in plastic cuvettes under conditions that avoid reaction between the two constituents. The assay is performed accordingly. Plasma (200 microliters) is diluted in 3.0 ml predispensed Tris buffer containing dextran sulfate 8000 to avoid losses of heparin due to platelet factor 4, for example. The diluted plasma (400 microliters) is added to a cuvette. The reagents are rapidly dissolved and the reactions start. The rate of Factor Xa inhibition is proportional to the heparin activity. During the incubation, the substrate is also hydrolyzed by the remaining enzyme. This combination of reactions is insensitive to changes in temperature making it possible to perform the assay at room temperature. After 10 minutes incubation, the reaction is stopped by adding 400 microliters of 5% acetic acid. The absorbance is read in a well-calibrated photometer and the results plotted on a graph available in the kit. Eleven commercially available heparins have been tested with the assay and they all fell within a narrow range, showing the relevance of using a standard plot for all heparins. The LMWH behaved differently and it may also be that they demand different therapeutic strategies.

Chromogenic peptide substrate assays and their clinical applications.

Chromogenic peptide substrates were first introduced into research laboratories in the early 1970s and were quickly utilised to develop assays for the determination of enzymes, proenzymes and inhibitors of the coagulation system. These assays were gradually introduced into coagulation and clinical chemistry laboratories as laboratory tools in the diagnosis and treatment of coagulation disorders. From the knowledge of the structures of the natural substrates attacked by enzymes other than those of the coagulation system or by synthesis and random screening, substrates for enzymes of the fibrinolytic, plasma and glandular kallikrein and complement systems were produced. These allowed various research groups to develop assays for components of these systems and subsequently led to the use of these assays in studies on various clinical conditions. Substrates for activated protein C ensured that assays for this enzyme and its inhibitors could be developed and introduced into the haematological routine. With the introduction of substrates for limulus lysate not only were assays for endotoxins in clinical samples produced but the control of all disposable products and injectables for endotoxin contamination can now be effected. Initially high costs and time-consuming manual assays were a hinderence to the general acceptance of the use of chromogenic peptide substrate assays and they were only used routinely in a few specialised laboratories. With the introduction of automated and microtitre plate methods however, these assays are are now available in most hospital laboratories. Since the first chromogenic peptide substrate was described thousands of articles have been published on the use of chromogenic substrate assays to measure proenzymes, enzyme activators, enzyme cofactors and inhibitors in blood and other body fluids in normal subjects and clinical material. We have endeavoured to cover as many of these as possible in this review.

The use of a quantitative assay in endotoxin testing.

By utilizing Limulus Amebocyte Lysate (LAL) and a chromogenic peptide substrate it is now possible to determine endotoxin concentrations quantitatively down to 10 EU/L (1 pg/mL) in a two stage assay. The optimal reaction conditions found for the two stages of the method, the endotoxin reaction with LAL and the measurement of the activation with the chromogenic substrate, are briefly described. The properties of the final kit reagents have also been investigated and the results are included. Finally, the usefulness of the present method is demonstrated by results obtained from testing therapeutical products as well as clinical plasmas. Some factors which may be critical in the performance of the assay are discussed.

Studies on synthetic peptide substrates for F XII enzymes.

Screening of chromogenic peptide substrates have shown that FXIIa readily splits substrates of D-Pro-Phe-Arg-pNA (S-2302) and -Gly-Arg-pNA (e.g. S-2222) types. The latter type is preferred in a system where kallikrein is present. By using the substrate S-2222 a method for the determination of beta FXIIa inhibitors has been designed. Chromatography data show that C1-esterase inhibitor is the major inhibitor of beta FXIIa in plasma. Preliminary studies have also been performed on the assay of FXII in human plasma. The procedure to obtain a complete activation of FXII has still to be studied.

Description and evaluation of a new chromogenic substrate assay kit for the determination of prekallikrein in human plasma.

Because of an increasing interest in the determination of prekallikrein a kit was made for the determination of this plasma proenzyme. The kit consists of 1) a prekallikrein activator of the cephalin-ellagic acid type containing Factor XII and HMW-kininogen to ensure a total activation of the prekallikrein even in pathological plasmas, 2) a buffer which is optimal for both activation and substrate hydrolysis and 3) the chromogenic substrate S-2302. A control plasma is also included. This kit was evaluated by thirteen research groups as well as by ourselves. Both normal and patient plasmas were analyzed. Good correlations were obtained for prekallikrein levels in plasma samples between the kit method and two other methods (immunochemical and functional). As well as in deficiency states the prekallikrein level was low in pancreatitis (n = 20), cancer (n = 16), early pregnancy with gestosis (n = 15), cirrhosis (n = 9) and cases with thromboembolic disorders (n = 5). The prekallikrein level was high in late pregnancy (n = 4).

Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line.

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80-90 kD as determined by affinity-labelling with [3H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5-8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.

The design of a reliable endotoxin test.

An endotoxin assay utilizing Limulus lysate (LAL) and a chromogenic peptide substrate is described. The assay of picograms of a compound, present in significant quantities almost everywhere, is obviously associated with a serious risk of contamination. Measures to avoid and detect contamination have been considered. When a quantitative assay like the present one is used, the inhibitory effects of samples analyzed are obvious and also possible to quantify. Methods to avoid such inhibitory effects have been studied and the simplest procedure seems to be dilution with endotoxin-free water, in some cases also combined with heat treatment (75 degrees C for 5 min).

Determination of prekallikrein in plasma by means of a chromogenic tripeptide substrate for plasma kallikrein.

A method for plasma prekallikrein determination utilizing a chromogenic tripeptic substrate is presented. The method has a good reproducibility and can easily be automized. Several parameters have been optimized. By using mixtures of deficient plasmas and pooled normal plasma or purified factors it was proved that prekallikrein was the factor determined and that more than 10% (of normal plasma concentration) of FXII and HMW kininogen were essential for the activation of prekallikrein in our method. Further experiments showed that the method was fairly selective and was not influenced by inhibitors present in normal plasma. The later finding was attributed to the high dilution of plasma made possible by using a potent activator and a sensitive substrate.

Methods for the determination of glandular kallikrein by means of a chromogenic tripeptide substrate.

A chromogenic peptide substrate H-D-Val-Leu-Arg-pNA (S-2266) has been used for the determination of glandular kallikrein derived from pancreas, urine and saliva. The conditions used have been optimized. The methods developed are simple and shown to have good reproducibility.

Studies on the duration of local anaesthesia: a possible mechanism for the prolonging effect of dextran on the duration of infiltration anaesthesia.

The mechanism of the prolonged effect of dextran on the duration of local anaesthesia has been studied. Using radio-active mepivacaine it was found that dextran prolonged the duration of infiltration anaesthesia in guinea-pigs by delaying the absorption of the local anaesthetic agent. Experiments in vitro indicate that molecular complexes between the local anaesthetic and dextran may be formed and it is assumed that the delayed absorption might be due to the formation of such molecular complexes. This hypothesis was strengthened by experiments in which dental infiltration anaesthesias were performed in healthy volunteers.