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PubMed1983

Assay for Hageman factor (factor XII) in human plasma by means of chromogenic substrate for plasma kallikrein.

Determination of factor XII was performed in a test system where plasma samples were diluted (1/400-1/3200) in buffer and factor XII deficient plasma. Diluted CephotestR was used as activator and after 10 min activation time chromogenic substrate H-D-Pro-Phe-Arg-pNA was added. The assay was adopted to an enzyme analyzer. The reproducibility of the method was acceptable with a coefficient of variation of 7.8% in the normal range. The correlation of this method to a one-stage clotting assay for factor XII was good, r 0.79. The preliminary reference interval was calculated to be 52-147% of normal (mean+ 2 S.D.).

Chromogenic Compounds↗
PubMed1984

Selective determination of alpha 2-plasmin inhibitor activity in plasma using chromogenic substrate.

In determination of antiplasmin activity of alpha 2-plasmin inhibitor (alpha 2 PI) using an amidolytic method, alpha 2-macroglobulin (alpha 2 M) in concentrations present in normal plasma inhibits the cleavage of chromogenic substrates by plasmin even if the reaction time between alpha 2M and plasmin is shortened as much as possible. The purpose of this study was to develop a new method to assay alpha 2 PI selectively with the chromogenic substrate S-2251 using an end-point method. The antiplasmin activity of alpha 2 M was destroyed by the addition of methylamine hydrochloride. In a case of congenital alpha 2 PI deficiency, antiplasmin activity determined with the previous method without the addition of methylamine was 20% of normal plasma, whereas the results obtained using this newly developed method was 5%. A statistically significant correlation between alpha 2 PI levels determined with this method and with a single radial immunodiffusion method was observed in plasma samples collected from 40 healthy subjects and 11 patients with disseminated intravascular coagulation (DIC). In two cases of DIC, in which markedly depleted levels of alpha 2 PI and normal concentrations of alpha 2 M were observed, antiplasmin activities measured without the addition of methylamine were higher than the ones measured immunologically, whereas the results obtained with the method described here were in accordance with the antigenicities of alpha 2 PI.

Aged↗
PubMed1984

Demonstration of the presence of plasminogen activator in human small intestine.

A cytosolic fraction of human small intestine was prepared. It contained esterase activity toward N-alpha-acetyl-lysine-methyl ester and amidolytic activities toward substrates S-2238, S-2288 and S-2251. In addition there was present a plasminogen activator activity which could cleave plasminogen to produce plasmin and the plasmin hydrolysed the same chromogenic substrates. Plasmin generation was also followed by a time-dependent hydrolysis of 125-I labeled plasminogen or monitored by fibrin-agar plate. The plasminogen activator was related to urinary urokinase immunologically. Anti-urokinase IgG cross-reacted with cytosolic fraction in double immunodiffusion. When the cytosolic fraction was electrophoresed in discontinuous polyacrylamide gel, two regions of hydrolytic activity toward the urokinase-specific substrate S-2444 were found. The activity of one of these regions could be completely inhibited by anti-urokinase while the other was not. The plasminogen activator was partially purified by ammonium sulfate precipitation and Concanavalin A-bound Sepharose chromatography.

Amides↗
PubMed1984

Thrombin-induced platelet reactions: effect of substrates and inhibitors on binding of thrombin and serotonin release.

Binding of 125I-thrombin to platelets and subsequent serotonin release were confirmed. The binding of unaltered thrombin to platelets was also measured by a new technique using a chromogenic substrate (S-2238). As compared to 125I-thrombin, this method gave similar constants for binding to the high affinity binding site, but lower for binding to the low affinity binding site. Furthermore, the results suggest that the platelets have two classes of independent binding sites. Substrates and inhibitors of thrombin inhibited thrombin induced serotonin release, suggesting that the release reaction depends on the proteolytic activity of thrombin. The serotonin release was more inhibited than the binding of thrombin, suggesting that the platelet binding site and the active site of thrombin are located in different parts of the thrombin molecule.

Alprostadil↗
PubMed1984

Properties of the clotting enzyme responsible for endotoxin-mediated Limulus coagulation.

A clotting enzyme, associated with the endotoxin-mediated activation of the cellularly based coagulation system of the American horseshoe crab (Limulus polyphemus), was considerably purified by a modification of the method employed to purify the corresponding enzyme from the Japanese horseshoe crab (Tachypleus tridentatus) (Nakamura, et al., 1982). This enzyme was inhibited by DFP, benzamidine, p-aminobenzamidine, antithrombin III, soybean trypsin inhibitor, and antipain, suggesting that it is a trypsin-type serine protease. The enzyme demonstrated amidolytic activity to Ac-Ile-Glu-Gly-Arg-pNA (S-2423) and related synthetic substrates (S-2222, S-2422, S-2337, and Boc-Leu-Gly-Arg-pNA) but not to other substrates (S-2160, S-2238, S-2251, S-2444, S-2266, and S-2302), indicating specificity similar to mammalian blood coagulation Factor Xa. These properties of the Limulus enzyme were identical with those of the corresponding Tachypleus enzyme. The structure and function of the enzymes in these two species probably have been highly conserved during the past few hundred million years of their evolution.

Animals↗
PubMed1984

Abolition by dextran sulfate of the heparin-accelerated antithrombin III/thrombin reaction.

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin. Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme. These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.

Animals↗
PubMed1984

Assay of unfractionated and LMW heparin with chromogenic substrates: twin methods with factor Xa and thrombin.

Utilizing two newly synthesized chromogenic substrates (CS), two different assay methods for heparin in plasma have been developed. The assay with bovine factor Xa and the highly reactive "Substrate FXa-1" (CH3 OCO-D-CHA-Gly-Arg-pNA X AcOH) measures both unfractionated (UF) heparin and low molecular weight (LMW) heparin within a single standard curve in the 0.05-1.5 U/ml plasma range. The very similar (and less expensive) assay with bovine thrombin and "Substrate Th-1" (2AcOH X H-D-CHG-Ala-Arg-pNA), measures UF heparin, but not LMW heparin. The standard curves are highly reproducible (CV 3.5-4.7%). For clinical work, a linear standard curve is obtained with three standards and lin-log plot. The "within run" SD was 0.007-0.026 U/ml. Mean recovery of 0.5 U/ml heparin added to 10 pathological plasma samples ranged 0.46-0.53 U/ml (SD 0.034-0.040). Activities of three UF heparin and three LMW heparin preparations are reported.

Animals↗
PubMed1984

Assay of dermatan sulfate cofactor (heparin cofactor II) activity in human plasma.

An assay measuring the thrombin inactivating effect of human plasma in the presence of dermatan sulfate (DS) is described. Test plasma, diluted 1/50, is incubated with human thrombin in the presence of DS. Remaining thrombin is determined with chromogenic substrate 2AcOH . H-D-CHG-Ala-Arg-pNA. Three dilutions of reference plasma suffice and the standard curve is linear. Antithrombin III (AT) exerts a small (3-8%) effect in the assay. When test plasma contains heparin above 0.05 U/ml, this unspecific effect of AT increases, but it may be abolished by antibodies against AT. In a normal material (n = 50), the SD of DS cofactor activity was greater (15%) than that of AT (8.7%). DS cofactor was normal in hereditary AT deficiency and in 15 patients with deep venous thrombosis. In liver cirrhosis and in DIC, both inhibitors were markedly depressed, to similar degrees (r = 0.84).

Antithrombin III Deficiency↗
PubMed1985

Action of crotalase, an enzyme with thrombin-like and kallikrein-like specificities, on tripeptide nitroanilide derivatives.

Since crotalase has thrombin-like and kallikrein-like functional and structural properties, we compared the actions of crotalase, thrombin and plasma kallikrein on 13 tripeptide nitroanilide substrates. Initial rates of hydrolysis were determined at 27 degrees C, pH 8.3, and used to construct Lineweaver-Burk plots from which Km and Vmax were determined. The ratio of kcat/Km was taken as a measure of enzymatic specificity. Crotalase yielded kcat/Km values for the following nitroanilide substrates in descending order of magnitude: H-D-NLeu-CHA-Arg, H-D-Pro-HHT-Arg, Tos-Gly-Pro-Arg, H-D-PhGly-Phe-Arg, Cbo-Glu(BuO)-Gly-Arg, H-D-But-CHA-Lys, H-D-CHG-But-Arg, H-D-NLeu-HHT-Lys, H-D-HHT-Ala-Arg, Bz-Pro-Phe-Arg, Tos-Gly-Pro-Lys, MeS-Leu-Gly-Arg, MeO-CO-CHG-Gly-Arg. This pattern of specificity correlated only roughly with those of thrombin and kallikrein.

Chromogenic Compounds↗
PubMed1986

Effects of EACA on thrombin generation as measured by the chromagen S2238.

In the presence of epsilon aminocaproic acid (EACA) thrombin generation in recalcified platelet rich plasma (PRP) was markedly stimulated, as measured by the cleavage of the synthetic substrate S2238. However, thrombin activity remaining after 30 minutes incubation was reduced when compared with control values. The residual activity was shown to be hirudin insensitive and to be associated with a species of higher molecular weight than free thrombin. These results suggested an inhibition of thrombin binding to the antithrombin, alpha 2-macroglobulin (alpha 2M). Preincubation of PRP with EACA reduced the concentration at which EACA elicited its dual effects. Similar results were obtained with the alpha 2M inhibitor, hydrazine. These experiments indicated that alpha 2M may play a more important role in regulating thrombin generation than has been previously recognized.

Aminocaproates↗
PubMed1986

A comparison of methods of removing inhibitors to the chromogenic Limulus assay in normal and jaundiced blood.

A comparative study was performed of methods of removing inhibitors to the quantitative chromogenic method of Limulus assay for endotoxin in normal and jaundiced plasma. Chloroform treatment failed to remove inhibitors in normal and jaundiced plasma and did not prevent inhibition of colorimetry by bilirubin. Perchloracetic acid (PCA) treatment decolourised jaundiced samples but gave unreliable results due to poor pH control. Boiling and dilution gave reliable results in jaundiced and normal plasma without significant loss of sensitivity and is the method of choice.

Chloroform↗
PubMed1986

Mechanism of the anticoagulant, Cerastase F-4, isolated from Cerastes cerastes (Egyptian sand viper) venom.

An anticoagulant enzyme, Cerastase F-4, from the venom of Cerastes cerastes was purified to homogeneity and was characterized (1). In the present report the mode of its fibrinogenolytic and fibrinolytic actions, and its effects on some other blood coagulation factors are described. Cerastes F-4 was shown to readily hydrolyze the alpha A chain of fibrinogen followed by the hydrolysis of the beta B chain. The gamma-chain was relatively resistant to hydrolysis. It also degrades the three chains of fibrin at different rates. The degradation products of the two substrates shown on SDS-polyacrylamide gel were quite different from those produced by plasmin, indicating different sites of cleavage by the enzyme. Using specific chromogenic substrates, Cerastase F-4 seems not to show thrombin-like, plasmin-like, kallikrein-like, antithrombin, or antiplasmin actions. Also, it does not activate prothrombin or plasminogen but degrades both of them slowly. It is concluded that the anticoagulation property of the purified enzyme, Cerastase F-4, is due to its destruction of fibrinogen.

Blood Coagulation↗