Rationale of 3,3′,5,5′-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis

SummaryFactor XII (FXII) levels were determined in plasma samples from 29 normal donors, 10 patients with inherited FXII deficiency (all lupus anticoagulant [LA] negative) and 67 LA positive patients, using clotting (FXIIct), chromogenic substrate (FXIIcs) and immunochemical (FXIIag) assays. Excellent correlations were obtained in the three FXII assays with the LA negative samples and between the FXIIcs and FXIIag assays in the LA positive samples. Correlations between both the FXIIcs and FXIIag with FXIIct in the LA positive patients were poor. Of 67 LA positive samples studied, 25 (37.3%) showed lower values in the FXIIct assay; 13 (19.4%) of these patients were pseudo FXII deficient with values of FXII below the lower limit of normal.These results indicate that a diagnosis of FXII deficiency can be made inappropriately in the presence of phospholipid antibodies and that such a diagnosis should not be made by FXIIct assay alone.

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Visualization of von Willebrand Factor Multimers by Enzyme-Conjugated Secondary Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642536 ◽

1986 ◽

Vol 55 (02) ◽

pp. 276-278 ◽

Cited By ~ 26

Author(s):

F Brosstad ◽

Inge Kjønniksen ◽

B Rønning ◽

H Stormorken

Keyword(s):

Von Willebrand Factor ◽

Chromogenic Substrate ◽

Agarose Electrophoresis ◽

Secondary Antibodies ◽

Von Willebrand ◽

Willebrand Factor ◽

Beta Galactosidase

SummaryA method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with a) primary vWF antiserum, b) peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.

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Plasma Thrombin Assay using a Chromogenic Substrate in Disseminated Intravascular Coagulation due to Snake Bite Envenomation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646807 ◽

1979 ◽

Vol 41 (03) ◽

pp. 544-552 ◽

Cited By ~ 3

Author(s):

R P Herrmann ◽

P E Bailey

Keyword(s):

Disseminated Intravascular Coagulation ◽

Degradation Products ◽

Snake Bite ◽

Chromogenic Substrate ◽

Coagulation Parameters ◽

Fibrinogen Degradation Products ◽

Intravascular Coagulation

SummaryUsing the chromogenic substrate, Tos-Gly-Pro-Arg-pNA-HCL (Chromozym TH, Boehringer Mannheim) plasma thrombin was estimated in six cases of envenomation by Australian elapid snakes. All patients manifested findings chracteristic of defibrination due to envenomation by these snakes. Fibrin-fibrinogen degradation products were grossly elevated, as was plasma thrombin in all cases.Following treatment with antivenene, all abnormal coagulation parameters returned rapidly towards normal by 24 hours and plasma thrombin disappeared.

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Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646601 ◽

1989 ◽

Vol 61 (03) ◽

pp. 386-391 ◽

Cited By ~ 6

Author(s):

Guido Tans ◽

Truus Janssen-Claessen ◽

Jan Rosing

Keyword(s):

Gel Electrophoresis ◽

Activated Protein C ◽

Factor Xa ◽

Coagulation Factors ◽

Product Formation ◽

Chromogenic Substrate ◽

Nitrocellulose Membrane ◽

Activation Products ◽

Factor Xia ◽

Prothrombin Activation

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

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Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650269 ◽

1996 ◽

Vol 75 (02) ◽

pp. 326-331 ◽

Cited By ~ 7

Author(s):

Unni Haddeland ◽

Knut Sletten ◽

Anne Bennick ◽

Willem Nieuwenhuizen ◽

Frank Brosstad

Keyword(s):

Conformational Changes ◽

Gel Permeation Chromatography ◽

Tissue Type ◽

Plasminogen Activation ◽

Chromogenic Substrate ◽

Type Plasminogen Activator ◽

Gel Permeation ◽

Self Association ◽

Fibrin Monomers ◽

Stimulation Of

SummaryThe present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5° C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during selfassociation.

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Lack of Agreement of Chromogenic and Glotting Assays for Factor X and Protein C in Warfarinised Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648152 ◽

1991 ◽

Vol 65 (04) ◽

pp. 360-363 ◽

Cited By ~ 2

Author(s):

P Han ◽

K P Fung ◽

U Rahdakrishnan

Keyword(s):

Serine Proteases ◽

Protein C ◽

Warfarin Therapy ◽

Intraclass Correlation ◽

Chromogenic Substrate ◽

Factor X ◽

Laboratory Automation ◽

Quantitative Results ◽

Assay Methods

SummaryCoagulation serine proteases can be measured with either a chromogenic substrate assay or a clotting assay using deficient plasmas. It is a concern whether both assays give similar quantitative results, in particular in plasma obtained fiom patients on long term warfarin therapy. If these two assay methods were interchangeable, then the chromogenic substrate assay has the advantages of precision as well as laboratory automation. We used the intraclass correlation coefficient (r1) to assess the agreement between the two methods in measuring factor X and protein C levels in warfarinised plasma. The results indicate that the extent and pattern of agreement of the two methods for the measurement of the two variables in warfarinised plasma are poor, despite high Pearson product moment coefficients of correlation.

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The Strong Positive Correlation between Factor VII Clotting Activity Using Bovine Thromboplastin and the Activated Factor VII Level

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653792 ◽

1995 ◽

Vol 73 (03) ◽

pp. 429-434 ◽

Cited By ~ 19

Author(s):

Kazuomi Kario ◽

Takefumi Matsuo ◽

Reiko Asada ◽

Toshiyuki Sakata ◽

Hisao Kato ◽

...

Keyword(s):

Risk Factor ◽

Bovine Brain ◽

Factor Vii ◽

Rabbit Brain ◽

Strong Positive Correlation ◽

Chromogenic Substrate ◽

Moderate Correlation ◽

Positive Correlation ◽

Activated Factor Vii

SummaryWe compared factor VII clotting activity (FVIIc) assays using different thromboplastins to determine which is the most sensitive for activated FVII (FVIIa) or for FVII antigen (FVIIag). FVIIc levels were measured using thromboplastins derived from bovine brain (FVIIc Bov), human placenta (FVIIc Hum), and rabbit brain (FVIIc Rab). FVIIa levels were measured by fluorogenic assays using human soluble tissue factor (rsTF) or bovine rsTF. We also measured FVII activity by an amidolytic assay (FVIIc:am Hum) using human thromboplastin and a chromogenic substrate for thrombin. FVIIag levels were determined by ELISA. In the FVIIa assay, the reaction time obtained from using bovine rsTF was shorter than that with human rsTF, suggesting that the interaction of plasma FVIIa with bovine rsTF was stronger than with human rsTF. The plasma FVIIa levels measured using human rsTF and bovine rsTF were almost the same (r=0.947, p<0.0001). Among the three FVIIc assays, FVIIc Bov had the strongest positive correlation with the plasma FVIIa level (r=0.886, p<0.000l), but had no correlation with FVIIag. An increase of 1 ng/ml in the plasma FVIIa level yielded a 27.9% increase of FVIIc Bov. Plasma FVIIc Hum and FVIIc:am Hum showed moderate correlations with both FVIIa (r=0.520, p<0.02 and r=0.569, p<0.01, respectively) and FVIIag (r=0.438, p<0.05 and r=0.468, p<0.05, respectively). FVIIc Rab had the lowest correlation with FVIIa (r=0.367, p<0.1), but had a moderate correlation with FVIIag (r=0.436, p<0.05). After in vitro cold activation, FVIIc Bov levels increased the most and FVIIc:am levels showed the least change. These findings indicate that consideration of the thromboplastin used for assay is necessary when assessing the clinical significance of FVII activity as a cardiovascular risk factor.

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On the Reliability of the Use of Heparin Immobilized on Agarose for the Study of the Interactions among Heparin, Thrombin and Antithrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657222 ◽

1982 ◽

Vol 48 (01) ◽

pp. 084-086

Author(s):

Wayne W Fish ◽

Ingemar Björk

Keyword(s):

Experimental Approach ◽

Antithrombin Iii ◽

Chromogenic Substrate ◽

Experimental Protocol ◽

Appreciable Difference ◽

Thrombin Inhibition ◽

Highly Sensitive ◽

Inhibition Of Thrombin

SummaryThe extent of inhibition of thrombin was re-examined as a consequence of the sequence of addition of thrombin and antithrombin III to a column of heparin immobilized on agarose. With the use of pure enzyme, pure inhibitor, and a highly sensitive chromogenic substrate, no appreciable difference in the extent of thrombin inhibition was observed between the two sequences of addition. These observations, together with a demonstrated sensitivity of the method to variations in experimental protocol, challenge the conclusions reached in an earlier work (Hatton and Regoeczi, Thromb. Res. 1977; 10:645) which utilized this experimental approach but which employed larger quantities of reactants and a less sensitive substrate.

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Factor X Activation by washed human platelets

10.1055/s-0038-1665669 ◽

1979 ◽

Author(s):

E van Wijk ◽

L Kahlé ◽

J ten Cate

Keyword(s):

Human Factors ◽

Trypsin Inhibitor ◽

Calcium Ions ◽

Factor Xa ◽

Human Platelets ◽

Soybean Trypsin Inhibitor ◽

Thrombin Inhibitor ◽

Chromogenic Substrate ◽

Factor X ◽

Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

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A Chromogenic Factor X Assay With New Standardized Reagents

10.1055/s-0038-1665663 ◽

1979 ◽

Author(s):

P Friberger ◽

C Lenne

Keyword(s):

Human Plasma ◽

Suitable Method ◽

Chromogenic Substrate ◽

Factor X ◽

Normal Human Plasma ◽

Normal Plasma ◽

Normal Human

A recently published method for Factor X (FX) assay (1) utilizing Russel's Viper Venom (RVV) and a chromogenic substrate has been further investigated by testing a large number of parameters. This method has been considered as a suitable method for monitoring coumarol treatment (Bergström et al).The conditions for the activation of FX by purified preparations of the RVV have been studied as well as the conditions for FXa determination with a new chromogenic substrate Bz-Ile-Glu(γ-piperidyl)-Gly-Arg-pNA (S-2337). Both purified factors and normal plasma have been used. The effect of plasma inhibitors as well as the selectivity of the method has been studied.The reproducibility and stability of the different reagents and standards have been studied and found to be good.The amount of FXa activity obtained from normal human plasma has been titrated with FXa inhibitors of known purity.1) Aurell L. et al, Thromb. Res., 11, 595 (1977)2) Bergström et al, Thromb. Res., 12, 531 (1978)

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