Development of a Rapid and Sensitive Chromogenic Heparin Assay for Clinical Use

Wagenvoord, Rob; Hendrix, H; Kolde, Hans-Jürgen; Hemker, H. Cœnraad

doi:10.1159/000216849

Cited by 10 publications

(9 citation statements)

References 34 publications

(44 reference statements)

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“…Factor IXa was assayed according to the principles worked out by van Dieyen et al (8) as further elaborated by Wagenvoord et al (9) We measured the enzymatic activity on factor X activation in a system where the non-factor IXa components of the tenase complex are present in excess: factorVllla The production of factor Xa was followed on subsamples taken into EDTA buffer, containing the chromogenic substrate 5-2337, as described above. Typically 1 nM of factor IXa will cause a reaction velocity of 200 nM qf factor Xa being lbrmed per minute in this system Because never more than 2 nM of draculin was added per nM of factor IXa, the inihibitory action of draculin on factor Xa would not significantly interfere with the outcome of the factor IXa determination Additionally, FIXa amidolytic activity was tested on thiobenzyl benzylocarbonyl-LJysinate as described by Green Protein determination was done using the method described by Bradford (11).…”

Section: Factor Ixa Activitymentioning

confidence: 99%

Purification and Partial Characterization of Draculin, the Anticoagulant Factor Present in the Saliva of Vampire Bats (Desmodus rotundus)

Apitz‐Castro

Béguin²,

Tablante

et al. 1995

Thromb Haemost

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SummaryFrom the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1–4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa.When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.

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Section: Factor Ixa Activitymentioning

confidence: 99%

Purification and Partial Characterization of Draculin, the Anticoagulant Factor Present in the Saliva of Vampire Bats (Desmodus rotundus)

Apitz‐Castro

Béguin²,

Tablante

et al. 1995

Thromb Haemost

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“…The relevance of anti-Xa measurements has been questioned and in some animal studies it has been shown that anti-IIa activity is better Correspondence to: Dr Bengt I Eriksson, Ortopediska kliniken CKO, Ostra sjukhuset, S 4 1 6 85 Goteborg, Sweden -FAX Number: +46 31374092 correlated with efficacy and safety than anti-Xa activity (4). Methods measuring the thrombin generation inhibition have also gained interest as a better predictor of clinical outcome (5).…”

Section: Introductionmentioning

confidence: 99%

A Comparative Study of Three Low-molecular Weight Heparins (LMWH) and Unfractionated Heparin (UH) in Healthy Volunteers

Eriksson

Söderberg²,

Widlund³

et al. 1995

Thromb Haemost

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SummaryThe levels of anti-IIa and anti-Xa activity, as reported in laboratory and clinical studies on low molecular weight heparin (LMWH) preparations, show a high degree of variability. This variation has been proposed as correlated to the variation in incidence of postoperative deep vein thrombosis (DVT) (8-30%) in different LMWH studies on comparable populations undergoing elective hip surgery. The aim of this study was to compare the ex vivo potency of Clexane® (enoxaparin), Fragmin® (dalteparin) and Logiparin® (tinzaparin), applying the concept of bioequivalence, although unknown which activity/activities are best correlated to efficacy. Unfractionated heparin (UH) was included in the study as a reference drug.The drugs were studied with a cross-over technique in 12 healthy subjects and given subcutaneously in the doses recommended for orthopedic surgery. Blood samples were drawn each hour up to 10 h and at 12 h after administration. Anti-Xa and anti-IIa activities were measured using chromogenic substrate methodsThe anti-Xa peak activity (Cmax) and the area under the curve (AUC) were highest for Clexane® and Fragmin® and lower for Logiparin® and UH. Clexane® and Fragmin® were considered bioequivalent in anti-Xa activity. Regarding anti-IIa activity, no bioequivalence was found between the products. Fragmin® was clearly different, with Cmax and AUC approximately twice as high as the other drugs. Whether the demonstrated differences in anti-Xa and anti-II activities are of any clinical significance remains unclear and can only be established by comparative clinical studies.

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“…Others have found no direct effect of aprotinin on platelets ( Wachtfogel et al , 1993 ; Ray et al , 1997 ) and it has been postulated that inhibition of fibrinolysis prevents the plasmin‐induced degradation of platelet glycoprotein Ib receptors ( Royston, 1991; Huang et al , 1993 ; Blauhut et al , 1994 ). Some clinical studies have found no effect of aprotinin on thrombocytopenia ( Bidstrup et al , 1989 ; Blauhut et al , 1991 ; Wagenvoord et al , 1993 ) and that the reduced bleeding observed due to aprotinin use in CPB is independent of an effect on platelets ( Boldt et al , 1993 ; Orchard et al , 1993 ; Ray et al , 1997 ). However, our experiments indicate some direct effect on platelets as fibrinolysis is minimal in an in vitro system.…”

Section: Discussionmentioning

confidence: 99%

Aprotinin complements heparin bonding in an in vitro model of cardiopulmonary bypass

Bannan

Martín

1998

Br J Haematol

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Summary.The relative contribution of full-dose aprotinin, used with heparin-bonded surfaces, to contact activation during cardiopulmonary bypass was examined. In vitro Carmeda-bonded cardiopulmonary bypass circuits were perfused with whole blood anticoagulated with heparin (3·3 U/ml). Aprotinin (300 kIU/ml) was added to the circuits of one set of experiments. Samples were taken prior to perfusion and at 30, 60, 120 and 360 min. The activated coagulation time was extended in the aprotinin experiments, significantly at 30 min (P ¼ 0·003) and 120 min (P ¼ 0·001). Thrombin-antithrombin complexes and prothrombin fragment F1þ2 were both higher in the nonaprotinin experiments at 120 min (P ¼ 0·02 each) and 360 min (P ¼ 0·005 and 0·001, respectively). Plasma leucocyte elastase was raised in the non-aprotinin experiments in comparison to the aprotinin experiments at each timepoint (30 min, P ¼ 0·04; 60 min, P ¼ 0·006; 120 min, P ¼ 0·001; 360 min, P ¼ 0·0001), as was interleukin-8 at 120 min (P ¼ 0·05) and 360 min (P ¼ 0·0001). No differences were found for the platelet activation marker P-selectin. Platelet and white blood cell counts fell significantly in the non-aprotinin experiments compared with the aprotinin experiments at 360 min (P ¼ 0·05 and 0·03, respectively). It would appear that the use of aprotinin has additional haemostatic beneficial effects to those found with heparin-bonded circuits in terms of effects on contact activation and inflammation.

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Development of a Rapid and Sensitive Chromogenic Heparin Assay for Clinical Use

Cited by 10 publications

References 34 publications

Purification and Partial Characterization of Draculin, the Anticoagulant Factor Present in the Saliva of Vampire Bats (Desmodus rotundus)

Purification and Partial Characterization of Draculin, the Anticoagulant Factor Present in the Saliva of Vampire Bats (Desmodus rotundus)

A Comparative Study of Three Low-molecular Weight Heparins (LMWH) and Unfractionated Heparin (UH) in Healthy Volunteers

Aprotinin complements heparin bonding in an in vitro model of cardiopulmonary bypass

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