Suitability of low-molecular-weight heparin(oid)s and a pentasaccharide for an in vitro human thrombosis model.

Lozano, María Luisa; Bos, Arjan A. van den; Groot, Philip G. de; Willigen, Gijsbert van; Meuleman, D.G.; Ordinas, Antonio; Sixma, Jan J.

doi:10.1161/01.atv.14.7.1215

Cited by 15 publications

(7 citation statements)

References 23 publications

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“…The other investigated aprotinin analogs 5 L I 5 , 6 L I 5 and 5 L 8 4 inhibited dot formation at plasma concentrations that are therapeutically feasible. The results are in line with the ob served inhibitory effect of tissue factor pathway inhibitor, heparin(oid)s and hirudin in similar How chamber experiments (30,31,36,43). A l though the aprotinin analogs had an high anti-factor X a and anti-factor Vlla-tissue factor activity they also significantly inhibited enzymes of the contact activation pathway.…”

Section: Effects Of 7122 5 L I5 6 L I 5 5ls4 and R-tap On In Vitrsupporting

confidence: 82%

“…A l though the aprotinin analogs had an high anti-factor X a and anti-factor Vlla-tissue factor activity they also significantly inhibited enzymes of the contact activation pathway. It is not clear to what extent this mechanism is involved since clot formation in this flow chamber set-up is mainly tissue factor dependent (29,30,36). The high doses o f apro tinin administered during extracorporeal circulation in cardiac surgery were shown to decrease the activation o f the coagulation significantly by a combined antiprotease activity towards thrombin, the factor V lla -T F complex, and the contact activation pathway (37,(45)(46)(47).…”

Section: Effects Of 7122 5 L I5 6 L I 5 5ls4 and R-tap On In Vitrmentioning

confidence: 97%

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Characterisation of a Novel Series of Aprotinin-Derived Anticoagulants

et al. 1995

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SummaryPrevious investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2,7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor Vlla-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.Thus the investigated aprotinin-derived coagulation inhibitors resulted in a series of combined coagulation inhibitors with a pharmacological behaviour, which justifies in vivo testing of their potential antithrombotic action, as reported in the accompanying paper.

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Section: Effects Of 7122 5 L I5 6 L I 5 5ls4 and R-tap On In Vitrsupporting

confidence: 82%

Section: Effects Of 7122 5 L I5 6 L I 5 5ls4 and R-tap On In Vitrmentioning

confidence: 97%

Characterisation of a Novel Series of Aprotinin-Derived Anticoagulants

et al. 1995

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“…In a previous study, our group reported that storage was associated with a decrease in the surface covered by thrombus platelets 10 . However, in that case, experimental conditions were different, as low‐molecular‐weight heparin‐anticoagulated blood was used 36 . Under these conditions, fibrin formation on perfused subendothelium is allowed, which probably interferes with platelet accretion to the aggregates.…”

Section: Discussionmentioning

confidence: 99%

Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage

et al. 1999

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Volume 39, September 1999 TRANSFUSION 951 BACKGROUND: The effect on platelets of two standard methods of platelet concentrate (PC) preparation was studied by flow cytometry. The findings were correlated with those obtained in an experimental in vitro perfusion model. STUDY DESIGN AND METHODS: PCs were prepared from whole blood by the platelet-rich plasma (PRP) or buffy coat (BC) method and placed on a flatbed platelet agitator at 22°C for up to 5 days. Platelet glycoproteins (GP)Ibα, GPIIb/IIIa, and GPIV, p-selectin and lysosomal integral membrane protein, and the binding of von Willebrand factor, fibrinogen, fibronectin, and coagulation factor Va were measured with the corresponding specific conjugated antibodies. Perfusions were carried out in an annular chamber with citrated blood depleted of platelets and white cells by filtration, to which samples from PCs were added. RESULTS: PRP-PC production provoked intense platelet activation. In contrast, in BC-derived PCs, platelet activation was milder, and only a significant increase in bound fibrinogen was seen. After 1 day of storage, differences between the methods that had been observed immediately after separation had almost disappeared. During the remaining storage period, increases in activation-dependent antigens and in procoagulant activity were measured. Of the studied platelet GPs, only GPIIb/ IIIa decreased by 25 percent in PRP-PCs. Differences in covered surface were not significant in perfusion studies performed on Day 0 and after 5 days of storage in PRP-PCs (26.8 ± 6.9 vs. 20.5 ± 5.8) or BC-PCs (23.8 ± 11 vs. 24.8 ± 10.2). CONCLUSION: Platelet activation occurred during the separation and storage of PCs prepared by both methods, and it was higher in PRP-PCs only in samples obtained immediately after preparation. Despite these changes, platelet adhesive and cohesive functions were similar in both types of PCs and remained basically unchanged after storage. ABBREVIATIONS: BC = buffy coat; FITC = fluorescein isothiocyanate; GP(s) = glycoprotein(s); LIMP = lysosomal integral membrane protein; MFI = mean fluorescence intensity; MoAb(s) = monoclonal antibody(ies); PC(s) = platelet concentrate(s); PE = phycoerythrin; PRP = platelet-rich plasma; vWF = von Willebrand factor.From the

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“…Blood was drawn from healthy volunteers by venipuncture, none of whom had taken drugs that might have interfered with platelet function. Blood was anticoagulated with citrate-phosphate-dextrose (final citrate concentration 19 mM) or with a LMWH (Dalteparin 20 IU/ml, Fragmin 1 , Kabi) (Lozano et al, 1994). Perfusion studies.…”

Section: Methodsmentioning

confidence: 99%

“…The purpose of the present study was to assess, separately, the effects of PC and APC on platelet deposition, and on fibrin formation on subendothelium exposed to human flowing blood. For this purpose, citrate and a low molecular weight heparin (LMWH) were used as anticoagulant; citrate in the perfusates prevents fibrin formation, and, at the concentrations used, LMWH allows fibrin formation on perfused subendothelium when the coagulation cascade is triggered (Zwaginga et al, 1990;Lozano et al, 1994). To examine the possible mechanisms involved in the effects on platelets during perfusions, additional flow cytometry and coagulation studies were performed on the perfusates.…”

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confidence: 99%

Activated protein C inhibits thrombus formation in a system with flowing blood

Lozano

Escolar

Schwarz³

et al. 1996

Br J Haematol

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We studied the effect of increasing concentrations of protein C (PC) and activated protein C (APC) on haemostasis in an in vitro thrombosis model. Blood from healthy donors was anticoagulated with citrate-phosphate-dextrose (final citrate concentration 19 mM) or a low molecular weight heparin (LMWH, 20 IU/ml). Enzymatically denuded rabbit aorta segments were exposed to flowing blood for 10 min in an annular perfusion chamber. PC and APC were added to the perfusate immediately prior to exposure. In citrated blood at a shear rate of 800/s, PC and APC induced a statistically significant decrease in platelet deposit at 16 micrograms/ml and 32 micrograms/ml. In perfusions performed with blood anticoagulated with LMWH, there was no effect on platelet deposition at 16 and 32 micrograms/ml either at shear rates of 300/s or 800/s. Addition of PC showed no effect on fibrin deposition at a shear rate of 300/s; in contrast, a nonstatistically significant 40% reduction was seen at a shear rate of 800/s, compared to controls. Addition of APC caused a 100% reduction in fibrin formation at 16 and 32 micrograms/ml at both shear rates studied. PC and APC inhibited platelet deposition on the exposed subendothelial surface, in a dose-dependent manner. Effects of PC and APC on platelet function might be mediated through inhibition of thrombin generation at the platelet microenvironment.

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Suitability of low-molecular-weight heparin(oid)s and a pentasaccharide for an in vitro human thrombosis model.

Abstract: There is much interest in in vitro thrombosis systems using exclusively human materials for evaluating new drugs. We have previously developed such a model using a perfusion chamber in which whole blood anticoagulated with low-molecular-weight heparin (LMWH) was circulated over

Cited by 15 publications

References 23 publications

Characterisation of a Novel Series of Aprotinin-Derived Anticoagulants

Characterisation of a Novel Series of Aprotinin-Derived Anticoagulants

Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage

Activated protein C inhibits thrombus formation in a system with flowing blood

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