SUITABILITY OF LOW-MOLECULAR-WEIGHT HEPARIN(OID)S AND A PENTASACCHARIDE FOR AN IN-VITRO HUMAN THROMBOSIS MODEL

There is much interest in in vitro thrombosis systems using exclusivel y human materials for evaluating new drugs. We have previously develop ed such a model using a perfusion chamber in which whole blood anticoa gulated with low-molecular-weight heparin (LMWH) was circulated over t he extracellular matrix of endothelial cells that had been stimulated with phorbol myristate acetate to cause tissue factor formation. Here we studied various LMWHs and a pentasaccharide to find out which was m ost useful in an in vitro thrombosis model. We compared unfractionated heparin, two commercial LMWHs (Fragmin and Fraxiparine), one commerci al heparinoid (Orgaran), and a chemically synthesized derivative of th e natural pentasaccharide (Org 31550). Blood was anticoagulated with t he concentration of each glycosaminoglycan that prevented thrombin for mation for at least 3 hours in the test tube (Fragmin, 20 anti-Xa U/mL ; Fraxiparine, 40 anti-Xa Institute Choay U/mL; Orgaran, 15 anti-Xa U/ mL; Org 31550, 200 anti-Xa U/mL; unfractionated heparin, 5 IU/mL) and recirculated over the matrix of unstimulated cells (no tissue factor i n the matrix) and phorbol-stimulated endothelial cells (tissue factor in the matrix). Platelet adhesion, aggregate formation, and fibrin dep osition were evaluated. In perfusions over the extracellular matrix of unstimulated cells, the highest platelet adhesion rates were observed with Orgaran. Fibrin deposition was absent with unfractionated hepari n in phorbol-stimulated matrix. Increasing amounts of fibrin were obse rved with Orgaran, Fragmin, and Fraxiparine. Most fibrin was found wit h the pentasaccharide Org 31550. Almost all fibrin that had been forme d was directly deposited on the coverslip at a shear rate of 300 s(-1) whereas only 10% was deposited at 1600 s(-1). An exception was the pe ntasaccharide Org 31550. Only 12% of formed fibrin was deposited at bo th shear rates. Unlike the other glycosaminoglycans, the pentasacchari de was unable to inhibit thrombin formation in the circulating blood w hen tissue factor was present in the matrix. The low-molecular-weight heparin(oid)s differed considerably in the flow model. These differenc es were not predicted by their anticoagulant activity in the test tube . Orgaran is most suitable for studying platelet adhesion under flow c onditions in which no tissue factor is present because it does not cau se platelet clumping in the circulating blood. The pentasaccharide Org 31550 showed the weakest inhibition of thrombin generation and may be the best anticoagulant for studying thrombosis when a single-pass per fusion system is used.