PERTURBATION OF ENDOTHELIAL CELLS INDUCES THROMBIN FORMATION IN FLOWING BLOOD RESULTING IN PLATELET AGGREGATION ON THEIR MATRIX
Studies which try to measure the contribution of the vessel wall to thrombin formation in flowing blood and the consequences of the formed thrombin for thrombus formation can not be performed with native blood because the perfusion system itself causes thrombin formation. Therefore, we have collected blood into^a solution of low molecular weight heparin (20 U/ml, Fragmin , Kabi Vitrum) and used this blood for perfusion studies over endothelial cells (EC) and their extracellular matrix (ECM) in a flat perfusiqn chamber (J.Lab.Clin.Med. 1983, 102, 522-532).Cells (2 x 105 ) and matrices (3.2 cm2) wetje perfused with 12 ml blood for 5 min at a shear rate of 1300 s . No fibrinopeptide A (FPA) generation in the perfusate was seen after perfusion over EC, ECM or empty coverslips. Treatment of EC for 4 hours with 4-phorbol 12-myristate 13-acetate (PMA 20 µg/ml) caused a strong increase in FPA generation (50 ng/ml) which was decreased again after a 16 hour incubation (25 ng/ml). ECM of EC's treated for 4 h with PMA showed a comparable FPA generation as their EC's (40 ng/ml), however, the FPA generation increased with prolonged incubation with PMA. FPA generation was completely blocked by hirudin and with a specific antibody against FVIIa (Dr. Mertens, CLB). By incubation of the matrix with an antitissue factor serum (Dr. Bertina, AZL), FPA generation could be blocked. The effect of the locally formed thrombin on platelet adhesion to ECM of 16 h PMA treated EC was evaluated and showed that 47.1 ± 9.3 (SD)% of the surface was covered with platelets of which 22.0 ± 9.3% consisted of small (< 5 µm) and 3.1 ± 3.4% of larger (5-10 µm) aggregates. EM studies showed degranulation of platelets and fibrin formation around the aggregates. ECM of untreated EC showed a similar coverage 41.5 ± 5.5% but only 6.7% small aggregates. Anti FVIIa and anti tissue factor decreased aggregate height and number to the values found with untreated ECM; adhesion remained unchanged. These data indicate that PMA perturbation of EC induces a tissue factor dependent thrombin formation which is associated with the induction of local thrombus formation. This system may serve as an in vitro model for thrombosis.