Selective thrombin inhibitor (argatroban): Amelioration of platelet adhesion to human brain microvascular endothelial cells in vitro: Observation with video-enhanced contrast microscopy

We examined whether argatroban (a selective thrombin inhibitor) inhibits adhesion of activated platelets to human brain microvascular endothelial cells (HBECs) in vitro using video-enhanced contrast (VEC) microscopy. In the control group (n = 5), HBECs were cultured on a coverglass and put in the observation chamber of VEC microscopy. Platelet-rich plasma (PRP) was then superfused on the HBECs with an infusion pump at a low shear rate (10/s) for 30 min, and platelet adhesion to HBEC was examined. In the ADP group (n = 9), PRP with ADP (2 muM) was superfused for 30 min and washed out. In the argatroban group (n = 9), PRP with ADP (2 muM) plus argatroan (5 mug/ml) was superfused, and platelet adhesion to HBECs was observed. In the control group, platelet adhesion to HBECs was rarely seen. In the ADP group, platelets adhered to HBECs in all experiments, and microaggregates of platelets were seen. In the argatroban group, platelet adhesion to HBECs was clearly suppressed. The average number of platelets adhering and aggregating to HBEC was 0.3 +/- 0.6/900 mum(2) in the control group, 25.5 +/- 11.3/900 mum(2) (P < 0.01, vs. control) in the ADP group, and 1.8 +/- 1.8/900 mum(2) in the argatroban group (P < 0.01, vs. ADP). These results showed that argatroban ameliorated adhesion and pile-up of activated aggregated platelets to HBECs in a low-flow state in vitro. It suggests that thrombin produced by platelet activation makes HBECs a procoagulant and is the most likely candidate subsequently to induce platelet adhesion to HBECs.