On the mechanism of thrombin-induced angiogenesis: inhibition of attachment of endothelial cells on basement membrane components

Human umbilical vein endothelial cells (HUVECs) placed on plastic plates coated with collagen type IV or laminin adhered within 60 min to an extent of about 32 and 39%, respectively. Brief exposure of HUVECs to thrombin caused a marked dose-dependent inhibition of adhesion. Thrombin at 1 IU/ml caused 50% inhibition even after 5 min of exposure of HUVECs. This effect was reversible since reincubation of thrombin-treated HUVECs with fresh growth medium for 15 min restored their ability for attachment. This short-term inhibitory effect of thrombin on the adhesion of HUVECs to extracellular matrix components was specific and depended on the activation of thrombin receptor. Hirudin abolished this effect of thrombin. Similarly, the proteolytically inactive PPACK-thrombin had no effect, but when used in combination with thrombin prevents the inhibitory effect of thrombin. In addition, the thrombin receptor agonist peptide (TRAP) mimicked the effect of thrombin on HUVEC adhesion. The transduction mechanism involved in this action of thrombin seems to be via cAMP, since forskolin or the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine restored the ability of HUVECs that had been exposed to thrombin to adhere. This novel cellular action of thrombin on endothelial cells may represent an important early event in activation of the normally quiescent endothelial cells and initiation of the angiogenic cascade.