Regulation of Thrombin Receptors on Human Umbilical Vein Endothelial Cells

Abstract

Activated thrombin receptors on human umbilical vein endothelial cells rapidly undergo homologous desensitization, leaving the cells unable to respond to thrombin. The present studies examine the fate of activated thrombin receptors on endothelial cells and the mechanisms that restore intact receptors to the cell surface. The results show that: 1) at biologically relevant concentrations, thrombin rapidly cleaves all of its receptors on the cell surface. 2) The cleaved receptors are cleared from the cell surface in a two-phase process, with 60% being internalized within 10 min, the remainder requiring several hours. 3) The restoration of intact, thrombin-responsive receptors on the cell surface initially occurs from an intracellular pool of receptors in a process that is independent of protein synthesis. 4) Recycling of cleaved receptors either does not occur on endothelial cells or is masked by receptor clearance. 5) Subconfluent endothelial cells re-express intact receptors on the cell surface at a slower rate than confluent cells. 6) The agonist peptide, SFLLRN, also causes receptor internalization, although at concentrations greater than those required for receptor activation and desensitization. These results are distinctly different from those observed with megakaryoblastic cell lines, where > 90% of the activated thrombin receptors are internalized rapidly, up to 40% of the cleaved receptors are recycled, and no intracellular pool of intact receptors has been detected. Since the primary structure of the thrombin receptor is the same in all the cell types studied, these results demonstrate that there can be substantial differences between cell types in the mechanisms involved in the clearance of activated receptors and the re-expression on the cell surface of intact receptors capable of responding to thrombin.

Protease activated receptors: theme and variations

“…In contrast, endothelial cells and ®broblasts can be repeatedly exposed to thrombin and are capable of responding more than once. Their ability to do so is supported by the presence of an intracellular pool of intact receptors that does not exist in platelets (Hein et al, 1994;Woolkalis et al, 1995b;Molino et al, 1997a). In the case of endothelial cells, this reservoir of intact receptors is capable of replenishing the cell surface with intact receptors in 1 ± 2 h, even in the presence of protein synthesis inhibitors.…”

“…In the case of endothelial cells, this reservoir of intact receptors is capable of replenishing the cell surface with intact receptors in 1 ± 2 h, even in the presence of protein synthesis inhibitors. In endothelial cells, this pool is associated with an intracellular tubulovesicular network that has not been precisely de®ned (Horvat and Palade, 1995;Woolkalis et al, 1995b). In ®broblasts Hein et al (1994) localized this pool to a golgi-like domain.…”

“…As already described, PAR family members represent an exception to this rule since once cleaved, they cannot be activated a second time by the same protease. Information about the internalization and tra cking of PAR1 and, to a lesser extent, PAR2 has been obtained by several groups of investigators (BoÈ hm et al, 1996;Hoxie et al, 1993;Woolkalis et al, 1995b;Hein et al, 1994;De ry et al, 1999). In most types of cells, PAR1 and PAR2 are rapidly internalized after agonist stimulation.…”

Re‐expression of functional P‐selectin molecules on the endothelial cell surface by repeated stimulation with thrombin

“…reported to cleave essentially all of its receptors on the endothelial cell surface (Woolkalis et al, 1995).…”

Antibody-mediated lung endothelium targeting: in vivo model on primates

“…One possibility is that the ACE molecule undergoes constitutive turnover and recycling in the endothelial plasma membrane, like some other endothelial membrane proteins. 33,34 If such constitutive ACE turnover occurs, ACE could serve as a carrier for intracellular transfer of anti-ACE mAbs bound to endothelial cells. The second possibility is that ACE itself is not normally internalized, but the interaction of mAbs to ACE induced internalization of the complex.…”