Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets

Abstract

The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of angiogenesis, such as endostatin and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin and VEGF from human platelets. Aggregation and endostatin release could be elicited by a specific PAR4 agonist (AYPGKF-NH(2)). The PAR4 agonist concentration dependently suppressed VEGF release. A selective PAR1 agonist (TFLLR-NH(2)) induced platelet aggregation and VEGF release but suppressed endostatin release. Thrombin did not affect endostatin or VEGF release. However, in the presence of a selective PAR1 antagonist (SCH79797), thrombin stimulated endostatin release and suppressed VEGF release. Conversely, in the presence of a selective PAR4 antagonist (transcinnamoyl-YPGKF-NH(2)), thrombin stimulated VEGF release. In vivo, treatment of rats with established gastric ulcers with a PAR1 antagonist each day for 1 wk resulted in a significant retardation of healing. We conclude that PAR1 and PAR4 counter-regulate the release of endostatin and VEGF from platelets. These protease-activated receptors could therefore play a crucial role in regulating angiogenesis and in turn could regulate the processes of wound healing and tumor growth.

Fig. 1.

Platelet aggregation (Upper) and endostatin release (Lower) induced by a PAR4 agonist (AYPGK-NH₂) and the effects of a selective PAR4 antagonist (tcYPGKF-NH₂; 400 μM). The platelets were exposed to vehicle (filled bars) or the PAR4 antagonist (hatched bars) for 5 min before exposure to the PAR4 agonist. *, P < 0.05 vs. the corresponding vehicle-treated group.

Fig. 2.

Release of VEGF and endostatin from human platelets in response to thrombin or to specific PAR1 and PAR4 agonists (TFFLR-NH₂ and AYPGK-NH₂, respectively). Each agonist was tested at concentrations producing 25%, 50%, 75%, or 100% aggregation and at a concentration 50% greater than that causing maximal aggregation (Supra). *, Significant (P < 0.05) increase in release of the growth factor as compared with the group not treated with an agonist (0). #, Significant (P < 0.05) decrease in release of growth factor as compared with the group not treated with an agonist (0).

Fig. 3.

Effects of selective PAR1 (SCH79797; 3 μM) and PAR4 (tcYPGKF-NH₂; 400 μM) antagonists on thrombin-induced release of VEGF and endostatin from human platelets. Platelets were incubated with one of the antagonists or vehicle for 5 min before stimulation with thrombin at the lowest concentration causing maximal aggregation (≈1 unit/ml). *, P < 0.05 vs. the corresponding vehicle-treated group.

Fig. 4.

Effects of selective PAR1 (SCH79797; 3 μM) and PAR4 (tcYPGKF-NH₂; 400 μM) antagonists on release of VEGF and endostatin from human platelets in response to selective PAR1 and PAR4 agonists (TFLLR-NH₂ and AYPGK-NH₂, respectively). Platelets were incubated with one of the antagonists or vehicle for 5 min before stimulation with the PAR1 or PAR4 agonist at the lowest concentration causing maximal aggregation (≈8–10 μM). *, P < 0.05 vs. the corresponding vehicle-treated group.

Fig. 5.

Gastric ulcer area before (day 3) and after 1 wk of twice-daily oral treatment with a PAR1 antagonist (SCH79797; 5 nmol in 0.5 ml of 0.9% saline) or vehicle. Although significant healing was observed in vehicle-treated rats (**, P < 0.01 vs. the day 3 group), the mean ulcer area in rats treated with the PAR1 antagonist was not significantly different from that in the rats killed at day 3.