To exclude a role for BK receptors, we tested whether the BK receptor antagonist, HOE140, affected KK-induced ERK1/2 activation. As shown in Fig. 2B, HOE140 had no effect on KK-stimulated ERK1/2 phosphorylation at a concentration sufficient to block the effect of exogenously supplied BK. FIGURE 2. Activation of ERK1/2 in primary rat aortic vascular smooth muscle cells by prekallikrein is this explanation independent of bradykinin receptors. A, serum-deprived R-VSMCs in six-well plates were treated with human plasma PK at increasing concentrations for 15 min (left … Plasma Kallikrein Activates PAR1 and PAR2 Leading to PAR-dependent ADAM Activation Plasma KK is a trypsin fold serine protease that cleaves substrates following Arg or Lys residues (41). As shown in Fig.

3A, the tethered ligand buried within the N terminus of PARs is bounded by Arg or Lys, and almost all known PAR activation is carried out by trypsin fold serine proteases. Moreover, several kallikrein-related peptidases have been shown to activate PARs with variable specificity (42,�C44), and kallikrein-related peptidase 4 was recently reported to activate PAR1 and PAR2, but not PAR4, in prostate cancer cells (21). To test whether plasma KK activates PARs, we assayed for KK-dependent internalization of GFP-tagged PAR1, PAR2, and PAR4 expressed in HEK293 cells. Activation-dependent internalization is a characteristic feature of most GPCRs (45). As shown in Fig. 3B, in vehicle-treated cells GFP-PAR1 was found primarily on the plasma membrane, with a lesser amount of GFP fluorescence, probably representing nascent GFP-tagged receptors, present in the cytosol.

Exposure to either thrombin or KK for 15 min caused a striking loss of GFP-PAR1 from the plasma membrane and accumulation within cytosolic puncta that partially colocalized with the early endosomal marker EAA1. Fig. 3C compares the ability of KK to stimulate internalization of GFP-PAR1, -PAR2, and -PAR4. GFP-PAR1 and GFP-PAR4 moved from the plasma membrane into the cytosol upon exposure to a known activator, thrombin, consistent with activation-dependent receptor internalization. GFP-PAR2 similarly internalized when exposed to trypsin, an endogenous activator of PAR2. When exposed to plasma KK, GFP-PAR1 and GFP-PAR2 but not GFP-PAR4 internalized, suggesting that plasma KK activates PARs with substrate specificity similar to that reported for kallikrein-related peptidase 4.

Fig. 3D presents these results quantitatively. FIGURE 3. Plasma kallikrein directly activates PAR1 GSK-3 and PAR2 receptors. A, schematic depicting the N-terminal proteolytic cleavage sites and internal ligand sequences of PAR1�C4. B, serum-deprived HEK293 cells expressing PAR1-GFP (P1) were treated for 15 … As shown in Fig. 4A, exposing R-VSMCs to plasma KK produced a dose-dependent increase in matrix metalloprotease activity assayed using the ADAM10/ADAM17-specific fluorogenic substrate, KPLGL-Dpa-AR-NH2.