Marina Molino

Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Protease activated receptors: theme and variations

The four PAR family members are G protein coupled receptors that are normally activated by proteolytic exposure of an occult tethered ligand. Three of the family members are thrombin receptors. The fourth (PAR2) is not activated by thrombin, but can be activated by other proteases, including trypsin, tryptase and Factor Xa. This review focuses on recent information about the manner in which signaling through these receptors is initiated and terminated, including evidence for inter- as well as intramolecular modes of activation, and continuing efforts to identify additional, biologically-relevant proteases that can activate PAR family members.

Endothelial Cell Thrombin Receptors and PAR-2 TWO PROTEASE-ACTIVATED RECEPTORS LOCATED IN A SINGLE CELLULAR ENVIRONMENT

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.

Thrombin Receptors on Human Platelets INITIAL LOCALIZATION AND SUBSEQUENT REDISTRIBUTION DURING PLATELET ACTIVATION

Platelet responses to thrombin are at least partly mediated by a G-protein-coupled receptor whose NH2 terminus is a substrate for thrombin. In the present studies we have examined the location of thrombin receptors in resting platelets and followed their redistribution during platelet activation. The results reveal several new aspects of thrombin receptor biology. 1) On resting platelets, approximately two-thirds of the receptors were located in the plasma membrane. The remainder were present in the membranes of the surface connecting system. 2) When platelets were activated by ADP or a thromboxane analog, thrombin receptors that were initially in the surface connecting system were exposed on the platelet surface, increasing the number of detectable receptors by 40% and presumably making them available for subsequent activation by thrombin. 3) Platelet activation by thrombin rapidly abolished the binding of the antibodies whose epitopes are sensitive to receptor cleavage and left the platelets in a state refractory to both thrombin and the agonist peptide, SFLLRN. This was accompanied by a 60% decrease in the binding of receptor antibodies directed COOH-terminal to the cleavage site irrespective of whether the receptors were activated proteolytically by thrombin or nonproteolytically by SFLLRN. 4) The loss of antibody binding sites caused by thrombin was due in part to receptor internalization and in part to the shedding of thrombin receptors into membrane microparticles, especially under conditions in which aggregation was allowed to occur. However, at least 40% of the cleaved receptors remained on the platelet surface. 5) Lacking the ability to synthesize new receptors and lacking an intracellular reserve of preformed receptors comparable to that found in endothelial cells, platelets were unable to repopulate their surface with intact receptors following exposure to thrombin. This difference underlies the ability of endothelial cells to recover responsiveness to thrombin rapidly while platelets do not, despite the presence on both of the same receptor for thrombin.

Differential Expression of Functional Protease-Activated Receptor-2 (PAR-2) in Human Vascular Smooth Muscle Cells

The protease-activated family of G protein-coupled receptors includes PAR-1 and PAR-3, which are activated by thrombin, and PAR-2, which is activated by trypsin and tryptase. PAR-2 has recently been shown to be expressed in human endothelial cells. In the present studies, we have examined the expression of PAR-2 in other cells, particularly vascular smooth muscle, and tested whether the receptors are functional. The results show that PAR-2 is present in human aorta and coronary artery smooth muscle cells, as well as in arteries traversing the walls of the small intestine. It was also detected in human keratinocytes, sweat glands, intestinal smooth muscle, and intestinal epithelium, but not at all in myocardial smooth muscle and only inconsistently in intestinal veins and venules. Activation of aortic smooth muscle cells in culture with PAR-2 peptide agonists caused a transient increase in the cytosolic Ca2+ concentration. In contrast, PAR-2 mRNA could not be detected in saphenous vein smooth muscle cells, and the same cells placed in culture showed little, if any, response to the PAR-2 agonist peptides. These observations show that PAR-2 is widely distributed in human vascular smooth muscle, particularly in arteries. However, this is not a universal finding and at least some venous smooth muscle cells, including those in saphenous veins, apparently do not express the receptor in detectable amounts.

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI(2), endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.

Polymorphonuclear leukocytes modulate platelet function

After Giulio Bizzozero in 1882 first reported the simultaneous involvement of leukocytes and platelets in thrombus formation [1], the presence of polymorphonuclear leukocytes (PMN) in hemostatic platelet plug and arterial thrombi has been repeatedly observed by microscopy. The general consensus however was that these cells were only playing a passive role in thrombus formation but were likely involved in the subsequent repair process.