Extracellular Mediators in Atherosclerosis and Thrombosis
Lessons From Thrombin Receptor Knockout Mice
It is well appreciated that thrombin as well as other proteases can act as signaling molecules that specifically regulate cells by cleaving and activating members of a novel class of protease-activated receptors (PARs). The utility of gene knockout strategies to define and better comprehend the physiological role of specific proteins is perhaps best exemplified in the field of thrombin receptors. The development of PAR knockout mice has provided the unique opportunity to identify and characterize new members of this novel family of GPCRs, evaluate the interaction of PARs jointly expressed in common cells and tissues, and better understand the role of PARs in thrombosis, restenosis, vascular remodeling, angiogenesis, and inflammation. Presently, 4 members of the PAR family have been cloned and identified. In this review, we examine experimental evidence gleaned from PAR−/− mouse models as well as how the use of PAR−/− mice has provided insights toward understanding the physiological role of thrombin in cells of the vascular system and vascular pathology.
Series Editor: Marschall S. Brass
ATVB In Focus Extracellular Mediators in Artherosclerosis and Thrombosis
Previous Brief Reviews in this Series:
•Brasier AR, Recinos A III, Eledrisi MS. Vascular inflammation and the renin-angiotensin system. 2002;22:1257–1266.
•Moser M, Patterson C. Thrombin and vascular development: a sticky subject. 2003;23:922–930.
The mouse thrombin receptor protease activated receptor-1 (PAR-1) was first cloned and sequenced in 1991 by Vu et al,1 ultimately leading to the elucidation of a novel class of G protein–coupled receptors (GPCRs) activated by proteolysis, the protease-activated receptors (PARs). With the discovery of PAR-1, mechanisms underlying thrombin-induced platelet activation and hemostasis became a renewed area of interest. Activation of PARs in vitro has largely been investigated using PAR-activating peptides (PAR-APs) derived from the tethered ligand sequence exposed after receptor cleavage.2 However, to define the role of PAR-1 in vivo, the PAR-1 gene was disrupted in mice by 2 independent groups using homologous recombination techniques.3,4⇓ Observations of normal thrombin-induced platelet activation in PAR-1−/− mice, coupled with normal hemostasis, provided the key evidence suggesting the existence of additional thrombin-sensitive receptors, leading to the identification of PAR-3 and PAR-4. The cloning of PAR-1,1,5,6⇓⇓ PAR-3,7 and PAR-48,9⇓ as well as thrombin-insensitive PAR-210 has proven to be a key advancement in the emerging theme that protease agonists differentially activate related yet distinct receptors (Table 1). Subsequent to their molecular cloning, PAR-3−/− and PAR-4−/− as well as PAR2−/− mice were generated.9,11,12⇓⇓ Properties of individual PAR receptors as well as gross phenotypes of PAR−/− mice are summarized in Table 1. Because several excellent reviews covering the cellular effects and signaling mechanism of PARs can be found elsewhere,2,13–16⇓⇓⇓⇓ this review will focus primarily on insights provided by use of PAR knockout mice, which have been instrumental in the functional analyses of specific PARs in platelet activation, as well as revealed potential roles for PARs in vascular development and pathophysiology.
PARs in Hemostasis and Thrombosis
It is well established that thrombin is the most effective activator of platelets ex vivo, inducing shape change, degranulation, and aggregation.17 Because the PAR-1-AP SFLLRN reproduced the effects of thrombin on human platelets, it was suggested that PAR-1 was the primary receptor on platelets.18,19⇓ However, species-specific differences in response to thrombin and PAR-1-APs, along with lack of PAR-1-AP responses in rodent platelets, suggested the possibility of a separate thrombin-sensitive receptor.20,21⇓ The subsequent generation of PAR-1−/− mice provided compelling evidence for the existence of an alternative thrombin responsive receptor.3,4⇓ Surprisingly, deletion of PAR-1 had no effect on thrombin-induced platelet activation, and PAR-1−/− mice were hemostatically normal.3,4⇓ Tail bleeding times, as well as thrombin-induced platelet aggregation and ATP secretion, were virtually identical in both wild-type and PAR-1−/− mice. In contrast, PAR-1−/−–derived fibroblasts were insensitive to both thrombin and SFLLRN, indicating that PAR-1 was the primary thrombin receptor on mouse fibroblasts. Hence, the persistence of thrombin-induced platelet responses supported the presence of a second thrombin receptor on mouse platelets.
Human and mouse PAR-3 were cloned from rat mRNA using a polymerase chain reaction–based strategy with primers corresponding to conserved sequences of both PAR-1 and the closely related yet thrombin-insensitive PAR-2.7 PAR-3 expression was demonstrated in mouse spleen by Northern analysis and in mouse splenic megakaryocytes by in situ hybridization, suggesting that PAR-3 might mediate thrombin responses in mouse platelets. Accordingly, PAR-3 expression in mouse platelets was demonstrated by immunoblot and flow cytometric analyses.22 Furthermore, PAR-3–blocking antibodies partially inhibited activation of mouse platelets by thrombin, indicating that PAR-3 contributes to thrombin-induced platelet activation in mice.22 Finally, the generation of PAR-3–deficient mice confirmed that PAR-3 is a thrombin receptor on murine platelets.9 PAR-3−/− platelets demonstrated a marked reduction in their response to thrombin. However, persistent thrombin responses suggested the presence of yet another thrombin-sensitive receptor in mice. A subsequent GenBank BLAST search for PAR-related sequences revealed an expressed sequence tag conserved among PAR family members, leading to the identification of PAR-4.8 Furthermore, because PAR-4-AP induced aggregation of PAR-3−/− platelets as well as SFLLRN desensitized human platelets, PAR-4 function in both mouse and human platelets was indicated.9
With the identification of human and murine PAR-4 and the subsequent generation of PAR-4−/− mice, a working model of thrombin-stimulated platelet activation has been derived.8,9,12⇓⇓ PAR-4−/−–derived platelets are completely insensitive to thrombin, indicating a requirement for PAR-4 in mouse platelets.12 In contrast, PAR-3 is necessary for responses to low concentrations of thrombin; however, because no thrombin-mediated responses are observed in PAR-4−/− mice, PAR-3 cannot mediate responses to thrombin in the absence of PAR-4. Heterologous overexpression studies have provided additional insight into the cooperative nature of PARs. Whereas overexpression of PAR-3 in COS cells fails to impart thrombin signaling, coexpression of PAR-3 and PAR-4 decreased the EC50 for thrombin by 6- to 15-fold, suggesting a cofactor role for PAR-3 in signaling by PAR-4.23 Thus, stemming from initial observations in PAR−/− mouse models, a novel paradigm of cofactor-assisted PAR activation has been forwarded. In concert with these findings, a dual-receptor paradigm has emerged in human platelets. Human platelets express PAR-1 and PAR-4.1,24⇓ The use of either PAR-specific antibodies or selective PAR-1 antagonists indicates that PAR-1 mediates responses to low concentrations of thrombin, whereas PAR-4 mediates responses to high thrombin concentrations, analogous to the differential responses observed in mouse platelets.24,25⇓ In contrast to mouse platelets, however, PAR-4 signals independently in human platelets. A schematic overview of the dual receptor signaling system in mouse and human platelets is depicted in Figure 1.
Platelet-dependent arterial thrombosis represents the underlying pathology of most heart attacks and strokes. The lack of PAR-1 in mouse platelets has confounded attempts to probe the potential role of PAR-1 in thrombosis. However, the comparable sensitivities of the dual receptor model indicate that mouse models may provide some insight into the potential effects of PAR antagonists in humans. One may predict that PAR-3−/− mice might serve as a model of PAR-1–inhibited human platelets. Accordingly, it has recently been reported that PAR-3 deficiency yields protection in 2 models of experimentally induced thrombosis.26 Arterioles from PAR-3−/− mice remained significantly more patent after ferric chloride–induced injury as well thromboplastin-induced pulmonary embolism. Indeed, in nonhuman primate models with a PAR expression profile comparable to humans, a PAR-1–selective antibody inhibited cyclic flow reductions in experimental arterial thrombosis.27 Moreover, the PAR-1–specific antagonist RWJ-58259 reduced platelet deposition in developing mural thrombi and significantly extended occlusion time in electrolytically injured carotid arteries.28 It has additionally been reported that PAR-4−/− mice are partially protected from occlusion.12 Therefore, inhibition of the primary thrombin receptor in mice and nonhuman primates is suggestive of PAR-1 inhibition in humans and supports the potential utility of a PAR-1 antagonist in the treatment of thrombosis.
Generation and use of PAR-deficient mice has been instrumental in elucidating the roles of PARs in platelet activation. The observation that PAR-1 was not expressed in mouse platelets led to the identification of a novel family of protease-activated receptors capable of mediating thrombin-dependent signaling. Furthermore, knockout technology has provided unique insights into strategies for drug development for the treatment of thrombosis. Despite differences in platelet PAR expression in human and mouse, evidence from PAR-deficient mice strongly argues that development of thrombin receptor antagonists will be beneficial in the treatment of thrombosis and that even partial PAR antagonism may have clinical significance. Finally, knockout technology has demonstrated a role for PARs beyond platelet function. Embryonic loss of PAR-1−/− mice is independent of platelet function, suggesting a role for PAR-1 in development, thus additionally expanding the putative roles of protease-activated receptors.
PARs in Vascular Development
Generation of PAR-1−/− mice led to partial embryonic lethality.3,4,29⇓⇓ Curiously, embryonic lethality was not associated with deletion of PAR-2, PAR-3, or PAR-4, suggesting a specific role for PAR-1 in development. Additional exploration of PAR-1−/− embryos has revealed fatal bleeding defects within the extracoelomic and pericardial cavities between E9.5 and E12.5.29 Reconstitution of endothelial PAR-1 in PAR-1−/− mice by using a TIE2 endothelial cell–specific promoter-enhancer rescued embryonic lethality, strongly suggesting that PAR-1 contributes to endothelial cell function in developing blood vessels.29 Additionally, thrombin has been implicated in vascular development,30,31⇓ exhibiting in vitro angiogenic activity via induction of endothelial tube formation in Matrigel as well as promotion of neovascularization in vivo.32 Furthermore, thrombin exerts indirect proangiogenic effects by upregulating vascular endothelial growth factor (VEGF) release as well as VEGF-receptor upregulation,33–35⇓⇓ consistent with a role for PARs in vascular development.
Interestingly, coagulopathy and angiogenesis are among the most consistent host responses associated with cancer.36 Because PAR-1 is directly implicated in vessel development and exogenous thrombin exerts proangiogenic activity, thrombin-induced vessel formation may be implicated in tumor neovascularization. In addition to a role in angiogenesis, PAR-1 is expressed on tumor cell lines derived from both mouse and human tissues as well as in cells in the surrounding tumor environment.37–40⇓⇓⇓ Despite accumulating evidence suggesting a role for thrombin and PAR-1 involvement in the tumor environment, angiogenesis and tumor invasion studies have not yet been reported in PAR−/− models. As such, angiogenesis and tumor formation remain tantalizing yet still-untapped frontiers to be explored.
The finding that PARs are involved in embryonic blood vessel development and stabilization may not have been possible without the initial observation of embryonic lethality in PAR-1−/− mice. That coagulation factors may initiate a cascade of angiogenic signals emphasizes the importance of thrombin and thrombin signaling in the vasculature, not only in the embryo but also in adults, where neovascularization is implicated in tumor formation, wound healing, and ischemic heart disease. Because PAR profiles are similar on mouse and human vascular endothelium, the continued exploration of PAR-deficient mice will likely yield novel insights into roles for PARs in tumor formation and heart disease.
PARs in Vasoregulation and Vascular Injury
Both endothelial cells and smooth muscle cells of the vasculature express PARs.1,41–43⇓⇓⇓ In vitro, thrombin and PAR-APs mediate endothelial-dependent relaxation of aortic and coronary blood vessels from diverse species, including rat,44 pig,45 and dog.46,47⇓ Furthermore, thrombin and PAR-APs generate strong contractile responses in endothelium-denuded vessels, consistent with constriction of underlying smooth muscle.47 These data support a role for PARs in modulation of vascular tone. The ability of endothelial cell PARs to alter vascular tone has recently been extended to humans, because thrombin and PAR-1-APs induce relaxation of contracted human pulmonary artery ring segments.42 In vivo, infusion of PAR agonists in mice results in rapid hypotension followed by a moderate, sustained hypertension.4,48⇓ Despite the ability of PAR agonists to stimulate both vasodilation and vasoconstriction in vitro and in vivo, baseline arterial pressure and heart rate were not significantly different in PAR-1−/− or PAR-2−/− mice.4,11,48⇓⇓ However, the hemodynamic responses to PAR agonists in vivo suggest putative roles for PARs in local vascular regulation after tissue damage.
In both mouse and primate models of vascular injury, PAR-1 is upregulated in the proliferating neointima, consistent with the generation of thrombin at sites of vascular injury.49,50⇓ PAR-1−/− mice are therefore an attractive model to probe the role of PARs in vascular injury, because PAR-1 deficiency does not affect mouse platelet responses and hemostasis. As reported by Cheung et al,50 PAR-1−/− mice displayed altered responses in an endothelial denudation model of vascular injury. Compared with wild-type mice, neointima formation and increases in medial area were lessened in PAR-1−/− mice. Furthermore, vessel and lumen diameter tended to increase in wild-type mice, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1−/− mice.50 Because platelets remain responsive to thrombin in PAR-1–deficient mice, the effects observed are clearly of vascular origin and support the potential efficacy of PAR-1 antagonism in vascular injury, such as restenosis. Accordingly, PAR-1 antagonism reduces vascular injury in rat models. In a balloon catheter–induced injury model, a PAR-1–specific antibody reduced neointimal smooth muscle cell accumulation and PAR-1 expression, suggesting PAR-1 is involved in the proliferation and accumulation of smooth muscle cells during vascular injury.51 Moreover, perivascular administration of the selective PAR-1 antagonist RWJ-58259 in rats produced a dose-dependent reduction in intimal area and thickness, as well as reduced percent stenosis, indicating that inhibition of thrombin-induced activation of PAR-1 in vivo can reduce vascular injury responses.52
The use of PAR-deficient mice has allowed the assessment of PARs in vascular responses independent of platelet activation and suggests that inhibition of PAR-1–mediated smooth muscle proliferation may be an effective strategy in reducing vascular injury in restenosis. This inference was confirmed in studies using a PAR-1–specific antagonist in a rat balloon injury model. Continued analysis of PAR-1−/− mice, perhaps in diet-induced or genetically induced atherosclerosis models, may prove additional roles of PARs in vascular injury. Because atherosclerosis bears many features of chronic inflammatory responses, PAR activation within plaques may also extend to the ensuing inflammatory processes.
PARs in Inflammation
PAR activation in vivo induces the classical hallmarks of inflammation, including upregulation of proinflammatory mediators and adhesion molecules, vasodilatation, and enhanced vascular permeability, suggesting the involvement of PARs in inflammation and tissue repair.53,54⇓ Thrombin stimulates production of the proinflammatory cytokines interleukin (IL)-1, IL-6, IL-8, and monocyte chemotactic protein-1, as well as platelet-derived growth factor within vascular endothelial cells.55–58⇓⇓⇓ Additionally, adhesion molecules such as E- and P-selectin, vascular cell adhesion molecule, and intercellular adhesion molecule-1 are upregulated on endothelial cells in response to thrombin.59–62⇓⇓⇓ This expression pattern suggests PARs contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence, and extravasation.63
The role of thrombin and PARs in inflammation has been evaluated in experimental models of skin and lung microvascular permeability, as well as renal inflammation.64–66⇓⇓ Thrombin and PAR-1-APs induced increases in pulmonary artery pressure and lung wet weight in isolated lung preparations.65 The effects of thrombin and PAR-1-AP were abrogated in PAR-1−/− mice, indicating permeability and vasoconstrictor effects of PAR-1 activation in pulmonary microvessels. A functional role for PAR-1 in renal inflammation has also been demonstrated.64 Hirudin, a selective inhibitor of thrombin, afforded protection against inflammation in a murine crescentic glomerulonephritis model. Because PAR-1−/− mice are protected to a similar degree, this indicates that receptor-mediated effects, rather than fibrin deposition and coagulant effects, are responsible for most of the thrombin contribution to renal injury in the glomerulonephritis model. PAR-1−/− mice were also protected against thrombin- or agonist-induced inflammation in a mouse paw edema model.66 In wild-type, but not PAR-1−/− mice, TFLLRN stimulated Evans blue extravasation in bladder, esophagus, stomach, intestine, and pancreas, additionally implicating a role for PAR-1 in inflammation. Interestingly, plasma extravasation occurs through a neurogenic mechanism involving PAR-mediated release of substance-P from sensory neurons.66,67⇓ These data provide compelling evidence that PAR-1 mediates thrombin-induced vascular permeability and subsequent edema, suggesting that PAR-1 antagonism may provide a new therapeutic modality in treatment of inflammatory diseases.
The PAR-1–dependent change in vascular permeability indicates that endothelial PAR-1 is the primary PAR responsible for this functional response. To date, no studies have been performed in PAR-1–deficient mice to assess the cellular events associated with the inflammatory response, ie, leukocyte infiltration. However, Vergnolle et al68 describe studies in a rat model using intravital microscopy to assess the ability of leukocytes to roll along and adhere to the vasculature. Surprisingly, these studies indicate that PAR-4, not PAR-1, is the primary PAR involved in leukocyte trafficking. By immunolabeling analysis, PAR-4 is expressed on rat leukocytes as well as endothelium; furthermore, the PAR-4-AP AYPGKF, but not the PAR-1–selective peptide, mimicked the actions of thrombin.68 Moreover, a PAR-1–selective antagonist, RWJ-56110, failed to block thrombin’s actions in this model. We await similar studies in PAR-1–deficient and PAR-4–deficient mice to fully understand the implications of these results in rodents and the potential relevance to inflammation in humans.
Because PAR-2 is also expressed on vascular endothelium as well as on leukocytes, its role in inflammation has received widespread attention. Studies in PAR-2−/− mice support a role for PAR-2 in early stages of inflammation, displaying increased leukocyte rolling velocity coupled with decreased adherence to cremasteric venules.69 Previous in vitro studies have demonstrated the upregulation of PAR-2 but not PAR-1 on vascular endothelial cells.70 Recently, IL-1–induced upregulation of both PAR-2 and PAR-4 in human coronary artery tissue has been reported, thus implicating PAR-4 in human inflammatory responses.71
Results thus far raise issues concerning species specificity and complement of PARs on particular cell types. With the development of PAR-deficient mice and continuing studies in human cells and tissues, a better understanding of thrombin-mediated inflammation will undoubtedly emerge.
PARs in Wound Healing and Tissue Remodeling
Tissue injury initiates coagulation, inflammatory, and reparative processes involving a panoply of cell types, including platelets, fibroblasts, keratinocytes, vascular endothelium, and inflammatory cells.72,73⇓ Although these processes are critical to achieve a normal host response to injury, excessive activation may lead to exacerbation of the response. The generation of thrombin at sites of injury, coupled with its ability to stimulate these many cell types through PAR-1, suggests that this pathway may participate in tissue remodeling and repair. Indeed, exogenous thrombin and SFLLRN accelerate wound healing in rodents.74,75⇓ Moreover, thrombin-stimulated connective tissue growth factor production is absent in PAR-1–deficient fibroblasts, indicating that thrombin may play a role in extracellular matrix deposition during tissue repair.76 Because many of these cell types (ie, platelets, endothelial cells, and inflammatory cells) express multiple PARs, PAR-1–deficient mice provide an important tool to explore the role of PAR-1 in tissue injury and repair. With the inability to modulate platelet function in PAR-1–deficient mice, the major focus of studies thus far has been with the tissue fibroblast, whose contractile, proliferative, and secretory capabilities are integral to wound closure.
In an effort to characterize the role of PAR-1 in wound healing, a panel of wound healing models has been explored in PAR-1−/− mice, including excisional punch biopsy to assess time to wound closure, incisional wounding to assess tensile strength and gross histology, and an implant model to determine the potential for cell infiltration and collagen synthesis within the wound.77 No differences between wild-type and PAR-1–deficient mice were observed among the parameters explored in these studies. The lack of effect on tensile strength, cellularity, and hydroxyproline content suggested that the fibroblast-dependent responses were not contingent on PAR-1. Because many of thrombin’s effects on fibroblasts are mediated indirectly through defined growth factors, be it from platelets or other cell types, it is not surprising that fibroblast-dependent responses are not significantly altered in these models of skin tissue repair. We have also explored the role of PAR-1 in a model of full excisional wounding in PAR-1–deficient mice. In PAR-1−/− mice, significant reductions in wound contraction, granulation tissue thickness, epidermal thickening, and infiltration of leukocytes were observed (Derian et al, unpublished observations, 2002). Immunohistochemical analysis revealed a reduction in the vascularity of the wounds as well as a reduction in the complement of leukocytes, suggesting PAR-1 activation plays a role in the inflammatory response to skin tissue injury. It is clear from these studies that some effects of thrombin are independent of platelet activation.
Although the expression of PAR-1 on dermal fibroblasts may not be critical to the process of normal wound healing, as would have been predicted from in vitro studies, the tissue specificity and the nature of the underlying pathology may be important in distinguishing the importance of PAR-1 in tissue repair. Studies are reportedly underway in a lung fibrosis model using PAR-1–deficient mice.78 Such studies, as well as others in additional tissues such as the heart, will likely provide critical information toward our understanding of the role of PAR-1 in wound repair and tissue fibrosis. The lack of PAR-1 on platelets has confounded the ability to fully assess the role of PAR-1 in wound repair, because platelets are a principal component in the initiation phase of healing. With the availability of PAR-3–deficient and PAR-4–deficient mice, as well as the potential development of compound PAR-KOs, essential studies can now be designed to address the overall response of thrombin-mediated processes in vivo.
Conclusions and Perspectives
The ability to manipulate the expression of specific genes in vivo has led to significant advances in our understanding of thrombin’s actions within distinct tissues, in particular the vasculature (Table 2). Perhaps most notably, generation of PAR-1−/− mice led to the identification and elucidation of a novel class of GPCRs activated by proteolysis. As such, exploration of PAR-deficient mice has provided an understanding of thrombin-mediated platelet activation that may not have been possible using standard pharmacological techniques. Furthermore, despite apparent differences between human and mouse PAR expression, gene deletion techniques have provided a convenient experimental platform to study vascular as well as extravascular diseases that will undoubtedly play a role in the development of novel therapeutics for the treatment of human pathology.
Evidence gleaned from PAR−/− mice suggests that modulating receptor/ligand pairing with receptor antagonists will have tremendous therapeutic potential in the areas of thrombosis, restenosis, and inflammation (as depicted in Figure 2). In particular, two approaches have now demonstrated the antithrombotic potential of PAR-1 antagonism in nonhuman primates, inhibition of thrombin/PAR-1 binding by PAR-1–specific antibodies and inhibition of receptor/ligand pairing with the small molecule antagonist RWJ-58259. Furthermore, because PAR-2 and PAR-4 are also expressed in a host of human tissues, particularly the vasculature, the additional utility of PAR-2–specific and PAR-4–specific antagonists remains an area primed for additional exploration. It should be noted that identifying small molecules that can effectively compete with the efficiency of tethered ligand binding is not a simple task. As such, PAR−/− mice continue to be important tools to probe the role of PARs in human disease. Additionally, the use of tools derived from PAR-deficient mice, namely primary cells derived from different tissues, vascular beds, and the tumor microenvironment, will illuminate the distinct roles of PAR subtypes within specific etiologies. Thus, despite giant steps in thrombin receptor biology afforded by KO mice, understanding the complex mechanisms of PAR activation and the cooperativity of the PAR receptors in humans remains a challenging yet potentially fruitful area for future research.
- Received December 20, 2002.
- Accepted March 21, 2003.
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