Sol Sherry Lecture in Thrombosis

How Thrombin `Talks' to Cells Molecular Mechanisms and Roles In Vivo

From the Cardiovascular Research Institute, Departments of Medicine and Cellular and Molecular Pharmacology, University of California at San Francisco.

Correspondence to Shaun R. Coughlin, Cardiovascular Research Institute, Departments of Medicine and Cellular and Molecular Pharmacology, University of California at San Francisco, 505 Parnassus Ave, San Francisco, CA 94143-0130. E-mail shaun-coughlin{at}quickmail.ucsf.edu

Key Words: thrombin • protease-activated receptors • platelets • embryonic development • knockout

This article is a summary of the Sol Sherry Lecture of the Council on Arteriosclerosis, Thrombosis, and Vascular Biology, which was presented at the 70th Scientific Sessions of the American Heart Association in November 1997. It highlights work from our laboratory addressing the molecular mechanisms by which the coagulation protease thrombin elicits cellular responses, notes some of the novel issues that protease signaling raises, and cites recent work on the role of thrombin signaling in vivo.

Thrombin is a multifunctional serine protease. In adult animals, active thrombin is generated in the context of vascular injury when activation of the coagulation cascade triggers conversion of the circulating zymogen prothrombin to active protease. Thrombin generation may also be important in other contexts, as will be shown below.

Several of thrombin's functions involve cleavage of circulating protein substrates, eg, conversion of fibrinogen to fibrin monomer or activation of protein C. However, thrombin also has important actions on cells. It is the most potent activator of platelets.¹ It causes endothelial cells to deliver the leukocyte adhesion molecule P-selectin to their surfaces,² to secrete von Willebrand factor,² and to elaborate growth factors and cytokines.^{3 4} It is also a mitogen for fibroblasts and vascular smooth muscle cells.⁵ Such cellular actions of thrombin raised several important questions. How does thrombin, a protease, act like a hormone to control cellular behaviors? And what are the roles of thrombin-regulated cellular events in vivo?

Thrombin's actions on platelets are of particular interest. Arterial thrombosis underlies most cases of unstable angina and myocardial infarction. Studies in animal models and clinical trials suggest that these events are both platelet dependent and thrombin dependent, but the relative contributions of and interactions between thrombin-induced platelet activation and fibrin formation in acute coronary syndromes are not known. By elucidating the mechanisms whereby thrombin activates platelets, we thus hoped to uncover new signaling mechanisms, provide tools for dissecting the pathophysiology of arterial thrombosis, and possibly, reveal new targets for therapeutic development.

We utilized an expression cloning strategy to identify a thrombin receptor.⁶ This receptor, now known as protease-activated receptor 1 (PAR1), is a member of the seven transmembrane domain G protein–coupled receptor family^{6 7} but is activated by a novel mechanism.

Mechanism of PAR Activation
PAR1's amino-terminal exodomain contained the putative thrombin cleavage site LDPR/S, which resembled a known thrombin cleavage site in protein C. Carboxyl to this site was the sequence DKYEPFWEDEE, which resembled a sequence in the thrombin inhibitor hirudin known to interact with thrombin's fibrinogen-binding exosite. These observations suggested that thrombin might recognize PAR1's amino-terminal exodomain and cleave the peptide bond between receptor residues R⁴¹ and S⁴² (the Figure).⁶ Mutation of the R⁴¹/S⁴² cleavage site to an "uncleavable" R/P site rendered the receptor unactivatable by thrombin.⁶ Replacement of the thrombin cleavage recognition sequence LDPR/S with DDDDK/S, the recognition site for enteropeptidase, switched receptor specificity; cells expressing this construct responded to enteropeptidase but not to thrombin.^{8 9} Thus, mutation studies suggested that cleavage of the R⁴¹/S⁴² peptide bond was both necessary and sufficient for receptor activation by thrombin.

View larger version (66K): [in this window] [in a new window]   Figure 1. Protease-activated receptors (PARS): cleavage site and tethered-ligand sequences and mechanism of activation. A, Amino acid sequences relevant to cleavage and activation of PAR1, 2, and 3 are shown. Thrombin recognizes PAR1 via the LDPR and DKYEPF sequences and cleaves the R41/S42 peptide bond (/). This serves to "unmask" the tethered-ligand sequence SFLLRN. The importance of the "hirudin-like" sequences found in thrombin receptors PAR1 and PAR3 (underlined) for receptor recognition by thrombin is discussed in the text. PAR2, which is activated by trypsin and tryptase but not thrombin, does not have such a sequence. B, Cleavage of the R41/S42 peptide bond by thrombin (green sphere) unmasks a new tethered ligand that binds intramolecularly to the receptor to effect transmembrane signaling. The thrombin receptor can thus be viewed as a peptide receptor that carries its own ligand. This ligand remains "silent" until revealed by receptor cleavage.

Biochemical studies supported this notion. Thrombin cleaved the soluble recombinant PAR1 amino-terminal exodomain efficiently and specifically at the R⁴¹/S⁴² peptide bond.¹⁰ Cleavage of PAR1 on intact cells was demonstrated by using antibodies to the receptor's activation peptide (the fragment cleaved from the receptor by thrombin) versus antibodies to receptor domains retained after cleavage.¹¹ Mutation of the R⁴¹/S⁴² site to R/P prevented receptor cleavage in these studies. The rates of receptor cleavage and second-messenger generation were well correlated.¹¹

PAR1 recognition by thrombin indeed appears to be mediated by two short sequences within PAR1's amino-terminal exodomain. X-ray crystallographic studies of thrombin cocrystallized with receptor peptides confirmed that PAR1 residues 39 to 41 (LDPR) can "dock" in thrombin's active center and that residues 50 to 55 (DKYEPF) can bind thrombin's fibrinogen-binding exosite.¹² The receptor region containing these sequences appears to be sufficient to account for thrombin-receptor interaction. It was cleaved with similar kinetics, whether displayed on the cell surface in its normal context in PAR1 or on an irrelevant membrane "tether."¹⁰ The importance of the DKYEPF interaction with thrombin has been demonstrated in functional studies with mutant receptors and in biochemical studies with receptor-based peptides.^{8 13 14} Mutation studies identified receptor residues Y52, E53, and F55 as key for interaction with thrombin and suggested that they might dock with thrombin's anion-binding exosite in a manner similar to residues F56, E57, and I59 of the leech anticoagulant hirudin^{8 15} (the Figure). This analogy was supported in the x-ray crystallographic studies mentioned above.¹² Occupancy of thrombin's fibrinogen-binding exosite by the DKYEPF sequence was associated with a conformational change in thrombin's active center,^{12 13} and the presence of the DKYEPF sequence in model peptide substrates was associated with both a lower K_m and higher k_cat.^{8 10} Studies with mutant receptors suggest that the binding of the DKYEPF sequence to thrombin may induce a conformational change in thrombin's active center that is important for thrombin's ability to bind and cleave the LDPR/S sequence.¹⁰ Thus, thrombin and PAR1 have evolved a rather intimate and cooperative protease-substrate relationship to enhance the efficiency and specificity of PAR1 cleavage at the R⁴¹/S⁴² peptide bond.

How might proteolysis within PAR1's amino-terminal extension cause transmembrane signaling? The synthetic peptide SFLLRN, which mimics the new amino terminus created when thrombin cleaves PAR1 (S⁴²FLLRN⁴⁷), was a PAR1 agonist and bypassed the requirement for receptor proteolysis.^{6 16 17} This key observation suggested two possible models.^{6 18} The first was the "tethered-ligand" hypothesis. In this model, the new amino-terminus SFLLRN was unmasked by receptor proteolysis functions as a tethered peptide agonist, docking intramolecularly with the body of the receptor to effect signaling (the Figure). The second was the "release-from-inhibition" hypothesis. In this model, PAR1 is tonically constrained in an off state by the amino-terminal exodomain, and receptor cleavage or competition by exogenous peptide releases the receptor from this tonic inhibition. This second model was refuted by the observation that a mutant PAR1 lacking an amino-terminal exodomain was not constitutively active, as would be predicted by the release-from-inhibition hypothesis. Moreover, this deletion mutant responded to the SFLLRN synthetic peptide like the wild-type receptor, consistent with the tethered ligand hypothesis. Other experiments confirmed that intramolecular as opposed to intermolecular ligand binding is the predominant mode of thrombin receptor activation.¹⁸

The thrombin receptor can thus be viewed as a peptide receptor that contains its own agonist. This "agonist-peptide" or tethered-ligand domain is kept "silent" in the naive receptor, to be unveiled only by receptor cleavage. How is this accomplished? Structure-activity studies with synthetic peptides representing the tethered-ligand domain revealed that adding residues at the amino terminus of the SFLLRN peptide or removing its N-terminal protonated amino group ablated its agonist activity.^{16 17 19} Cleavage of the R⁴¹/S⁴² peptide bond in PAR1 would both remove sequence amino terminal to S42 and create the critical protonated amino group at the tethered ligand's amino terminus. These actions presumably constitute the proteolytic "switch" that allows the tethered ligand to express activity.

Where within PAR1 does the tethered ligand dock? Available data suggest that PAR1's tethered ligand is recognized at least in part by the receptor's extracellular "face," in particular a section of PAR1's second extracellular loop and a region just outside PAR1's first transmembrane domain.^{20 21} Interestingly, mutations that have caused constitutive activation of PAR1 were found in these same regions,²² consistent with the notion that alteration of these extracellular structures by ligand binding might effect transmembrane signaling. As for other G protein–coupled receptors, the details of how agonist binding effects movement of the receptor's transmembrane domains and G protein activation are unknown.

How Does a Cell Accommodate the Irreversibility of PAR1's Activation Mechanism?
Classically, G protein–coupled receptor signaling is terminated by dissociation from ligand or by phosphorylation of activated receptor by G protein–coupled receptor kinases. The phosphorylated receptor then binds arrestin, which prevents the receptor from signaling by blocking its interaction with G proteins. After their initial uncoupling, most activated G protein–coupled receptors are subsequently internalized into endosomes, where it is thought that they dissociate from their ligands, become dephosphorylated, and then return to the cell surface in a state capable of responding again to ligand.²³

The proteolytic mechanism by which PAR1's tethered ligand is unmasked and its tethered status make for an irreversible activation mechanism, in contrast to the reversible agonist binding that mediates activation of classic G protein–coupled receptors. This begs the question of how desensitization and resensitization are accomplished for an irreversibly activated receptor.

Like other activated G protein–coupled receptors, activated PAR1 becomes rapidly phosphorylated, and PAR1 phosphorylation appears to promote its uncoupling from downstream signaling pathways.^{11 24} In fibroblasts and endothelial cells, PAR1 also undergoes rapid activation-triggered internalization.^{25 26 27 28} However, unlike classic G protein–coupled receptors, which sequester and recycle, activated PAR1 is sorted predominantly to lysosomes.^{25 27 29} If PAR1 were to recycle like classic G protein–coupled receptors, would this alter its signaling behavior? Is PAR1's distinctive trafficking pattern—the sorting of activated and internalized PAR1 to lysosomes—critical for termination of PAR1 signaling? Studies to address these questions are ongoing. If disposal of PAR1 is indeed a solution to the irreversibility of its activation mechanism, this would represent an interesting connection between trafficking and signaling and would raise the possibility that naturally occurring mutations that defeat PAR1's sorting to lysosomes might result in gain of function in signaling.

The finding that activated PAR1 is internalized and degraded begs the question of how a cell maintains or regains the ability to respond to thrombin. In fibroblasts and endothelial cells, PAR1 resides both on the plasma membrane and in an intracellular compartment. Intracellular PAR1 is protected from cleavage by thrombin. PAR1 appears to cycle tonically between these two compartments, and delivery of naive PAR1 to the cell surface is correlated with the recovery of sensitivity to thrombin.^{27 29} Studies with PAR1 mutants have suggested that agonist-triggered internalization requires receptor phosphorylation, but tonic internalization does not. Determining the mechanism by which tonic internalization and recycling of naive thrombin receptors occurs and the relative importance of this pathway for maintaining sensitivity to thrombin remain a challenge.

Role of PAR1 In Vivo
Which of thrombin's known cellular actions are mediated by PAR1, and what is the importance of these actions in vivo? Toward answering these questions, a PAR1-deficient mouse was generated. This mouse strain revealed an unexpected role for PAR1 in development and provided definitive evidence for a second thrombin receptor on mouse platelets and for tissue-specific roles for distinct thrombin receptors.^{30 31}

Mouse embryos lacking PAR1 developed normally through the first 8.5 days (E8.5). By E9.0, delayed development was generally evident, and by E9.5, embryos lacking PAR1 were markedly smaller and less developed than their wild-type or heterozygous littermates. Half or more of PAR1-deficient embryos die at this time. Thus, PAR1 is important during a critical "window" between E8.5 and E9.5 of mouse embryonic development. What is the mechanism of embryonic loss in PAR1 deficiency? Organogenesis, development of the vasculature and yolk sac circulation, early hematopoiesis, and other important events occur between E8.5 and E9.5. At E9.5, PAR1 is expressed by the endocardium and endothelium, a circulating hematopoietic precursor of unknown identity, in the developing nervous system, and in mesenchymal cells. A preliminary histological examination of PAR1-deficient embryos at E9.5 revealed general developmental delay without any characteristic single abnormality. On the basis of the relatively high level of PAR1 mRNA expression in the endocardium, endothelium, and hematopoietic cells at E9.5, it is tempting to postulate a role for PAR1 in vascular development or hematopoiesis. Delayed maturation of the yolk sac circulation was noted in some PAR1-deficient embryos but was associated with general developmental delay. It is thus not possible to say whether this defect is primary or secondary. It is interesting to note that platelets are not yet present at E9.5³² and indeed, are not required for normal development throughout this period.³³ Moreover, there is no defect in hemostasis in PAR1-deficient adult mice (see below). It is thus likely that the mechanism of failed development in PAR1-deficient embryos does not involve hemostasis in the usual sense. It is also interesting to note that the factor V knockout mouse displays an embryonic phenotype similar to that of PAR1.³⁴ This is consistent with the notion that factor V and PAR1 may act in the same developmental process. Factor V is necessary for normal thrombin generation; thus, these observations raise the exciting possibility that the "coagulation cascade" is playing an important role in embryonic development that is distinct from its role in hemostasis. Understanding the cellular basis of the defective development of PAR1-deficient embryos remains an important goal.

Despite the developmental phenotype described above, a significant fraction of PAR1-deficient embryos survived to term, were born without apparent defects, and developed normally postnatally. There was no evidence of spontaneous bleeding, and tail bleeding times in these mice were indistinguishable from those of the wild type. Moreover, aggregation, secretion, and calcium mobilization to thrombin were identical in wild-type and PAR1-deficient platelets. PAR1-activating peptides did not activate mouse platelets^{30 35 36} but did activate cells transfected with mouse PAR1 cDNA.³⁰ These data strongly suggest that PAR1 plays only a minor role in mouse platelet activation and provide definitive evidence for a second thrombin receptor in mouse platelets (see below). In contrast to mouse platelets, all known thrombin responses sought were ablated in fibroblasts derived from PAR1-deficient mice—clear evidence for tissue-specific roles for distinct thrombin receptors. Examination of thrombin signaling in other cell types derived from these mice is ongoing.

What is the identity of the second thrombin receptor in mouse platelets? We recently identified a candidate designated protease-activated receptor 3 (PAR3).³⁷ PAR3 displayed the structural features of a thrombin receptor with an obvious "hirudin-like domain" for thrombin recognition. It was specifically cleaved by thrombin at a site analogous to that in PAR1 and mediated thrombin signaling when expressed heterologously in Xenopus oocytes or Cos7 cells. In situ hybridization revealed it to be highly expressed in mouse megakaryocytes, with little signal seen in other cell types. Antibodies to PAR3 inhibited mouse platelet activation by thrombin, but this inhibition was overcome at high thrombin concentrations.³⁸ PAR3 thus appears to be an important mediator of thrombin signaling in mouse platelets, but we certainly cannot exclude an additional thrombin receptor in mouse platelets. Analysis of PAR3-knockout mice will provide a definitive answer.

The apparent species differences in thrombin receptor utilization by mouse and human platelets may be summarized as follows. Human platelets express PAR1 and are activated by the PAR1 agonist SFLLRN.^{6 16 17 39 40} PAR1 antibodies inhibit human platelet activation by thrombin, but as for PAR3 in mouse platelets, this inhibition is overcome at high thrombin concentrations. Thus, PAR1 seems to be an important contributor to human platelet activation by thrombin but plays little if any role in mouse platelet activation. As discussed above, PAR3 seems to be important for mouse platelet activation; its role in humans is unknown.

In conclusion, characterization of PAR1 provided one answer to the question of how a protease can function as a hormone to activate cells. PAR1 is prototypical of a small family of PARs. These now number three: PAR1 and PAR3 as discussed above, and PAR2,⁴¹ which has not been discussed here because it is activated not by thrombin but by trypsin and tryptase.^{41 42} Time will tell how large this family becomes and the extent of its repertoire in mediating protease signaling. The irreversibility of the PARs' activation mechanism is revealing new links between receptor signaling and intracellular trafficking. Last, PARs provide an important tool for dissecting the roles of protease signaling in physiology and pathophysiology. Whether PAR antagonists will play a role in antiplatelet therapy or find more novel applications remains to be seen.

Acknowledgments

This work was supported by NIH grants HL44907, DK50267, and HL59202 and the Daiichi Research Center at UCSF (to S.R.C.). Thanks are due to my many colleagues who contributed to this work.

Received January 16, 1998; accepted February 3, 1998.

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