Active site-inhibited seven: Mechanism of action including signal transduction

Active Site-Inhibited Seven: Mechanism Including Signal Transduction

of Action

Lavs C. Petersen Vascular injury brings tissue factor (TF) into contact with blood and its natural ligands, factors VII (FVII) and Vlla (FVlla). This results in localized FVlla activity on TF-expressing cells, initiating coagulation, and nonhemostatic activities. Activation of transcription factors, expression of genes for inflammation, tissue remodeling, and wound healing follow, but these mechanisms for maintaining vascular integrity may lead to pathophysiologic states. Recombinant FVlla is converted into a catalytically inert protein by reactive site residues reacting with Phe-Phe-Argchloromethyl ketone. Active site-inhibited FVlla (ASIS) retains its affinity for TF and competes for FVlla and FVII binding to TF, blocking FVlla activity and FVII to FVlla activation. It therefore acts as an antithrombotic agent and has been shown in previous studies on animal models of sepsis to prevent organ failure associated with fibrin deposition. Mitigation of inflammatory response and prolonged survival were remarkable and additional effects of TF blockage by ASIS not observed with inhibitors of downstream coagulation factors Xa and thrombin. This suggests that FVlla/TF exerts a noncoagulopathic effect on cellular activities, attenuated by ASIS blocking FVlla-induced signaling. The precise mechanism remains elusive but blockade of TF/FVlla activity provides an attractive possibility for pharmaceutical intervention. In vitro measurements of ASIS-TF binding and FVlla/TF inhibition are described, together with investigation of the FVlla-induced signaling pathway and gene expression. Additionally, possible implications of ASIS blockage for hemostatic and nonhemostatic aspects of the pathophysiology associated with vascular stress and injury are discussed. Semin Hematol38(suppl12):39-42. Copyright 0 2001 by W.B. Saunders Company.

T

ISSUE FACTOR (TF) is the transmembrane, cellular receptor, and cofactor for factor VII (FVII)/ activated FVII (FVIIa) that functions as the primary initiator of blood coagulation. TF is constitutively expressed at extravascular sites, for example in the tunica adventitia, where it is supposed to limit hemorrhage by initiation of blood coagulation in the event of vessel injury. Binding of FVII/FVIIa is a key event for the initiation of coagulation and results in the generation of active proteolytic enzymes such as FVIIa, FIXa, FXa, and thrombin. In the past, a number of studies have emphasized that localization of activated coagulation factors to the surface of plasma membranes enhances blood coagulation and plays a pivotal role in controlling this extracellular process. However, it has become increasingly clear that cells exposed to such localized activity may also experience a series of events that traverses the plasma membrane, propagates signals to intracellular compartments, and imposes pleiotropic cellular responses with specific physiological consequences. The extent to which these responses are due to FVIIa-mediated signal transduction or to signal transduction mediated by the downstream coagulation factors, Xa and thrombin, is currently an issue of intense investigation. Comparative studies with specific coagulation inhibitors are obvious sources of information in these matters. Thus, effects obtained by blockage of FVIWTF but not by inhibition of Seminars

in Hematology,

Vol38,

downstream sent cellular

coagulation factors are likely to repreresponses directly induced by FVIIa/TF.

Blockage of TF by Active Inhibited FWIa

Site-

Before describing the effects obtained by TF-blockage by active site-inhibited FVIIa (ASIS) or tissue factor pathway inhibitor (TFPI) compared with those obtained with hirudin, heparin/antithrombin III, tick anticoagulant protein, and others, it is of interest to review current knowledge on the binding of ASIS to TF. Derivatization of FVIIa in the catalytic site with Phe-Phe-Arg-chloromethyl ketone to obtain ASIS induces a conformational change. As demonstrated by plasmon resonance,2o this change implies that ASIS binds with a higher affinity than FVIIa to TF. Binding of ASIS to TF is characterized by a relatively slow 4 X lo5 molLr . s-i) but owes its on-reaction (k,,= high affinity to TF (K,=0.6 nmolL) to its very slow off-rate. ASIS bound to TF dissociates from TF with a From the Department of Protein Biotechnology, Nova Nordisk A/S, M&v, Denmark. Address reprint requests to Lars C. Petersen, DSc, Novo Nordisk A/S, Department of Protein Biotechnology, Building G8.2.101, M&v, Denmark. Copyright 0 2001 by W.B. Saunders Company 0037-1963/01/3804-1209$35.00/O doi:10.1053/shem.2001.29510

No 4, Supp1 12 (October),

2001:

pp 39-42

39

40

Lars C. Petersen

half-life of approximately 50 minutes. The binding kinetics have important implications for the physiological effect of ASIS. In particular, the inhibitory effect depends strongly on the conditions under which ASIS encounters TF, ie, whether the encounter occurs in competition with FVII/FVIIa. The difference in binding between FVIIa and ASIS also applies to cell surface-expressed TF. It is therefore surprising that the mean effective concentration (EC50) for rVIIa measured in the presence of ASIS (1 nmol/L) is identical to the mean inhibitory concentration (I&,) for ASIS in the presence of an equimolar concentration of FVIIa when determined in a TF-dependent FXa generation assay with the same cells20 This indicates that binding to functional TF sites on the cell surface is the same for ASIS as for FVIIa and is not affected by blockage of the catalytic site. This may be explained by an inhomogeneous composition of TF sites in the membrane where the functional sites constitute a smaller fraction and a larger fraction appears to be “cryptic” in the sense that it allows for binding of FVIIa but is inactive when it comes to activation of X. This is in line with previous findings 11 showing that the appearance of the maximal functional Xa-generating activity on the cell surface upon binding of FVIIa is nearly immediate and occurs long before surface-exposed TF is saturated with FVIIa. Possible explanations for the cryptic TF include dimerization of TF1 and sequestration in specific membrane domainsI’ just as the cryptic characteristics of TF may occur as the result of a phospholipid composition unfavorable for binding of FX.9,17 Recent studies have shown that both FVIIa and ASIS, when bound to TF, can be internalized and recycled back to the cell surface as an intact protein.8J0 Experiments with human fibroblasts suggested the existence of two parallel internalization pathways. ASIS is internalized by a basal TF-dependent mechanism that also internalizes FVIIa in the absence of TFPI. However, FVIIa is also internalized as a complex with TFPI via an additional pathway involving clathrin-coated pits and the low density lipoprotein receptor-related protein (LRP) scavenger receptor in the presence of TFPI.lO The basal internalization mechanism does not involve clathrincoated pits or the LRP receptor. Further studies are needed to determine whether or not internalization/ recycling may have any implications for the inhibitory effect of ASIS under physiological conditions. Comparison

Between the Effects TFPI on TF Blockage

of ASIS

and

Although both antagonists block FVIIa/TF-mediated FX activation, the mode of action of ASIS is distinctly different from that of TFPI. This is well illustrated

when the two inhibitors are compared in a flow experiment where FVIIa and FX are passed over surface-exposed TF in the presence of either antagonist.23 Whereas ASIS causes a competitive inhibition of the TF-mediated activity from the very start of the encounter with TF, this is not the case with TFPI. Exposure of FVIIa and FX to TF in the presence of TFPI causes a burst of FXa, and the FX activation is only blocked after a specific amount of a TFPI/FXa complex has formed. A limited amount of FXa generation, which may be sufficient also for the triggering of cellular responses, is therefore required for an efficient down-regulation of FVIIa/TF. It is obvious that, after the formation of the FVIIa/TF complex, the inhibitory action of ASIS cannot proceed faster than the dissociation of FVIIa from TF, whereas dissociation of FVIIa is not required for the inhibitory action of TFPI/FXa. The implication of these differences in inhibition kinetics on the cellular response has not yet been established. The Effect of ASIS Intracellular

on FVIIa-Induced Signaling

Since ASIS blockage of FVIIa binding to TF prevents activation of FX and prothrombin, it also prevents cellular responses induced by FXa and thrombin. The question remains, however, whether ASIS prevents any cellular responses that are not prevented by inhibitors of FXa and thrombin. To answer this question we have performed a thorough comparison of FVIIa-, FXa-, and thrombin-induced signal transduction in baby hamster kidney (BHK) cells stably transfected with TF.15J9 The conclusion from these studies was that stimulation induced by the three proteases resulted in distinctly different intracellular signaling patterns. Thrombin induced a prominent CaZf release from intracellular stores, whereas FVIIa and FXa did not. In contrast, FVIIa-induced signaling resulted in a marked activation of p44/42 mitogenactivated protein kinase (MAPK) with a maximum at about 10 minutes, whereas thrombin and FXa induced small and short-lived MAPK activation. In addition to the original observations on Ca2+ release4 and MAPK activation,19 FVIIa has recently been reported to activate signaling pathways involving protein kinase C (PKC), l8 PI3K, and PKB.24 However, the intracellular response appears to vary widely with the cell type. The catalytic activity is a prerequisite for FVIIainduced signaling just as thrombin-induced signaling requires the catalytic activity of this protease. The cellular responses elicited by the catalytic activity of thrombin involve changes in cell morphology, increased expression of adhesion molecules, and release of cytokines.5 Of the four known PARS, three (PAR-l, -3, and -4) have been implicated in throm-

ASIS: Mechanism of Action

bin-induced signaling. The fourth receptor, PAR-2, is activated by trypsin, FXa, and tryptase. All four belong to a subgroup of the G-coupled receptor family. PAR-l is best known and serves as a general prototype for PARS. Studies on this receptor suggest that the PARS are activated by a mechanism in which a protease recognizes and cleaves an extracellular Nterminal exodomain of the receptor to reveal a new N-terminus, which acts as a tethered ligand. Binding of this ligand induces a conformational change of the receptor resulting in signal transduction across the membrane. Given the characteristics of FVIIa-induced signaling with its requirement for catalytic activity and membrane localization, it is obvious to assume that a PAR might be involved in FVIIa-induced signaling. It was evident though, from our experiments with BHK(TF) and other cell lines, that the intracellular signaling pattern induced by FVIIa was distinctly different from that of thrombin, FXa, and trypsin. From these data and data from desensitization experiments with agonist peptides, it was concluded that FVIIa-induced signaling was not transmitted by any of the known PARs.i5 However, results from a recent study by Camerer et al3 appear to be at variance with this conclusion since experiments with PAR-2 and TF expression in a xenopus system suggest that FVIIa may directly activate PAR-2. Further experiments are needed to resolve this apparent discrepancy and to finally establish the identity of the physiologically relevant receptor cleaved by FVIIa. ASIS efficiently blocks all FVIIa-induced signaling presumably by preventing FVIIa binding to TF, and it has been convincingly demonstrated that specific inhibitors of FXa and thrombin do not affect FVIIa signaling. This shs ws that binding to TF per se is not responsible for signaling and that it is not caused by an indirect mechanism involving generation of FXa orthrombin. Effect

of ASIS

on Gene Regulation

Recent studies have demonstrated that several genes are regulated by stimulation of TF-expressing cells with FVIIa. The pattern of genes induced by FVIIa resembles a wound-type response including upregulation of transcription factors, growth factors, proinflammatory cytokines, and proteins involved in cellular reorganization and cell migration.2,13J4,21 A number of these genes are also regulated by thrombin activation and the physiologic relevance of FVIIa signaling relative to thrombin signaling has yet to be completely understood. This also applies to the possible differentiation with respect to cellular localization and tissue distribution of FVIIa- and thrombininduced responses. ASIS compared to inhibitors of downstream effecters, Xa, and thrombin, might help

41

to clarify this picture. It is interesting to stimulation of fibroblasts with FVIIa, but FXa or thrombin, strongly enhances the derived growth factor-induced chemotaxis cells.i* Effect

of ASIS in Pharmacologic

note that not with plateletof these

Models

TF has been implicated in the pathogenesis of a number of disease states. This has prompted the widespread use of ASIS for testing in various pharmacological models. ASIS was shown to have an antithrombotic effect in vascular surgery with minimal bleeding complications, and similarly was shown to decrease neointima hypoplasia upon femoral balloon angioplasty. Furthermore, the use of ASIS not only mitigated sepsis-induced disseminated intravascular coagulation (DIG) and glomerular thrombosis but also prolonged the survival of the animals.16 Recent pharmacological studies have shown that blockage of TF either by ASIS or a TF antibody” can limit the infarct size after myocardial ischemia-reperfusion. Three separate effects of TF-blockage may account for these remarkable results. First, targeting with an inhibitor at the initiation of the coagulation cascade may account for the beneficial antithrombotic effect with minor bleeding. Second, an anti-inflammatory effect caused by inhibition of FVIIa- and/or thrombin-induced signaling may account for the protection against tissue damage in sepsis and ischemia-reperfusion. Third, attenuation of intima thickening may be caused by an inhibitory effect of ASIS on fibroblast migration.18 Conclusion In summary, the high affinity of ASIS for TF serves to competitively block the formation of the rFVIIa/TF complex and prevent subsequent FVIIa- and thrombin-induced cell signaling, thus obstructing both coagulopathic and noncoagulopathic mechanisms. Evaluation of ASIS in pharmacologic models has confirmed that TF-blockage has important implications for the treatment of a number of pathophysiological states associated with vascular stress and injury. References 1. Bach RR, Moldow CF: Mechanism of tissue factor activation on HL-60 cells. Blood 89:3270-3272, 1997 2. Camerer E, Gjernes E, Wiiger MT, et al: Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem 275:6580-6585, 2000 3. Camerer E, Huang W, Caughlin SR: Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Nat1 Acad Sci USA 97:5255-5260, 2000 4. Camerer E, Rmttingen J-A, Iversen J-G, et al: Coagulation factors VII and X induce CaLf oscillations in Madin-Darby

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canine kidney cells only when proteolytically active. J Biol Chem 271:29034-29042, 1996 D&y 0, Corvera CU, Steinhoff M, et al: Protease-activated receptors: Novel mechanisms of signaling by serine proteases. Am J Physiol274:C1429-C1452,1998 Erlich EH, Boyle EM, Labriola J, et al: Inhibition of the tissue factor-thrombin pathway limits infarct size after myocardial ischemia-reperfusion injury by reducing inflammation. Am J Path01 157:1849-1862, 2000 Golino P, Ragni M, Cirillo P, et al: Recombinant human, active site-blocked factor VIIa reduces infarct size and noreflow phenomenon in rabbits. Am J Physiol Heart Circ Physiol278:H1507-H1516, 2000 Hansen CB, Pyke C, Petersen LC, et al: Tissue factor-mediated endocytosis, recycling and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor. Blood 97:1712-1720, 2001 Hansen CB, van Deurs B, Petersen LC, et al: Discordant expression of tissue factor and its activity in polarized epitheha1 cells. Asymmetry in anionic phospholipid availability as a possible explanation. Blood 94:1657-1664, 1999 Iakhiaev AV, Pendurthi UR, VoigtJ, et al: Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor. J Biol Chem 274: 3699537003, 1999 Le DT, Rapaport 51, Rao LVM: Relations between factor VIIa binding and expression of catalytic activity on cell surfaces. J Biol Chem 267:15447-15454, 1992 Mulder AB, Smit JW, Born VJJ, et al: Association of smoothmuscle cell tissue factor with caveolae. Blood 88:1306-1313, 1996

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Pendurthi UR, Allen KE, Ezban M, et al: Factor VIIa and thrombin induce the expression of Cyrbl and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa-induced signal transduction. J Biol Chem 275:1463214641,200O Pendurthi UR, Alok D, Rao LMV: Binding of factor VIIa to tissue factor induces alterations in gene expression in human

fibroblast cells: Upregulation of poly(A) polymerase. Proc Nat1 Acad Sci USA 94:12598-12603, 1997 15. Petersen LC, Thastrup 0, Hagel G, et al: Exclusion of known protease activated receptors in factor VIIa-induced signal transduction. Thromb Haemost 83:571-576, 2000 16. Rao LVM, Ezban M: Active site-blocked activated factor VII as an effective anti-thrombotic agent: Mechanism of action. Blood Coagul Fibrinolysis ll:S135-S143, 2000 (suppl 1) 17 Rao LVM, Pendurthi UR: Tissue factor on cells. Blood Coagul Fibrinolysis 9:S27-S35, 1998 (suppl 1) 18. Siegbahn A, Johnell M, Rorsman C, et al: Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis. Blood 96:3452-3458, 2000 BB, Freskgard P, Nielsen LS, et al: Factor VIIa19. Sorensen induced p44/42 mitogen-activated protein kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain. J Biol Chem 274:21349-21354, 1999 20. Sorensen BB, Persson E, Freskgard P-O, et al: Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor. J Biol Chem 272:11863-11868, 1997 21. Taniguchi I, Kakkar AK, Tuddenham EGD, et al: Enhanced expression of urokinase receptor induced through the tissue factor-factor VIIa pathway in human pancreatic cancer. Cancer Res 58:4461-4467, 1998 22. Taylor FB, Chang ACK, Peer G, et al: Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD,,, Eschetichia coli. Blood 5:1609-1615, 1998 23. Valentin S, Reutlingsberger CPM, Nordfang 0, et al: Inhibition of factor X activation at extracellular matrix of fibroblasts during flow conditions: A comparison between tissue factor pathway inhibitor and inactive factor VIIa. Thromb Haemost 74:1478-1485, 1995 24. Versteeg HH, Hoedemaeker I, Diks SH, et al: Factor VIIa/ tissue factor-induced signaling via activation of Src-like kinase, phosphoinositol 3-kinase, and Rat. J Biol Chem 275: 28750-28756,200O