Biomedical subjects
Publications and source records attributed to C T Esmon.
The identification of the blood-clotting inhibitor, protein S, as a ligand for a previously 'orphan' family of receptor tyrosine kinases demonstrates a new role for clotting and anti-clotting proteins in regulating cell proliferation.
The influence of phospholipid vesicle concentration and size on the affinity and the kinetics of assembly of the prothrombin activation complex are examined. Activation of prethrombin 1 was used to monitor complex formation between factors Va and Xa. When activation rates were measured immediately after the addition of the reactants, the rate of activation increased, and subsequently decreased, as a function of increasing vesicle concentration. Larger vesicles did not inhibit the reaction to a comparable extent until much higher phospholipid concentrations were present. The inhibition by high vesicle concentrations was significantly reduced by a prolonged incubation period. These results are interpreted as an initial step of factors Va and Xa binding independently to separate phospholipid vesicles, followed by a slow redistribution between vesicles to maximize complex formation. These experiments indicated that the Kd < or = 25 pM, much tighter than previously reported. Two-dimensional binding on the membrane surface was investigated under conditions where all of the proteins were membrane bound. The complex formation was independent of the surface density of the reactions, indicating a near complete complex formation at the lowest surface density of the reactants. Thus, we conclude that (i) the overall affinity of factor Va-factor Xa interaction in the presence of vesicles is higher than previously appreciated, and (ii) factor Va and factor Xa complex once they bind to the same vesicle.
Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein. This raised the possibility that CCD41 was the murine homologue of EPCR. To address this possibility, to better understand structure-function relationships, and to facilitate physiological experiments on EPCR function, we cloned and sequenced murine and bovine EPCR from endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR and CCD41 exhibited five differences corresponding to one base change, three single-base insertions, and one base deletion in the protein coding region. As a result, the predicted structures of EPCR and CCD41 differed in their amino and carboxyl termini but were identical in the central portion of the coding sequence. Based on comparison of the murine, bovine, and human EPCR sequences and the regions where discrepancies between murine EPCR and CCD41 were detected, we believe that CCD41 is probably identical to murine EPCR and that the reported sequence differences are likely the result of compression on the sequencing gel. Compared with human EPCR, the murine and bovine sequences were 69 and 73% identical, respectively, and 57% of the residues were identical between all three species. Both bovine and murine EPCR could bind human activated protein C when the cDNA clones were transfected into 293T cells. Like human EPCR, of the cell lines tested, the murine EPCR message was restricted to endothelium. Cloning of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C pathway.
The protein C anticoagulant pathway regulates thrombin formation. The pathway is triggered when thrombin binds to the endothelial cell proteoglycan, thrombomodulin. Unlike thrombin, this complex is a potent activator of the protein C zymogen, but it cannot clot blood. Activated protein C binds to protein S on cell surfaces where it proteolytically inactivates coagulation factors Va and VIIIa. Activated protein C also binds to a newly identified endothelial protein C receptor. Congenital deficiencies in this pathway are associated with thrombotic disease, and inflammation can cause acquired deficiencies. Activated protein C appears to inhibit inflammation. Thus, this pathway modulates both coagulation and inflammation.
The protein C anticoagulant system generates an "on demand" physiologic anticoagulant response. The pathway is initiated when thrombin binds to the endothelial cell thrombin binding protein, thrombomodulin. The complex exhibits dramatically altered macromolecular specificity. It rapidly cleaves the protein C zymogen to form the anticoagulant, activated protein C. Complex formation between thrombin and thrombomodulin also prevents thrombin, the enzyme responsible for clot formation and a potent platelet activator, from being able to clot fibrinogen or to activate platelets. Structural, kinetic, and competition studies suggest that thrombomodulin blocks these clotting reactions by masking the binding sites for fibrinogen and the platelet thrombin receptor. Stimulation of protein C activation appears to occur through conformational changes in the extended binding pocket of thrombin. This prevents repulsive interactions with protein C that exist when the free enzyme attempts to dock with this substrate. In addition to protein-protein interactions, thrombomodulin has a covalently associated chondroitin sulfate moiety. Chondroitin sulfate binds to a basic surface on thrombin that is also involved in heparin interaction. The chondroitin sulfate enhances the affinity of thrombin for thrombomodulin approximately 10- to 20-fold, making thrombomodulin a more potent inhibitor of coagulation, altering thrombin's conformation and specificity, and accelerating thrombin inhibition by the serpin, antithrombin. These properties make thrombomodulin a molecular switch ideally suited to trigger an anticoagulant response when too much thrombin is generated. The importance of the system is documented by the clinical observation that patients deficient in protein C often die of massive thrombotic complications that can be reversed or prevented by infusion of protein C.
Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies.
Human protein C and activated protein C are shown to bind to endothelium specifically, selectively and saturably (Kd = 30 nM, 7000 sites per cell) in a Ca(2+)-dependent fashion. Expression cloning revealed a 1.3-kilobase pair cDNA that coded for a novel type 1 transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/major histocompatibility complex superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor function and message are both down-regulated by exposure of endothelium to tumor necrosis factor. Identification of endothelial cell protein C receptor as a member of the CD1/major histocompatibility complex superfamily provides insights into the role of protein C in regulating the inflammatory response.
To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys. These mutants, E2FXD70K and E2FXE80K, allow examination of the Ca(2+)-dependent assembly of activation complexes involving substrate and enzyme forms of factor X that do not bind Ca2+. E2FXD70K and E2FXE80K share similar properties. They retain functional activity, but the D70K mutant no longer binds Ca2+, and neither mutant exhibits a Ca(2+)-dependent increase in peptide chromogenic substrate activity. Activation of E2FXD70K by the soluble tissue factor-factor VIIa complex was still Ca(2+)-dependent (Kd(app) = 133 +/- 38 microM), but the Kd(app) for Ca2+ was decreased relative to E2FX (Kd(app) = 205 +/- 53 microM). Prothrombin or prethrombin 1 activation by the E2FXaD70K in the presence of factor Va was also Ca(2+)-dependent (Kd(app) = 124 +/- 47 microM and 102 +/- 38 microM, respectively), and the Kd(app) was again lower than that of E2FXa (272 +/- 79 microM and 179 +/- 54 microM, respectively). In the absence of factor Va, activation of prethrombin 1 by E2FXaD70K was not influenced by Ca2+, but activation by E2FXa was enhanced (Kd(app) = 161 +/- 32 microM). The results suggest the presence of functionally important Ca2+ binding sites in components of the factor X and prothrombin activation complexes that do not involve factor X. Furthermore, the Ca2+ binding sites in factor X and trypsin appear to be structurally similar. The Asp or Glu-->Lys mutations within this site may create an internal salt bridge that conserves factor X (Xa) function even in the absence of Ca2+.
Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3' residue is Asn. To determine the functional significance of these Asp-->Asn substitutions, the Asp in the P3 or P3' position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin-thrombomodulin complex. We conclude that, unlike activation of the Asp-->Gly mutants by thrombin, activation of the P3 and P3' Asp-->Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained.
Modeling studies have ascribed the remarkable resistance of thrombin to inhibition by the Kunitz type inhibitors, bovine pancreatic trypsin inhibitor (BPTI), and tissue factor pathway inhibitor (TFPI), to steric inhibition by the 60-loop insertion, especially Trp60D (in the chymotrypsin numbering system). Indeed, deletion of Pro60B, Pro60C, and Trp60D from this loop (des-PPW) enhances BPTI inhibition (Ki = 16 nM) (Le Bonniec, B. F., Guinto, E. R., MacGillivray, R. T. A., Stone, S. R., and Esmon, C. T. (1993) J. Biol. Chem. 268, 19055-19061). Activated protein C, however, lacks an equivalent insertion loop but is nevertheless resistant to inhibition by these Kunitz inhibitors. A unique feature of thrombin and activated protein C is the presence of Glu at position 192. Substitution of Glu192 with Gln in activated protein C dramatically enhances inhibition by BPTI and TFPI (Rezaie, A. and Esmon, C. T. (1993) J. Biol. Chem. 268, 19943-19948). We now demonstrate that thrombin E192Q (the Glu192-->Gln mutant) is inhibited by BPTI (Ki = 24 nM) or TFPI (Ki = 14 nM) much more effectively than wild type thrombin (Ki > 1 microM for both inhibitors). A thrombin mutant having both the des-PPW deletion and E192Q substitution binds BPTI (Ki = 35 pM) and TFPI (Ki = 25 pM) even tighter. BPTI can displace dansylarginine N-(-3-ethyl-1,5-pentanediyl)-amide from the active site of thrombin E192Q (Ki = 19 nM), indicating that BPTI interacts directly with the S1 binding site in thrombin. The E192Q mutation and PPW deletion contribute comparably and additively to the binding energy of thrombin with the Kunitz inhibitors. We suggest that access to the active center of thrombin is less restricted than predicted from previous studies.
Proteoglycans play pivotal roles in the regulation of thrombin. Thrombomodulin (TM) binds thrombin through protein-protein contacts and a chondroitin sulfate moiety. The complex activates the anticoagulant zymogen, protein C. Thrombin and a thrombin mutant with Arg93, Arg97, and Arg101 changed to Ala bind soluble TM lacking the chondroitin sulfate with comparable affinities, but the mutant binds TM containing chondroitin sulfate 45-fold weaker than thrombin. A simple hyperbolic relationship describes the Ca2+ dependence of protein C activation with the thrombin mutant-TM complex whether or not the TM contains chondroitin sulfate. A similar Ca2+ dependence is observed with wild type thrombin only when the TM contains chondroitin sulfate. Thus, charge neutralization of Arg93, Arg97, and Arg101 mimics the functional effects of the chondroitin sulfate. The mutant and wild type thrombin are inhibited at comparable rates by antithrombin +/- the pentasaccharide capable of inducing the "active" antithrombin conformation, but heparin acceleration of antithrombin inhibition of the mutant is reduced by more than 95%. Binding studies revealed that the mutant has a > or = 20-fold decrease in heparin affinity. We conclude that heparin and chondroitin sulfate interact with one or more of these Arg residues. These basic residues appear to play a critical role in the regulation of thrombin activity.
Protein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca(2+)-dependent, but Ca2+ inhibits protein C activation by thrombin alone. In most proteases, specificity is determined primarily by the residues that lie near the scissile bond. In protein C, the P2 position is Pro, whereas in the fibrinogen A chain, P2 is Val. We have expressed a Pro-->Val mutant of protein C (P168V) in mammalian cells. At saturating Ca2+, the P168V and wild-type proteins were activated by the T-TM complex equivalently, but half maximal rates of activation were obtained at 50 mumol/L Ca2+ for wild type and approximately 5 mmol/L Ca2+ for the P168V mutant. In the absence of TM, Ca2+ no longer inhibited the activation of the P168V mutant. These results indicate that Pro168 influences the Ca(2+)-dependent conformational changes in protein C that control activation. Recently, a patient with thrombotic complications has been identified with a Pro168-->Leu substitution. Both the P168V and the P168L mutation lead to impaired secretion caused by retention within the cell.
Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alters its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's structure [Le Bonniec, B. F., Guinto, E. R., & Esmon, C. T. (1992) J. Biol. Chem. 267, 19341-19348]. In an attempt to localize conformational change in this thrombin mutant (des-ETW), its affinity for 43 site-directed mutants of hirudin have been compared with that of native thrombin. The inhibition constants (KI) of recombinant and natural hirudin with des-ETW were found to be approximately 2300-fold higher than those with thrombin. The reduced affinity was predominantly the result of an increase in the dissociation rate constant rather than a decrease in the association rate constant. For most of the hirudin mutants tested, the difference in binding energy between thrombin and des-ETW [delta delta Gbo(IIa-ETW)] was about 19.9 kJ mol-1 and comparable to that of hirudin; in particular, all mutations in the C-terminal region of the inhibitor did not affect delta delta Gbo(IIa-ETW). Thus, the conformation of thrombin's anion-binding exosite seems unaltered by the des-ETW mutation. In contrast, hirudin substitutions involving Val1', Val2', Thr4', Asp5', and Ser19', as well as the addition of an N-terminal glycine, resulted in values of delta delta Gbo(IIa-ETW) which were 2 to 10 kJ mol-1 lower than that for hirudin. Furthermore, a Leu15'-->Ala hirudin mutant behaved with des-ETW, as a partial competitive inhibitor rather than a tight-binding inhibitor, in contrast to all other hirudin variants tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X. In thrombin, which does not bind Ca2+, Glu-70 is replaced by Lys, creating an internal salt bridge with Glu-80. We constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant is accelerated by thrombomodulin in a Ca(2+)-independent fashion. Unlike wild type Gla-domainless protein C, Ca2+ no longer inhibits activation of the mutant by free thrombin, and Ca2+ stimulation of chromogenic activity is also absent. The characteristic Ca(2+)-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca(2+)-binding site in protein C critical for zymogen activation involves Glu-80. The Glu-80 to Lys mutation probably results in a salt bridge with Glu-70 that stabilizes protein C zymogen in a conformation similar, if not identical, to the Ca(2+)-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.
Membrane surfaces accelerate the proteolytic inactivation of factor Va by activated protein C. In most coagulation complexes, the most active membrane phospholipid is believed to be phosphatidylserine. In this study, we demonstrate that with phosphatidylserine-containing vesicles, incorporation of phosphatidylethanolamine increased the rate of factor Va inactivation approximately 10-fold at all concentrations of factor Va studied and at all vesicle concentrations at or below the optimum for prothrombin activation. In contrast, phosphatidylethanolamine had very little influence on prothrombin activation. Phosphatidylethanolamine was a critical component of vesicles for the vesicles to support activated protein C anticoagulant activity optimally in plasma. These results demonstrate that the membrane requirements for the different coagulation/anticoagulation complexes differ much more than previously appreciated.
Inflammation and the cytokines clearly affect the coagulation system. Less clear are the specific influences of the coagulation system on inflammation. In this chapter only some of the coagulation systems affected by the cytokines are discussed, and the influences on the fibrinolytic system, which is also downregulated by selected cytokines are not mentioned; see Schneiderman and Loskutoff (1991) for a brief review. The major focus is on possible models by which inflammation and coagulation are linked, and examples where in vitro studies have led to correct in vivo predictions and where the results remain ambiguous. Finally, this chapter is clearly a biased perspective with the primary emphasis on the components and pathways with which the author has personal experience.
BACKGROUND: The thrombomodulin-dependent protein C anticoagulant pathway plays a major physiologic role in the down-regulation of the coagulation process. In cell culture, inflammatory cytokines or endotoxin can down-regulate endothelial thrombomodulin (TM) suggesting that suppressed TM expression may contribute to thrombotic complications noted in Gram-negative sepsis. EXPERIMENTAL DESIGN: In the present study, we have examined TM expression in the kidneys of septic rats utilizing indirect immunofluorescence and have quantified TM antigen and TM activity in extracts of the same kidneys by enzyme-linked immunosorbent assays and protein C activation assays, respectively. Conscious Sprague-Dawley rats were injected intravenously with LD95 doses of live E. coli (N = 30), or endotoxin (N = 30). Control animals (N = 30) were injected with equivalent volumes of saline. The rats were killed 30, 90, 180, 360, and 720 minutes after the initiation of sepsis. RESULTS: Glomerular capillary thrombosis developed by 180 minutes in approximately half of the animals after the initiation of sepsis. We failed to demonstrate suppressed TM expression in the kidneys of septic animals using immunofluorescence. Neither enzyme-linked immunosorbent assays, nor protein C activation assays showed decreased levels in TM antigen expression or activity at different time points during the sepsis. CONCLUSIONS: These results indicate that suppressed TM expression does not contribute to the development of the glomerular capillary thrombosis in this septic rat model.