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A new antithrombotic strategy: inhibition of the C‐terminal active site of protein disulfide isomerase

2017; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês

10.1111/jth.13634

ISSN

1538-7933

Autores

Congrong Wang, David W. Essex,

Tópico(s)

Advanced Fluorescence Microscopy Techniques

Resumo

Protein disulfide isomerase (PDI) was discovered over 60 years ago as an enzyme that forms disulfide bonds in newly synthesized proteins in the endoplasmic reticulum 1.Essex D.W. Redox control of platelet function.Antioxid Redox Signal. 2009; 11: 1191-225Crossref PubMed Scopus (90) Google Scholar. Twenty‐five years ago PDI was shown to be released from activated platelets 2.Chen K. Lin Y. Detwiler T.C. Protein disulfide isomerase activity is released by activated platelets.Blood. 1992; 79: 2226-8Crossref PubMed Google Scholar, opening up a new field of research on extracellular functions of this enzyme. PDI was subsequently localized to the platelet surface 3.Essex D.W. Chen K. Swiatkowska M. Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane.Blood. 1995; 86: 2168-73Crossref PubMed Google Scholar and shown to mediate platelet aggregation 4.Essex D.W. Li M. Protein disulphide isomerase mediates platelet aggregation and secretion.Br J Haematol. 1999; 104: 448-54Crossref PubMed Scopus (118) Google Scholar, 5.Lahav J. Jurk K. Hess O. Barnes M.J. Farndale R.W. Luboshitz J. Kehrel B.E. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange.Blood. 2002; 100: 2472-8Crossref PubMed Scopus (167) Google Scholar. PDI binds to and regulates activation of the αIIbβ3 integrin, the fibrinogen receptor that supports platelet aggregation and thrombosis 6.Cho J. Kennedy D.R. Lin L. Huang M. Merrill‐Skoloff G. Furie B.C. Furie B. Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.Blood. 2012; 120: 647-55Crossref PubMed Scopus (104) Google Scholar, 7.Kim K. Hahm E. Li J. Holbrook L.M. Sasikumar P. Stanley R.G. Ushio‐Fukai M. Gibbins J.M. Cho J. Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.Blood. 2013; 122: 1052-61Crossref PubMed Scopus (125) Google Scholar, 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar. Using inhibitory antibodies and targeted knockout mice, PDI has been shown to have a role in platelet accumulation and fibrin generation in vivo 7.Kim K. Hahm E. Li J. Holbrook L.M. Sasikumar P. Stanley R.G. Ushio‐Fukai M. Gibbins J.M. Cho J. Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.Blood. 2013; 122: 1052-61Crossref PubMed Scopus (125) Google Scholar, 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar, 9.Cho J. Furie B.C. Coughlin S.R. Furie B. A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.J Clin Invest. 2008; 118: 1123-31PubMed Google Scholar, 10.Reinhardt C. von Bruhl M.L. Manukyan D. Grahl L. Lorenz M. Altmann B. Dlugai S. Hess S. Konrad I. Orschiedt L. Mackman N. Ruddock L. Massberg S. Engelmann B. Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.J Clin Invest. 2008; 118: 1110-22PubMed Google Scholar. Potential substrates for PDI in coagulation include tissue factor 10.Reinhardt C. von Bruhl M.L. Manukyan D. Grahl L. Lorenz M. Altmann B. Dlugai S. Hess S. Konrad I. Orschiedt L. Mackman N. Ruddock L. Massberg S. Engelmann B. Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.J Clin Invest. 2008; 118: 1110-22PubMed Google Scholar, 11.Langer F. Spath B. Fischer C. Stolz M. Ayuk F.A. Kroger N. Bokemeyer C. Ruf W. Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase.Blood. 2013; 121: 2324-35Crossref PubMed Scopus (88) Google Scholar and factor XI 12.Giannakopoulos B. Gao L. Qi M. Wong J.W. Yu D.M. Vlachoyiannopoulos P.G. Moutsopoulos H.M. Atsumi T. Koike T. Hogg P. Qi J.C. Krilis S.A. Factor XI is a substrate for oxidoreductases: enhanced activation of reduced FXI and its role in antiphospholipid syndrome thrombosis.J Autoimmun. 2012; 39: 121-9Crossref PubMed Scopus (42) Google Scholar, 13.Zucker M. Seligsohn U. Yeheskel A. Mor‐Cohen R. An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase.J Thromb Haemost. 2016; 14: 2202-11Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar. PDI also influences platelet‐dependent thrombin generation by regulating binding of coagulation factors to the platelet surface 14.Jurk K. Lahav J. VAN Aken H. Brodde M.F. Nofer J.R. Kehrel B.E. Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding.J Thromb Haemost. 2011; 9: 2278-90Crossref PubMed Scopus (59) Google Scholar. PDI contains four thioredoxin‐like domains arranged in order of a‐b‐b′‐a′, with a 19 amino acid linker between the b′ and a′ domains termed x (Fig. 1) 15.Hatahet F. Ruddock L.W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid Redox Signal. 2009; 11: 2807-50Crossref PubMed Scopus (504) Google Scholar. The a and a′ domains contain the Cys‐Gly‐His‐Cys (CGHC) active sites that catalyze the reversible oxidation and isomerization of disulfide bonds. The b and b′ domains are non‐catalytic domains, with the b′ domain providing the principle substrate binding domain. Targeting PDI is a current focus for the development of antithrombotic agents and several classes of small‐molecular‐weight molecules that selectively inhibit PDI have been recently described. Quercetin‐3‐rutinoside, a commonly ingested compound in fruits, vegetables, teas and other food, was selected from a 5000‐compound library by the ability to inhibit PDI 16.Jasuja R. Passam F.H. Kennedy D.R. Kim S.H. van Hessem L. Lin L. Bowley S.R. Joshi S.S. Dilks J.R. Furie B. Furie B.C. Flaumenhaft R. Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.J Clin Invest. 2012; 122: 2104-13Crossref PubMed Scopus (224) Google Scholar. This membrane‐impermeable molecule inhibits PDI activity by reversibly binding to the b′x domain of PDI 17.Lin L. Gopal S. Sharda A. Passam F. Bowley S.R. Stopa J. Xue G. Yuan C. Furie B.C. Flaumenhaft R. Huang M. Furie B. Quercetin‐3‐rutinoside inhibits protein disulfide isomerase by binding to its b'x domain.J Biol Chem. 2015; 290: 23543-52Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar and inhibits platelet accumulation and fibrin formation in a laser‐induced cremaster arteriole injury model 16.Jasuja R. Passam F.H. Kennedy D.R. Kim S.H. van Hessem L. Lin L. Bowley S.R. Joshi S.S. Dilks J.R. Furie B. Furie B.C. Flaumenhaft R. Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.J Clin Invest. 2012; 122: 2104-13Crossref PubMed Scopus (224) Google Scholar. A phase II/III study is currently underway evaluating the ability of isoquercetin to inhibit thrombosis in patients with cancer 18.Flaumenhaft R. Furie B. Vascular thiol isomerases.Blood. 2016; 128: 893-901Crossref PubMed Scopus (52) Google Scholar. More recently, another reversible class of PDI inhibitors called bepristats were developed and shown to inhibit platelet aggregation, and platelet accumulation and fibrin formation at the site of vessel injury 19.Bekendam R.H. Bendapudi P.K. Lin L. Nag P.P. Pu J. Kennedy D.R. Feldenzer A. Chiu J. Cook K.M. Furie B. Huang M. Hogg P.J. Flaumenhaft R. A substrate‐driven allosteric switch that enhances PDI catalytic activity.Nat Commun. 2016; 7: 12579Crossref PubMed Scopus (73) Google Scholar. The target of bepristats is also the hydrophobic pocket of the b′ domain of PDI. In addition to targeting the substrate binding b′ domain of PDI, the active sites of PDI are potential targets for antithrombotic activity. Although there is 36.8% sequence similarity between the a and a′ domains 15.Hatahet F. Ruddock L.W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid Redox Signal. 2009; 11: 2807-50Crossref PubMed Scopus (504) Google Scholar, the active sites are not functionally equivalent. Using reduced RNase as the substrate, the a domain or N‐terminal active site has higher catalytic activity and functions primarily as a disulfide isomerase, whereas the a′, C‐terminal, active site contributes more to the Km of PDI and functions as a disulfide oxidase 20.Lyles M.M. Gilbert H.F. Mutations in the thioredoxin sites of protein disulfide isomerase reveal functional nonequivalence of the N‐ and C‐terminal domains.J Biol Chem. 1994; 269: 30946-52Abstract Full Text PDF PubMed Google Scholar, 21.Kulp M.S. Frickel E.M. Ellgaard L. Weissman J.S. Domain architecture of protein‐disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p‐mediated disulfide formation.J Biol Chem. 2006; 281: 876-84Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar. Similarly, the active sites have different abilities to rearrange specific disulfide bonds in bovine pancreatic trypsin inhibitor 22.Darby N.J. Penka E. Vincentelli R. The multi‐domain structure of protein disulfide isomerase is essential for high catalytic efficiency.J Mol Biol. 1998; 276: 239-47Crossref PubMed Scopus (152) Google Scholar, and the C‐terminal active site is a better oxidase in the Ero1‐mediated oxidative folding pathway 23.Wang L. Li S.J. Sidhu A. Zhu L. Liang Y. Freedman R.B. Wang C.C. Reconstitution of human Ero1‐Lalpha/protein‐disulfide isomerase oxidative folding pathway in vitro. Position‐dependent differences in role between the a and a' domains of protein‐disulfide isomerase.J Biol Chem. 2009; 284: 199-206Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar. Although these in vitro studies indicate distinct functions for each active site of PDI, until recently nothing was known about the relative importance of each active site in physiologic reactions, or in thrombosis. A critical role for the C‐terminal active site of PDI in platelet activation and thrombosis was recently reported 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar. Mice lacking PDI in vessel wall cells and platelets, and transgenic mice harboring PDI that lacks a functional C‐terminal CGHC motif, had defective thrombosis in several vascular injury models. Decreased platelet accumulation and fibrin generation in a laser‐induced cremaster arteriole injury were rescued by infusion of recombinant PDI containing only a functional C‐terminal CGHC motif. The C‐terminal active site mediated platelet aggregation and αIIbβ3 activation, as well as ATP secretion and P‐selection expression through a non‐αIIbβ3 substrate. The whole body knockout of PDI was lethal in embryogenesis but the PDI transgene with only a functional N‐terminal active site rescued the lethal phenotype. These studies indicate the C‐terminal CGHC active motif is pivotal for platelet function and thrombosis, whereas the N‐terminal CGHC motif is required for murine survival. These findings suggested that inhibiting the C‐terminal active site of PDI might prove useful for antithrombotic therapy while sparing the function of the N‐terminal active site. In this issue, Sousa et al. 24.Sousa H.R. Gaspar R.S. Lena E.M.L. Sda Silva S.A. Fonelles J.L.d.L. Araujo T.L.S. Mastrogiovanni M. Fries D.M. Azevedo‐Santos A.P.S. Laurindo F.R.M. Torostchansky A. Paes A.M. Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: evidence of covalent binding to C‐terminal CGHC redox motif.J Thromb Haemost. 2017; 15: 774-84Abstract Full Text Full Text PDF Scopus (17) Google Scholar demonstrate that a 12 amino acid peptide (VEFYAPWCGHCK) covalently binds to the C‐terminal CGHC active motif of PDI and inhibits platelet aggregation. This PDI‐targeted peptide, called CxxC, contains the C‐terminal CGHC active site sequence of PDI and was previously shown by this group to inhibit PDI in the insulin reductase assay and to strongly inhibit PDI‐mediated superoxide generation by leukocyte Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 25.de A Paes A.M. Verissimo‐Filho S. Guimaraes L.L. Silva A.C. Takiuti J.T. Santos C.X. Janiszewski M. Laurindo F.R. Lopes L.R. Protein disulfide isomerase redox‐dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation.J Leukoc Biol. 2011; 90: 799-810Crossref PubMed Scopus (70) Google Scholar. CxxC dose‐dependently inhibited ADP and thrombin‐induced aggregation, as well as αIIbβ3 activation. A scrambled control peptide or a peptide with the Cys residues mutated to Ala did not inhibit. CxxC did not inhibit P‐selectin expression, in contrast with a previous report that found a role for PDI in this process 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar; the cause of these differences is not clear. An impressive finding came from mass spectrometry analysis where the investigators demonstrated the CxxC peptide covalently linked to the reduced form of Cys400 in the C‐terminal CGHC motif of PDI through a mixed disulfide bond (Fig. 1). Although the exact mechanism by which CxxC reacts with Cys400 is not clear, the authors 'speculate that Cys397 attacks CxxC, or vise‐versa, to form a mixed disulfide that is stabilized at Cys400'. The apparent stability of the covalent bond between CxxC and Cys400 raises the possibility of designing an inhibitor of PDI that reacts similarly. Because the authors didn't retrieve the fragment containing Cys53 and Cys56 from the a domain N‐terminal active site of PDI in their mass spectrometry analysis, it is not clear whether the CxxC peptide also reacts with the N‐terminal CGHC motif of PDI. However, the C‐terminal active site of PDI has already been demonstrated to support thrombosis 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar and therefore the strategy to target the C‐terminal active site and, in particular, Cys400 provides a model for development of site‐specific antithrombotic agents. The active sites of PDI can exist in both dithiol and disulfide forms 15.Hatahet F. Ruddock L.W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid Redox Signal. 2009; 11: 2807-50Crossref PubMed Scopus (504) Google Scholar but little is known about the redox state of PDI that catalyzes reactions in platelets. The CxxC peptide reacted with reduced but not oxidized PDI. This suggests CxxC targets the reduced form of the C‐erminal active site on the platelet surface, and that the reduced form of PDI is important in platelet function. Because the dithiol form of PDI catalyzes isomerization and cleavage of disulfide bonds 1.Essex D.W. Redox control of platelet function.Antioxid Redox Signal. 2009; 11: 1191-225Crossref PubMed Scopus (90) Google Scholar, 15.Hatahet F. Ruddock L.W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid Redox Signal. 2009; 11: 2807-50Crossref PubMed Scopus (504) Google Scholar, these reactions are likely to have a role in PDI regulation of platelet function. CxxC also decreased 3‐N‐maleimidyl‐propionyl biotin (MPB) labeling of thiols in PDI on the surface of resting platelets without decreasing the labeling of other surface proteins, suggesting some specificity of CxxC for PDI. The inhibition of thiol labeling in PDI by CxxC suggests that PDI has free thiols in the C‐terminal active site. The impermeant reducing agent, TCEP, increased MPB labeling of PDI on the surface of resting platelets by several fold. Therefore, the majority of platelet surface PDI on resting platelets is in the oxidized form, as previously reported 26.Burgess J.K. Hotchkiss K.A. Suter C. Dudman N.P. Szollosi J. Chesterman C.N. Chong B.H. Hogg P.J. Physical proximity and functional association of glycoprotein 1balpha and protein‐disulfide isomerase on the platelet plasma membrane.J Biol Chem. 2000; 275: 9758-66Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar. Activation of platelets is known to increase thiols in platelet surface PDI 26.Burgess J.K. Hotchkiss K.A. Suter C. Dudman N.P. Szollosi J. Chesterman C.N. Chong B.H. Hogg P.J. Physical proximity and functional association of glycoprotein 1balpha and protein‐disulfide isomerase on the platelet plasma membrane.J Biol Chem. 2000; 275: 9758-66Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 27.Manickam N. Sun X. Li M. Gazitt Y. Essex D.W. Protein disulphide isomerase in platelet function.Br J Haematol. 2008; 140: 223-9PubMed Google Scholar, possibly through a transmembrane electron transport system 26.Burgess J.K. Hotchkiss K.A. Suter C. Dudman N.P. Szollosi J. Chesterman C.N. Chong B.H. Hogg P.J. Physical proximity and functional association of glycoprotein 1balpha and protein‐disulfide isomerase on the platelet plasma membrane.J Biol Chem. 2000; 275: 9758-66Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 28.Essex D.W. Li M. Feinman R.D. Miller A. Platelet surface glutathione reductase‐like activity.Blood. 2004; 104: 1383-5Crossref PubMed Scopus (33) Google Scholar. A previously reported inhibitor of PDI, PACMA‐31, also interacts with Cys397/Cys400 in the C‐terminal active site of PDI 29.Xu S. Butkevich A.N. Yamada R. Zhou Y. Debnath B. Duncan R. Zandi E. Petasis N.A. Neamati N. Discovery of an orally active small‐molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment.Proc Natl Acad Sci USA. 2012; 109: 16348-53Crossref PubMed Scopus (155) Google Scholar. However, this reagent was recently found to inhibit ERp57, ERp5 and thioredoxin, and to inhibit both active sites of PDI 19.Bekendam R.H. Bendapudi P.K. Lin L. Nag P.P. Pu J. Kennedy D.R. Feldenzer A. Chiu J. Cook K.M. Furie B. Huang M. Hogg P.J. Flaumenhaft R. A substrate‐driven allosteric switch that enhances PDI catalytic activity.Nat Commun. 2016; 7: 12579Crossref PubMed Scopus (73) Google Scholar. In the Sousa study, the CxxC peptide didn't enhance inhibition of platelet aggregation by the anti‐PDI antibodies BD34 and RL90, suggesting that CxxC mainly targets PDI. However, whether the CxxC peptide covalently binds to the CGHC motif in other PDI family members remains to be elucidated. If CxxC turns out to be specific for PDI it could provide a useful research tool. Other members of the PDI family are also potential targets for antithrombotic therapy. ERp5 and ERp57, the closest homologue of PDI in platelets, are important for platelet function and thrombosis 30.Jordan P.A. Stevens J.M. Hubbard G.P. Barrett N.E. Sage T. Authi K.S. Gibbins J.M. A role for the thiol isomerase protein ERP5 in platelet function.Blood. 2005; 105: 1500-7Crossref PubMed Scopus (120) Google Scholar, 31.Holbrook L.M. Sasikumar P. Stanley R.G. Simmonds A.D. Bicknell A.B. Gibbins J.M. The platelet‐surface thiol isomerase enzyme ERp57 modulates platelet function.J Thromb Haemost. 2012; 10: 278-88Crossref PubMed Scopus (78) Google Scholar, 32.Wu Y. Ahmad S.S. Zhou J. Wang L. Cully M.P. Essex D.W. The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis.Blood. 2012; 119: 1737-46Crossref PubMed Scopus (76) Google Scholar, 33.Wang L. Wu Y. Zhou J. Ahmad S.S. Mutus B. Garbi N. Hammerling G. Liu J. Essex D.W. Platelet‐derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the alphaIIbbeta3 integrin.Blood. 2013; 122: 3642-50Crossref PubMed Scopus (70) Google Scholar, 34.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The disulfide isomerase ERp57 is required for fibrin deposition in vivo.J Thromb Haemost. 2014; 12: 1890-7Crossref PubMed Scopus (26) Google Scholar, 35.Passam F.H. Lin L. Gopal S. Stopa J.D. Bellido‐Martin L. Huang M. Furie B.C. Furie B. Both platelet‐ and endothelial cell‐derived ERp5 support thrombus formation in a laser‐induced mouse model of thrombosis.Blood. 2015; 125: 2276-85Crossref PubMed Scopus (54) Google Scholar. As with PDI, the C‐terminal active site of ERp57 supports platelet aggregation 33.Wang L. Wu Y. Zhou J. Ahmad S.S. Mutus B. Garbi N. Hammerling G. Liu J. Essex D.W. Platelet‐derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the alphaIIbbeta3 integrin.Blood. 2013; 122: 3642-50Crossref PubMed Scopus (70) Google Scholar, and platelet accumulation and fibrin generation in vivo 34.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The disulfide isomerase ERp57 is required for fibrin deposition in vivo.J Thromb Haemost. 2014; 12: 1890-7Crossref PubMed Scopus (26) Google Scholar. It is not known whether inhibiting one PDI or several PDIs would provide optimal antithrombotic therapy, as the efficacy in inhibiting several PDIs will have to be balanced with potential toxicities. Inhibiting only extracellular PDIs should provide optimal antithrombotic benefit while minimizing toxicity 16.Jasuja R. Passam F.H. Kennedy D.R. Kim S.H. van Hessem L. Lin L. Bowley S.R. Joshi S.S. Dilks J.R. Furie B. Furie B.C. Flaumenhaft R. Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.J Clin Invest. 2012; 122: 2104-13Crossref PubMed Scopus (224) Google Scholar. Because the N‐terminal active site is important in murine viability 8.Zhou J. Wu Y. Wang L. Rauova L. Hayes V.M. Poncz M. Essex D.W. The C‐terminal CGHC motif of protein disulfide isomerase supports thrombosis.J Clin Invest. 2015; 125: 4391-406Crossref PubMed Scopus (64) Google Scholar, targeting only the C‐terminal active site may be another way to provide antithrombotic efficacy while also minimizing toxicity. Further studies of the detailed mechanisms by which PDIs support thrombosis should help elucidate optimal antithrombotic strategies. L. Wang and D. W. Essex wrote the manuscript. The authors have no conflict of interest.

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