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Role of Protein Kinase C in Ischemic Preconditioning: Player or Spectator?

1996; Lippincott Williams & Wilkins; Volume: 79; Issue: 3 Linguagem: Inglês

10.1161/01.res.79.3.628

1524-4571

Gavin Brooks, David J. Hearse,

Anesthesia and Neurotoxicity Research

HomeCirculation ResearchVol. 79, No. 3Role of Protein Kinase C in Ischemic Preconditioning: Player or Spectator? Free AccessResearch ArticleDownload EPUBAboutView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticleDownload EPUBRole of Protein Kinase C in Ischemic Preconditioning: Player or Spectator? Gavin Brooks and David J. Hearse Gavin BrooksGavin Brooks Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK. and David J. HearseDavid J. Hearse Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK. Originally published1 Sep 1996https://doi.org/10.1161/01.RES.79.3.628Circulation Research. 1996;79:628–631Although it may be debatable whether preconditioning will ever fulfill its clinical expectations, the hope that a new therapeutic modality may emerge from this fascinating phenomenon has given great impetus to the search for its underlying mechanism. Preconditioning is unquestionably a powerful phenomenon, it is readily and consistently demonstrable under many experimental conditions, and it has just celebrated its 10th birthday.1 In view of all this, it is perhaps surprising that the precise mechanism of preconditioning remains elusive and recently has become the subject of controversy.An observer of the preconditioning literature could, at first sight, be forgiven for concluding that the precise mechanism is established and that protein kinase C (PKC) activation is a pivotal common factor that links a spectrum of receptor-mediated triggers of preconditioning. Downey and colleagues, now supported by other investigators (reviewed in References 2 and 3),23 have developed the compelling hypothesis that stimulation of a variety of G protein–coupled receptors (eg, adenosine A1, α1-adrenergic, muscarinic, bradykinin, and endothelin-1 receptors) results in the activation of PKC. This, in turn, leads to the physical translocation of PKC from the cytoplasm to the sarcolemma, where it phosphorylates a substrate protein (possibly the ATP-sensitive K+ [KATP] channel4 ) and thereby confers resistance to ischemia. Support for this attractive hypothesis comes from a wealth of studies (most of which rely upon indirect evidence in the absence of PKC activity measurements) with activators and inhibitors of PKC or its translocation process, with receptor agonists and antagonists, and with agents that interfere with the signaling pathways between various receptors and PKC (reviewed in References 5 and 6).Considering the substantial body of evidence supporting the involvement of PKC activation in the preconditioning phenomenon, the observer could again be forgiven for wondering why this issue of Circulation Research is publishing one article (by Vahlhaus et al7 ) claiming that PKC activation is not the mechanism of preconditioning whilst also publishing two articles (by Light et al8 and Mizumura et al9 ) claiming that it is. Why should clouds of doubt and controversy be forming over the "Downey hypothesis"? In this Editorial we will consider the current evidence against the PKC hypothesis and try to identify factors that might be contributing to the growing controversy over the involvement of PKC in preconditioning. We shall restrict our consideration to the putative signal transduction pathways involved in "acute" preconditioning and will not discuss the "second window," since we believe that this most likely involves different mechanisms (a conclusion supported by the study of Tang et al10 also in this issue).Current Evidence For and Against the "Downey Hypothesis"Although the involvement of PKC in preconditioning is supported by numerous experiments using PKC activators and inhibitors in rat, pig, rabbit, dog, and human tissues (reviewed in Reference 2), some controversy exists, since the concept does not seem to apply consistently in all species and under all conditions. This issue of Circulation Research includes several articles that illustrate this point.Thus, Vahlhaus et al7 were unable to block preconditioning in pigs with the potent PKC inhibitor staurosporine, despite the fact that the dose used was able to block the increase in coronary artery resistance induced by a PKC-activating phorbol ester. Although the authors did not measure PKC activity per se and used staurosporine in both control and preconditioning groups (they did not include a control group in which staurosporine was not present), the results do seriously question whether activation of PKC is mandatory for effective preconditioning. A preliminary study previously reported by Vogt et al11 supports the conclusions of Vahlhaus et al. Using a method of intramyocardial microinfusion in open-chest pigs (a method that ensures delivery of high local concentrations of drug to the myocardium in the absence of associated systemic effects), these authors demonstrated that infusion of two PKC inhibitors (staurosporine or bisindolylmaleimide) failed to block ischemic preconditioning.11 Interestingly, bisindolylmaleimide by itself exerted a protective effect, suggesting that PKC inhibition as opposed to PKC activation may play a role in protecting the ischemic myocardium! This conclusion was also reached in a previous study by Przyklenk et al,12 who showed that administration of a PKC inhibitor (1-[5-isoquinolinylsulfonyl]-2-methylpiperazine) afforded additional protection in the preconditioned canine heart. This study was notable in that the authors also used a fluorescein probe conjugated to bisindolylmaleimide, which selectively binds to active PKC.12 Using this probe and immunofluorescence, they were unable to show any difference in the total amount, or subcellular distribution, of PKC during ischemic preconditioning in dogs. In addition, these investigators determined PKC activation directly by measuring 32P incorporation into a PKC-specific peptide in subcellular fractions obtained from canine myocardium and showed that preconditioning failed to activate the enzyme. Similarly, the conclusion that preconditioning does not cause PKC activation also has been drawn from studies in the rabbit13 and in the rat.1415 Furthermore, we recently have shown that the novel diacylglycerol and phorbol ester–activated protein kinase, protein kinase D (PKD),16 is not activated during ischemic preconditioning in the isolated rat heart (authors' unpublished data, 1996). In contrast, perfusion with a phorbol ester (phorbol 12-myristate 13-acetate) in place of the ischemic episode induced a significant activation of PKD. Interestingly, evidence now exists to show that PKC can directly activate PKD (E. Rozengurt, personal communication, 1996); therefore, the fact that ischemic preconditioning failed to activate PKD provides additional support to our finding that PKC activation failed to protect the heart.14Maintaining support for the "Downey hypothesis" are two studies published in this issue of Circulation Research. Mizumura et al9 provide indirect support for the involvement of PKC by manipulating the A1 receptor in dogs with a compound that increases agonist binding to the receptor and enhances functional A1-mediated responses in the heart. Their results show that this compound reduces the duration of preconditioning ischemia required to achieve protection. Also in this issue, Light et al8 show, for the first time, that the application of purified PKC to inside-out isolated rabbit myocyte membrane patches results in the activation of the KATP channel at physiological levels of ATP. These authors demonstrate that application of active PKC to the intracellular surfaces of the patches increases the probability of KATP channel opening by threefold. In addition, Light et al show that the response could be blocked completely with the specific peptide inhibitor PKC(19-31). These authors hypothesize that PKC catalyzes the phosphorylation of the KATP channel (or some other associated protein) and that it is this that leads to preconditioning.Clearly, there is growing controversy over the role of PKC in preconditioning, and in the following sections, we examine some factors that might contribute to the conflicting conclusions that are appearing in this and other journals.Factors That May Contribute to the PKC ControversyOur starting point is that PKC is not a simple enzyme; instead, it is an immensely complex protein that can exist in at least 11 different isoforms. PKC still is not fully understood biochemically; it is very difficult to manipulate specifically and, as a result, is even more difficult to study.1718 It is not surprising, therefore, that experimental pitfalls and potential misinterpretations may arise and act as seeds of controversy. In the following sections, we will address such problems in the context of preconditioning, with special emphasis on (1) the dangers of indirect or inappropriate measurements of PKC activity, (2) the use and abuse of activators and inhibitors, (3) the multitude of distinct isoforms of PKC, and (4) the problems of models, species, and end points.Dangers of Indirect or Inappropriate Measurements of PKC ActivityCorrectly measuring PKC activity in the manner of the classical enzymologist is not easy, and as a consequence, it is very tempting to use indirect methods of assessment. Many studies of preconditioning fall into the trap of applying various agonists or antagonists of receptors that are known to be linked to PKC and then assuming that complete signal transduction and consequent PKC activation or inactivation is actually accomplished. The same criticism can be applied to the liberal use of putative activators or inhibitors of PKC; rarely have investigators demonstrated biochemically a direct activation or inhibition of the enzyme under the conditions of their studies.Furthermore, in the rare instances that PKC activity is measured biochemically, it is not always carried out appropriately, since as with any enzyme assay, it is essential that the correct substrates are used. Thus, histone IIIS often is used, and although it is certainly a good substrate for measuring the activity of calcium-dependent PKCs, it is not a good substrate for calcium-independent isoforms, which prefer myelin basic protein as a substrate. Although unrelated to preconditioning, the study by Rybin and Steinberg19 in this issue elegantly illustrates the importance of using appropriate substrates for the assay of PKC activity. Some of the problems of substrate choice can be circumvented, however, if a peptide based on the PKCε pseudosubstrate domain were to be used (as in the Amersham PKC assay kit), since this acts as a good substrate for both calcium-dependent and calcium-independent PKC isoforms.For those investigators who do venture into the minefield of direct PKC activity measurements, other potential pitfalls exist. Thus, many protein phosphorylation and/or immunoreactivity assays for PKC activity are carried out using cardiac biopsies that have been rapidly frozen in liquid nitrogen, stored for a period of time, and then homogenized for the preparation of PKC fractions. Great care must be exercised when using such procedures, since relocalization and/or (in)activation of PKC could occur during these processes, since PKC is unstable until purified to homogeneity.20 A further complication to consider is that the biochemical determination of PKC activity provides a measure of the maximum rate of phosphorylation that can be detected in the presence of the optimal (compared with intracellular) concentrations of calcium/phosphatidylserine/diacylglycerol. Thus, the biochemical activity measurement represents the maximum rate of PKC-mediated phosphorylation and, as such, gives no information on basal intracellular PKC activity or the extent of stimulation over preexisting basal activity caused by preconditioning.Use and Abuse of Inhibitors and Activators"No one thing does only one thing," and the use of pharmacological agents to inhibit, stimulate, or mimic biological events is fraught with the dangers of secondary nonspecific pharmacological actions. Much of the evidence for the involvement of PKC in preconditioning is indirect and based on the use of receptor agonists and antagonists or a bewildering array of putative modifiers of PKC activity and/or translocation. The choice of agent used in preconditioning studies varies widely between investigators, and often no account is taken of secondary effects, such as the ability of adenosine agonists to cause cardiac arrest, the ability of several PKC activators and inhibitors (at pharmacologically active doses) to induce cardiac depression, vascular spasm, and leukocyte activation, and the fact that many inhibitors of PKC are not specific to PKC and inhibit, to varying degrees, other protein kinases.The "gold standard" inhibitor for many PKC studies is staurosporine, which is a potent inhibitor of all PKC isoforms. However, at increasing concentrations, staurosporine becomes nonspecific with respect to PKC, since it also inhibits other kinases, such as cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. This low specificity for PKC is due to the fact that, like many other PKC inhibitors (such as bisindolylmaleimide), it competes with ATP for binding within the catalytic domain of the enzyme. In addition, although bisindolylmaleimide displays better specificity toward PKC, it has low potency in vivo because of the high ATP content of many cell types. Polymyxin B has been used in some studies, but this is not a specific inhibitor of PKC, and furthermore, it blocks the KATP channel,21 which is one of the putative target proteins for PKC! Thus, polymyxin B is not well suited for dissecting the specific role of PKC in preconditioning. More selective inhibitors of PKC do exist (eg, calphostin C and chelerythrine), and these interact with the regulatory domain of the enzyme. However, these compounds also have problems, such that calphostin C requires activation with UV light in the presence of PKC, thus limiting its use in whole-animal experiments, and chelerythrine suffers from poor solubility in aqueous solution, especially those containing significant salt concentrations. By far the most specific inhibitors of PKC are synthetic peptides based on the pseudosubstrate domain of PKC, such as the one used by Light et al8 in this issue. However, it should be noted that the use of such inhibitory peptides is useful only for in vitro studies. Until similar specific inhibitors are available for in vivo studies, it would be a wise precaution to use several of the currently available PKC inhibitors in any in vivo study.Finally, it is worth noting that many of the studies that have used PKC inhibitors to support a role for PKC activation in preconditioning have been in the rat and rabbit. However, it has not been confirmed that blockade of PKC activity actually occurs in these species in vivo, although one study22 has determined the effects of the PKC inhibitor, calphostin C, on infarct size and incidence of ventricular tachycardia in the rat heart in vivo although no assessment of effects on PKC activity were measured. In vivo studies have been carried out in pigs and dogs, and interestingly, these are the species in which evidence against an involvement of PKC activation in preconditioning is strongest.The Family ProblemAnother factor undoubtedly contributing to the confusion over the role of PKC in preconditioning is the fact that the protein exists as at least 11 different isoforms that differ from each other, not only in relation to their cofactor requirements for activation but also in their substrate specificities. To date, six isoforms (α, β, δ, ε, η, and ζ) have been detected in heart232425 ; however, it is almost inevitable that various PKC inhibitors will be more specific and more potent for certain isoforms, although no in vivo data are yet available to demonstrate this point. An additional complication is that although some selectivity in terms of potency and degree of activation of different isoforms by different phorbol esters has been reported,26 phorbol esters are able to activate both calcium-dependent (α, βI, βII, and γ) and calcium-independent (δ, ε, μ, η, and θ) PKC isoforms. Specific activators of individual isoforms are not yet available, and until they are, conclusions about the role of specific isoforms should be made with caution.One pitfall of conventional PKC activity measurements is that by measuring total PKC activity, no assessment of the relative involvement of different isoforms can be made. In an attempt to address this problem, some studies have used specific antibodies to assess PKC isoform activity by immunofluorescence. Such an approach has been used by Mitchell et al,27 who showed that PKCδ translocates to the sarcolemma after exposure of rat hearts to the α1-adrenergic agonist, phenylephrine, diacylglycerol, or ischemic preconditioning. However, the results from such studies must be interpreted with caution, since isoform-specific antibodies are not always able to distinguish between an activated or an inactive PKC isoform.Models, Species, and End PointsAnother factor contributing to the difficulty in reconciling the many studies that attempt to link PKC activation and preconditioning is wide variability in experimental design. Key variables include the following: species (in vitro, ex vivo, or in vivo); end points of protection (infarct size, function, arrhythmias, metabolism, or contracture), and differences in the choice of the preconditioning protocol (frequency and duration) or triggering event (ischemia, rapid pacing, or one of a host of receptor agonists). However, even when very similar protocols have been used in the same species, different research groups can come to opposite conclusions.1228The Way ForwardPossible Ways to Resolve the ControversyOne possible way to definitively determine the role of specific PKC isoforms in mediating preconditioning would be to use knockout technology to develop transgenic animals that lack one or more PKC isoforms. Similarly, studies might exploit forced expression, homologous recombination, and gene transfer technologies. An alternative approach might be to study enzyme activity in vivo by measuring phosphorylation of one of the specific substrate proteins of PKC, such as the myristoylated alanine-rich C kinase substrate.2930 However, such studies could pose significant safety concerns in view of the high levels of radioactive isotopes that would be required for an in vivo study.If PKC Is Not the Central Mediator of Preconditioning, What Might Be the Alternative?At the present time, we cannot exclude the possibility that PKC might be an epiphenomenon or secondary to a more central mechanism—what might this be? The rapid, but transient, cardioprotective effect that characterizes preconditioning would certainly be consistent with the involvement of a kinase. The fact that PKC has been implicated in many studies may be due to the fact that cross talk can occur between various signaling pathways. Thus, in instances in which, for example, α1-adrenergic agonists or ischemia has been used to precondition the heart, it is possible that PKC has been stimulated as a secondary response in addition to the central mechanism of preconditioning, since these stimuli are known to initiate multifactorial signaling cascades. Furthermore, the fact that endogenous mediators such as bradykinin, which leads to the release of NO and subsequent activation of guanylate cyclase, can protect the heart via a PKC-independent pathway31 suggests that PKC is unlikely to be the common mechanism for preconditioning. An alternative kinase pathway worthy of consideration is the mitogen-activated protein kinase (MAPK) cascade, since this pathway is known to be functional in cardiac myocytes and is activated in response to stress and hypertrophic agents.32 Transmembrane receptors that are coupled to Gi can ultimately stimulate the MAPK pathway. Interestingly, Raf-1 (which acts upstream from MAPK) can be activated both by PKC-dependent and -independent pathways in vitro and in vivo. Thus, if MAPK were the common pathway in preconditioning, this could explain why, in some studies, PKC activation was shown to occur with preconditioning, whereas in others it was not. Unfortunately, the current lack of selective activators and inhibitors of MAPK means that any investigation of its role in preconditioning would encounter similar difficulties to those that exist for PKC.So, Is PKC a Player or Spectator in Ischemic Preconditioning?From the evidence presented above, it is clear that the role of PKC activation in mediating the cardioprotective effects of preconditioning is controversial and that, in large animals at least, PKC probably is not an obligatory mediator. Although it is tempting to take refuge in the possibility that PKC mediates preconditioning in small animals, we do not think that this is a tenable argument. Instead, it seems likely that PKC activation is an epiphenomenon rather than a mandatory or exclusive means of preconditioning the heart. This conclusion is based not so much on the negative studies discussed above but on the belief that a powerful adaptive phenomenon that is induced so easily and reproducibly in so many models, laboratories, and species is almost certain to be mediated by a universal mechanism.FootnotesCorrespondence to Dr Gavin Brooks, Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK. References 1 Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circ Res..1986; 74:1124-1136.LinkGoogle Scholar2 Downey JM, Cohen MV. Mechanisms of preconditioning: correlates and epiphenomena. In: Marber MM, Yellon DM, eds. Ischaemia: Preconditioning and Adaptation. Oxford, UK: UCL Molecular Pathology Series, BIOS Scientific Publishers Limited; 1996:21-34.Google Scholar3 Lawson CS, Downey JM. Preconditioning: state of the art myocardial protection. Cardiovasc Res..1993; 27:542-550.CrossrefMedlineGoogle Scholar4 Speechly-Dick ME, Grover GJ, Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel?: studies of contractile function after simulated ischemia in an atrial in vivo model. Circ Res..1995; 77:1030-1035.CrossrefMedlineGoogle Scholar5 Marber MM, Yellon DM, eds. Ischaemia: Preconditioning and Adaptation. Oxford, UK: UCL Molecular Pathology Series, BIOS Scientific Publishers Limited; 1996.Google Scholar6 Gho BCG, Eskildson-Helmond YEG, de Zeeuw S, Lamers JMJ, Verdouw PD. Does protein kinase C play a pivotal role in the mechanisms of ischemic preconditioning? Cardiovasc Drugs Ther. In press.Google Scholar7 Vahlhaus C, Schulz R, Post H, Onallah R, Heusch G. No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ Res..1996; 79:407-414.CrossrefMedlineGoogle Scholar8 Light PE, Sabir AA, Allen BG, Walsh MP, French RJ. Protein kinase C–induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels: a possible mechanistic link to ischemic preconditioning. Circ Res..1996; 79:399-406.CrossrefMedlineGoogle Scholar9 Mizumura T, Auchampach JA, Linden J, Bruns RF, Gross GJ. PD 81,723, an allosteric enhancer of the A1 adenosine receptor, lowers the threshold for ischemic preconditioning in dogs. Circ Res..1996; 79:415-423.CrossrefMedlineGoogle Scholar10 Tang X-L, Qiu Y, Park S-W, Sun J-Z, Kalya A, Bolli R. Time course of late preconditioning against myocardial stunning in conscious pigs. Circ Res..1996; 79:424-434.CrossrefMedlineGoogle Scholar11 Vogt A, Barancik M, Weihrauch D, Arras M, Podzuweit T, Schaper W. Protein kinase C inhibitors reduce infarct size in pig hearts in vivo. Circulation. 1994;90(suppl I):I-647. Abstract.Google Scholar12 Przyklenk K, Sussman MA, Simkhovich BZ, Kloner RA. Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation..1995; 92:1546-1557.CrossrefMedlineGoogle Scholar13 Simkhovich BZ, Przyklenk K, Hale SL, Kloner RA. Subcellular distribution of protein kinase C is not altered by brief preconditioning ischemia in rabbit myocardium. Circulation. 1995;92(suppl I):I-137. Abstract.Google Scholar14 Galin˜anes M, McGill C, Brooks G, Hearse DJ. Can the protein kinase C activator diacylglycerol precondition the rat heart? J Mol Cell Cardiol..1996; 28:A73.288. Abstract.Google Scholar15 Moolman JA, Genade S, Tromp E, Lochner A. No evidence for mediation of ischemic preconditioning by alpha1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart. Cardiovasc Drugs Ther..1996; 10:125-136.CrossrefMedlineGoogle Scholar16 Valverde AM, Sinnett-Smith J, Van Lint J, Rozengurt E. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci U S A..1994; 91:8572-8576.CrossrefMedlineGoogle Scholar17 Newton AC. Protein kinase C: structure, function and regulation. J Biol Chem..1995; 270:28495-28498.CrossrefMedlineGoogle Scholar18 Nishizuki Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J..1995; 9:484-496.CrossrefMedlineGoogle Scholar19 Rybin V, Steinberg SF. Thyroid hormone represses protein kinase C isoform expression and activity in rat cardiac myocytes. Circ Res..1996; 79:388-398.CrossrefMedlineGoogle Scholar20 Parker PJ, Stabel S, Waterfield MD. Purification to homogeneity of protein kinase C from bovine brain: identity with the phorbol ester receptor. EMBO J..1984; 3:953-959.CrossrefMedlineGoogle Scholar21 Harding EA, Jaggar JH, Squires PE, Dunno MJ. Polymyxin B has multiple blocking actions on the ATP-sensitive potassium channel in insulin-secreting cells. Pflugers Arch..1994; 426:31-39.CrossrefMedlineGoogle Scholar22 Li Y, Kloner RA. Does protein kinase C play a role in ischemic preconditioning in rat hearts? Am J Physiol..1995; 268:H426-H431.CrossrefMedlineGoogle Scholar23 Bogoyevitch MA, Parker PJ, Sugden PH. Characterization of protein kinase C isotype expression in adult rat heart: protein kinase C-ε is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin. Circ Res..1993; 72:757-767.CrossrefMedlineGoogle Scholar24 Rybin VO, Steinberg SF. Protein kinase C isoform expression and regulation in the developing rat heart. Circ Res..1994; 74:299-309.CrossrefMedlineGoogle Scholar25 Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform βII and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci U S A..1992; 89:11059-11063.CrossrefMedlineGoogle Scholar26 Ryves WJ, Evans AT, Olivier AR, Parker PJ, Evans FJ. Activation of the PKC-isotypes α, βI, γ, δ and ε by phorbol esters of different biological activities. FEBS Lett..1991; 288:5-9.CrossrefMedlineGoogle Scholar27 Mitchell MB, Meng X, Ao L, Brown JM, Harken AH, Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res..1995; 76:73-81.CrossrefMedlineGoogle Scholar28 Kitakaze M, Node K, Minamino T, Komamura K, Funaya H, Shinozaki Y, Chujo M, Mori H, Inoue M, Hori M, Kamada T. Role of activation of protein kinase C in the infarct size–limiting effect of ischemic preconditioning through activation of ecto-5′-nucleotidase. 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J Biol Chem..1995; 270:28092-28096.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByHochrainer K and Yang W (2022) Stroke Proteomics: From Discovery to Diagnostic and Therapeutic Applications, Circulation Research, 130:8, (1145-1166), Online publication date: 15-Apr-2022. 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