The platelet-activating factor signaling system and its regulators in
`syndromes of inflammation and thrombosis
`
`Guy A. Zimmerman, MD; Thomas M. McIntyre, PhD; Stephen M. Prescott, MD; Diana M. Stafforini, PhD
`
`Objectives: To review the platelet-activating factor (PAF) sig-
`naling system, its regulation, and its dysregulation in acute in-
`flammation and thrombosis and in syndromes that involve these
`cascades, including sepsis.
`Data Sources: A summary of published literature from MED-
`LINE search files and published reviews.
`Data Extraction, Synthesis, and Summary: PAF, a phospholipid
`signaling molecule, transmits outside-in signals to intracellular
`transduction systems and effector mechanisms in a variety of cell
`types, including key cells of the innate immune and hemostatic
`systems: neutrophils, monocytes, and platelets. Thus, the PAF
`signaling system is a point of convergence at which injurious
`stimuli can trigger and amplify both acute inflammatory and
`thrombotic cascades. The biological activities of PAF are regu-
`lated by several precise mechanisms that, together, constrain and
`control its action in physiologic inflammation. Unregulated syn-
`thesis of PAF or defects in the mechanisms that limit its biological
`activities have the potential to cause pathologic inflammation and
`thrombosis.
`In addition, nonenzymatic generation of oxidized
`phospholipids that are recognized by the PAF receptor can trigger
`inflammatory and thrombotic events. There is evidence that the
`
`PAF signaling system is dysregulated in sepsis, shock, and trau-
`matic injury and that interruption or termination of its effector
`responses leads to beneficial outcomes. Plasma PAF acetylhydro-
`lase, an enzyme that hydrolyzes PAF and structurally related
`oxidized phospholipids, yielding products that are no longer rec-
`ognized by the PAF receptor, may be a particularly important
`signal terminator.
`Conclusion: The PAF signaling system can trigger inflamma-
`tory and thrombotic cascades, amplify these cascades when
`acting with other mediators, and mediate molecular and cellular
`interactions (cross talk) between inflammation and thrombosis.
`Evidence from in vitro experiments, studies of experimental ani-
`mals, and clinical observations in humans indicates that the PAF
`signaling system is important in sepsis and other syndromes of
`inflammatory injury and that therapeutic strategies to interrupt or
`terminate signaling via the PAF signaling system may be useful in
`these conditions. (Crit Care Med 2002; 30[Suppl.]:S294 –S301)
`KEY WORDS: inflammation; platelet-activating factor; platelet-
`activating factor acetylhydrolase; platelet-activating factor–like
`lipid; platelet-activating factor receptor; sepsis; thrombosis
`
`T his focused review outlines
`
`key features of the platelet-
`activating factor (PAF) signal-
`ing system that are relevant to
`unregulated inflammation and thrombo-
`sis in sepsis, septic shock, and related
`syndromes of critical illness.
`
`PAF SIGNALING SYSTEM
`
`PAF. PAF is the central component in
`a system that has evolved to transmit
`
`From the Program in Human Molecular Biology and
`Genetics (GAZ, TMM), the Huntsman Cancer Institute
`(SMP, DMS), and the Departments of Internal Medicine
`(GAZ, TMM, SMP, DMS) and Pathology (TMM), Univer-
`sity of Utah, Salt Lake City, UT.
`Presented, in part, at the Margaux Conference on
`Critical Illness, Sedona, AZ, November 14 –18, 2001.
`Supported, in part, by the National
`Institutes of
`Health, the American Heart Association, and the Nora
`Eccles Treadwell Foundation.
`Address requests for reprints to: Guy A. Zimmer-
`man, MD, 15 North 2030 East, Building 533, Room
`4220, University of Utah, Salt Lake City, UT 84112.
`E-mail: guy.zimmerman@hmbg.utah.edu
`Copyright © 2002 by Lippincott Williams & Wilkins
`
`S294
`
`juxtacrine and paracrine signals between
`cells (1, 2). In some cases, PAF may also
`have endocrine, autocrine, and intercrine
`signaling roles (3– 6). A variety of cell
`types, many of which are central to the
`inflammatory and hemostatic systems,
`synthesize PAF. The enzymatic synthesis
`of PAF is highly regulated and most com-
`monly involves a two-step mechanism
`that has been characterized in endothe-
`lial cells and other inflammatory and vas-
`cular cells; additional synthetic mecha-
`nisms operate in other tissues (5, 6).
`PAF Receptor. The PAF receptor, a
`plasma membrane receptor that specifi-
`cally recognizes PAF and related PAF-like
`lipids, is a central component in the PAF
`signaling system. The structural features
`of PAF and related lipids that confer their
`selective recognition by the PAF receptor
`have been defined (6, 7). The plasma
`membrane receptor for PAF is a member
`of the seven membrane–spanning do-
`main G protein–linked superfamily that
`has been extensively studied in pharma-
`cologic experiments (8). The receptors
`
`from several species have been cloned
`and characterized at the molecular level
`(5, 6, 8, 9). The murine PAF receptor has
`been deleted by homologous recombina-
`tion and also overexpressed, yielding im-
`portant biological and pathophysiologic
`insights into the PAF signaling system
`(9 –13). Recently, a single nucleotide
`polymorphism in the human PAF recep-
`tor that has a significant frequency in the
`Japanese population was identified. The
`amino acid substitution, which occurs in
`the putative third cytoplasmic domain,
`partially impairs coupling of the receptor
`to intracellular signal transduction cas-
`cades (14). This may account for interin-
`dividual variation in responses to PAF,
`which occur in human subjects and in-
`bred mouse strains (15–18). The PAF re-
`ceptor undergoes homologous desensiti-
`zation (19 –22), a control mechanism
`that potentially limits its signaling ac-
`tions. Homologous desensitization was
`also used to characterize specific actions
`of PAF before the development of highly
`selective competitive antagonists (23). Al-
`
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`terations in expression of PAF receptor
`mRNA and protein occur in response to
`inflammatory and developmental stimuli
`in isolated cells and, presumably, in vivo
`(6, 24).
`PAF Receptor-Induced Signaling. The
`PAF receptor is coupled via G proteins to
`intracellular signaling enzymes and path-
`ways that regulate cytoplasmic calcium
`concentration, phosphatidylinositol turn-
`over, cyclic AMP levels, and phosphoryla-
`tion states of critical proteins (6, 9). The
`diversity of signaling cascades linked to
`the PAF receptor explains the varied and
`pleiotropic effector responses and func-
`tional changes in cells that are induced
`when it is engaged; this pattern of func-
`tional responses is cell specific and can be
`modulated by co-engagement of adhesion
`molecules or other surface receptors (2,
`25, 26).
`Many signaling events, and conse-
`quent functional responses, triggered by
`the PAF receptor occur in seconds to
`minutes and do not require new gene
`expression. However, ligation of the PAF
`receptor can also lead to nuclear signal-
`ing and transcriptional
`induction of
`genes involving nuclear factor-␬B and
`other transcription factors (3, 27). In ad-
`dition, we recently found that PAF in-
`duces activation of signal-dependent
`translation pathways in human platelets
`(28) and neutrophils (S Lindemann et al.,
`unpublished observations). The latter ob-
`servations demonstrate that the PAF re-
`ceptor modulates new gene expression by
`signaling to key posttranscriptional
`checkpoints,
`in addition to activating
`transcription.
`
`PAF IS A PIVOTAL MEDIATOR
`THAT LINKS THE HEMOSTATIC
`AND INNATE IMMUNE
`SYSTEMS
`
`The intimate relationship between the
`hemostatic and innate immune systems
`has been recognized for many years, and
`new mechanisms of convergence between
`these systems continue to be discovered.
`Acute inflammation is a requisite re-
`sponse mediated by the innate immune
`system that is critical in defense against
`microbial infection and in wound surveil-
`lance and repair. Hemostasis is an equally
`critical homeostatic response to injury.
`The PAF receptor is constitutively ex-
`pressed on human platelets and on key
`effector cells of the innate immune sys-
`tem—monocytes and polymorphonu-
`clear leukocytes (PMNs; neutrophils)—
`
`Crit Care Med 2002 Vol. 30, No. 5 (Suppl.)
`
`establishing PAF as a signaling molecule
`with the capacity to trigger both throm-
`botic and acute inflammatory events (Fig.
`1). These responses may be particularly
`important in sepsis, shock, and traumatic
`tissue injury, in which unregulated in-
`flammation and pathologic thrombosis
`are central pathophysiologic mechanisms
`and interplay between the two systems
`may amplify systemic manifestations and
`tissue injury (29).
`Platelet Aggregation. One of PAF’s
`earliest identified activities was in vitro
`activation of platelets isolated from ex-
`perimental animals, and this led to its
`trivial name (8). Intravenous infusion of
`PAF into baboons causes acute thrombo-
`cytopenia and neutropenia and was one of
`the earliest observations suggesting that
`it may have thrombotic and pro-inflam-
`matory effects in vivo (30). PAF was sub-
`sequently shown in other species to trig-
`ger in vivo aggregation and accumulation
`of platelets and consequent changes in
`local blood flow at sites of experimental
`thrombosis and vascular injury (31). PAF
`also activates human platelets (32) and
`induces aggregation at nanomolar con-
`centrations (33, 34). Platelet aggregation
`is mediated by inside-out signaling of in-
`tegrin ␣IIb␤3 and consequent binding of
`fibrinogen (35), indicating that the PAF
`receptor is linked to this prothrombotic
`intracellular pathway. Engagement of in-
`tegrin ␣IIb␤3 can then, in turn, mediate
`outside-in signaling and additional am-
`plification responses (35).
`Cytokine Synthesis by Platelets. Rapid
`aggregation, release of preformed media-
`tors, and synthesis of eicosanoids are re-
`sponses of platelets that have been recog-
`nized for many years. In addition to these
`“traditional” activities, our recent exper-
`iments indicate that platelets have a pre-
`viously unrecognized synthetic repertoire
`that can be activated by PAF and other
`agonists. Freshly isolated human plate-
`lets carry many constitutive messenger
`RNAs (mRNAs) that were transcribed at
`the nucleated megakaryocyte stage. This
`had been recognized earlier, but more
`recently, we further documented the
`presence of constitutive mRNAs by using
`interrogation of arrayed complementary
`DNA libraries (28, 36). In response to
`appropriate activating signals, some of
`these mRNAs are translated to their cor-
`responding proteins in a highly regulated
`manner (28, 36, 37). Intracellular com-
`partmentalization and cytoskeletal asso-
`ciation of critical translation control fac-
`tors is an important mechanism (38).
`
`Figure 1. The platelet-activating factor (PAF) sig-
`naling system is a point of convergence and am-
`plification of the thrombotic and inflammatory
`cascades. PAF and oxidatively modified phospho-
`lipids (Ox-PLs) are recognized by a signal-
`transducing receptor (PAF receptor) that is con-
`stitutively expressed by key cells of
`the
`hemostatic and innate immune systems: human
`platelets, neutrophils, and monocytes. Pathologic
`inflammatory and thrombotic responses can be
`triggered if mechanisms that generate PAF or
`other ligands recognized by the PAF receptor are
`dysregulated or impaired. Signaling through the
`PAF receptor can also indirectly induce proco-
`agulant events. Because the PAF signaling system
`can induce, amplify, and mediate cross talk be-
`tween hemostasis and inflammation, it is a point
`of convergence that may be critical when these
`cascades are induced in pathologic syndromes.
`PMNs, polymorphonuclear leukocytes.
`
`mRNA for interleukin (IL)-1␤ is one of
`the transcripts that is translated in a
`rapid and sustained fashion. Signal-
`dependent translation of pro–IL-1␤ and
`processing of this precursor to the ma-
`ture protein can be induced by PAF,
`thrombin, and certain other agonists
`(28). Newly synthesized IL-1␤ is released
`from activated platelets in microvesicles
`and accumulates in the fibrin matrix in a
`model of platelet-fibrin clot formation,
`suggesting that
`the platelet-fibrin
`thrombi may be reservoirs for cytokines
`and other inflammatory signaling mole-
`cules. In addition, IL-1␤ released from
`stimulated platelets induces human en-
`dothelial cells to become adhesive for
`PMNs (28), a response previously shown
`to be dependent on new gene expression
`and synthesis of E selectin and chemo-
`kines (39, 40). Thus, this sequence of
`events represents a new mechanism link-
`ing the thrombotic and inflammatory
`cascades (28) and adds to the ways in
`which PAF can mediate interplay between
`these systems (41). Because IL-1 induces
`
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`expression of a variety of genes in endo-
`thelial cells (42– 44), additional
`func-
`tional alterations in the endothelium, in-
`cluding
`procoagulant
`and
`pro-
`inflammatory phenotypic changes, may
`be stimulated by its synthesis and release
`from activated platelets (28). In addition,
`IL-1 stimulation of endothelial cells in-
`duces genes that are involved in the
`switch from neutrophil accumulation to
`mononuclear leukocyte trafficking and
`chronic inflammation (43, 44).
`PAF-Induced PMN Responses. The ac-
`tivation responses of myeloid leukocytes
`that are triggered by signals delivered
`through the PAF receptor are even more
`diverse than those in platelets. When PAF
`is rapidly produced and displayed on the
`surfaces of human endothelial cells that
`have been stimulated with thrombin and
`certain other agonists, it acts as a juxta-
`crine signal for spatially regulated activa-
`tion and adhesion of neutrophils (1, 2, 23,
`45). This provided the first evidence that
`human endothelium can synthesize and
`locally express signaling molecules for
`leukocytes (1, 23, 33). PAF has the poten-
`tial to trigger a variety of responses in
`PMNs, whether it is locally displayed by
`endothelial cells in physiologic inflam-
`mation or synthesized by endothelium or
`other cells in a dysregulated fashion in
`pathologic syndromes (5, 41, 46). Activa-
`tion of PMNs via the PAF receptor in-
`duces inside-out signaling of ␤2 (CD11/
`CD18)
`integrins with consequent
`adhesiveness and aggregation, priming
`for enhanced inflammatory responses,
`polarization and directional migration,
`degranulation, and oxygen radical gener-
`ation. Each of these effector activities is
`rapidly triggered with constitutive signal
`transduction systems (8). Priming for
`augmented release of granular factors
`may be a critical neutrophil response to
`PAF (13, 25) because granular enzymes,
`such as elastase, mediate inflammatory
`tissue injury and also have the capacity to
`induce coagulation when they are locally
`released (41, 47). It is likely that one or
`more of these PAF-mediated activation
`responses is particularly important in
`syndromes of inflammatory tissue dam-
`age, such as acute lung injury (13). In
`addition to these effector responses,
`which do not require new gene expres-
`sion, PAF induces transcriptional events
`in PMNs; furthermore, it is a remarkably
`potent stimulus for signal-dependent
`translation of a subset of mRNAs in hu-
`man neutrophils (S Lindemann et al.,
`unpublished observations) (48). Thus,
`
`S296
`
`previously unrecognized activation re-
`sponses of neutrophils continue to be
`identified, and some are induced by PAF.
`PAF-Induced Monocyte Responses.
`Human monocytes also bear the PAF re-
`ceptor on their plasma membranes and
`are activated when it is ligated (8). A
`critical function of these cells, which are
`ubiquitous in inflammatory syndromes
`ranging from sepsis to atherosclerosis
`(27, 49, 50), is synthesis of chemokines,
`cytokines, tissue factor, and other medi-
`ators. In addition to the PAF receptor,
`monocytes express P selectin glycopro-
`tein ligand-1, which is a ligand for P
`selectin that mediates adhesive interac-
`tions with stimulated endothelial cells
`and platelets (Fig. 2). Human monocytes
`adherent to P selectin respond to PAF
`with enhanced translocation of nuclear
`factor-␬B to the nucleus and dramatically
`increased synthesis of monocyte che-
`moattractant protein-1, IL-8, tumor ne-
`crosis factor-␣, and other inflammatory
`gene products (27). Nuclear signaling
`and altered gene expression is prominent
`when monocytes establish stable adhesive
`interactions with thrombin-stimulated
`platelets (51), a cell-cell interaction that
`occurs in thrombosis and a variety of
`vascular syndromes (52).
`PAF Synthesis by Critical Cells of the
`Hemostatic and Inflammatory Systems.
`An additional feature that indicates that
`PAF is an important mediator at points of
`convergence between the hemostatic and
`acute inflammatory cascades is its syn-
`thesis by critical cells that are involved in
`these systems. As outlined above, human
`endothelial cells rapidly synthesize PAF
`and use it as a juxtacrine signal for neu-
`trophils (1, 2, 4 – 6, 23, 33, 41, 45). In this
`context, it acts coordinately with P selec-
`tin, which is rapidly translocated to the
`surfaces of
`inflamed endothelial cells
`from intracellular storage granules (Fig.
`2) (2, 25, 45). These observations have
`been confirmed by many laboratories,
`and there is evidence that PAF at the cell
`surface can perform its signaling func-
`tion in vitro and in vivo under flow con-
`ditions when leukocytes are subjected to
`shear forces (45). Notably, PAF synthesis
`by endothelial cells is stimulated by an-
`other pivotal hemostatic and inflamma-
`tory agonist: thrombin (23, 33, 53). A
`variety of other inflammatory mediators,
`oxidants, and bacterial toxins that induce
`thrombosis and inflammation in patho-
`logic syndromes also induce PAF synthe-
`sis (8, 41, 45, 46). In addition, PAF is
`synthesized by adherent, activated plate-
`
`Figure 2. Platelet-activating factor (PAF) mediates
`juxtacrine signaling at the surfaces of inflamed en-
`dothelial cells and adherent, activated platelets. A,
`PAF mediates juxtacrine signaling of neutrophils in
`vitro when it is rapidly synthesized and translocated
`to the plasma membranes of human endothelial
`cells stimulated with thrombin. Neutrophil re-
`sponses that are triggered by the PAF juxtacrine
`signal include activation of ␤2 integrins, which con-
`tribute to tight adhesion of the leukocytes to the
`endothelial surface together with tethering initially
`provided by P selectin. Increased intracellular cal-
`cium, shape change and polarization, and priming
`for enhanced granular secretion and oxygen radical
`generation are additional effector responses that are
`induced via the PAF receptor. B, the P selectin–PAF
`juxtacrine system provides a molecular basis for
`rolling, tight adhesion, and emigration of neutro-
`phils in vivo (45). Bottom, P selectin and PAF are
`displayed on the surfaces of adherent, activated
`platelets and mediate juxtacrine signaling, which
`triggers activation of ␤2 integrins on the leukocytes
`in a fashion analogous to that occurring at the
`surfaces of thrombin-stimulated endothelial cells (A
`and B). This system also provides a molecular basis
`for rolling, tight adhesion, and localization of neu-
`trophils. Juxtacrine signaling of neutrophils by PAF
`displayed by activated platelets is one of the mech-
`anisms of cross talk between the thrombotic and
`inflammatory cascades that is mediated by the PAF
`signaling system. PMN, polymorphonuclear leuko-
`cyte; PSGL-1, P selectin glycoprotein ligand-1.
`Adapted with permission from Dixon et al (52).
`
`lets. A tethering and juxtacrine signaling
`system at the platelet surface analogous
`to that on the plasma membranes of en-
`dothelial cells mediates rolling and tight
`
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`

`adhesion of PMNs (Fig. 2) (45, 54, 55).
`Thus, considerable evidence indicates
`that PAF is strategically located and sig-
`nals target cells at key sites in the molec-
`ular topography of
`inflammation and
`thrombosis by virtue of its synthesis by
`endothelial cells and platelets acting in
`response to relevant stimuli.
`
`PAF MAY BE A CRITICAL
`MEDIATOR IN SHOCK, SEPSIS,
`AND TRAUMA
`
`The synthesis of PAF is highly regu-
`lated. In addition, several other mecha-
`nisms have evolved to control its biolog-
`ical activities. These include spatial
`regulation of signaling (juxtacrine activa-
`tion of leukocytes and other target cells
`by membrane-associated PAF), cell-
`specific expression of its receptor, recep-
`tor desensitization, and degradation of
`PAF by plasma and cellular hydrolases
`(Fig. 3). Evolution of a precise set of
`regulatory checks and balances for the
`PAF signaling system strongly indicates
`that it has physiologic roles. A corollary is
`that PAF, and related molecules that are
`recognized by the PAF receptor, may be
`mediators of injury if their generation is
`
`Figure 3. The platelet-activating factor (PAF) sig-
`naling system is regulated by multiple control
`mechanisms. The plasma PAF acetylhydrolase is
`a particularly important regulator of the PAF
`signaling system because it limits the half-life of
`PAF to minutes in whole human blood and also
`terminates signaling upstream from the PAF re-
`ceptor. Plasma PAF acetylhydrolase degrades PAF
`at cellular surfaces and in solution. PMN, poly-
`morphonuclear leukocyte.
`
`Crit Care Med 2002 Vol. 30, No. 5 (Suppl.)
`
`inappropriate or unregulated or if control
`mechanisms that limit their biological
`activities are inoperative or circum-
`vented. Evidence that PAF has injurious
`actions in shock and syndromes of tissue
`damage is consistent with this idea. In-
`jection of PAF into experimental animals
`causes hypotension and other features of
`anaphylactic or septic shock and induces
`sequelae that include gastric mucosal
`erosions, ischemic bowel necrosis, and
`cerebral ischemia (56). The latter obser-
`vations suggest that PAF, or PAF-like lip-
`ids, contributes to multiple organ dys-
`function or failure, in addition to shock
`states. Furthermore, PAF activity is in-
`creased in blood or tissue samples in
`some of these models, and PAF receptor
`antagonists ameliorate tissue injury in
`many of these experimental syndromes
`(56). Activation of target leukocytes via
`the PAF receptor may be a mechanism of
`injury in experimental hemorrhagic
`shock (57) and in models of anaphylaxis
`and other shock states (56).
`PAF and PAF-like Lipids as Mediators
`of Sepsis. Many observations in experi-
`mental animals suggest that PAF or PAF-
`like lipids are mediators of septic shock
`and the sepsis syndrome (56, 58). Among
`these is evidence that PAF contributes to
`acute sequestration of neutrophils and
`their adhesion to endothelial cells after
`endotoxin (lipopolysaccharide) adminis-
`tration (59) and to induction of nitric
`oxide synthase in experimental endotox-
`emia (60). A provocative observation is
`that overexpression of the PAF receptor
`increases lethality in response to lipo-
`polysaccharide administration in mice
`(10).
`In humans, an initial observation was
`the presence of intravascular PAF activity
`in children with sepsis (61). Another
`early study demonstrated that platelets
`from patients with sepsis have reduced
`numbers of binding sites for PAF, indi-
`cating receptor occupancy; in addition,
`there was increased bioactivity character-
`istic of PAF in samples from septic pa-
`tients when compared with controls (62).
`In subsequent studies, increased PAF bio-
`activity was also reported in plasma sam-
`ples from patients with bacteremia com-
`pared with blood from control subjects
`(63) and in samples from patients with
`septic shock and trauma (64). Plasma
`PAF acetylhydrolase (PAF AH) activity
`was reported to be reduced in critically ill
`patients with the clinical diagnosis of sep-
`sis (65, 66). In addition, reduced PAF AH
`activity was correlated with multiple or-
`
`gan failure in patients with critical ill-
`nesses, some of whom had sepsis (67).
`Recently, neutrophils from septic pa-
`tients were found to have substantially
`increased adhesion to immobilized plate-
`lets (68) under conditions in which PAF
`and P selectin are co-expressed (Fig. 2)
`(54, 55). Together, these findings suggest
`that PAF may be a critical mediator in
`human sepsis and its complications.
`PAF Receptor Antagonists in Sepsis.
`Preliminary data from therapeutic trials
`with PAF receptor antagonists supported
`a role for the PAF signaling system in
`sepsis caused by Gram-negative organ-
`isms (69, 70). Although no regimen based
`on blockade of the PAF receptor that is
`clearly efficacious in human sepsis has
`subsequently emerged, neither have
`there been persuasive observations indi-
`cating that this strategy is not rational
`(71). However, it is possible that termi-
`nation of signals upstream in the signal-
`ing cascade may be more effective than
`competing with PAF and PAF-like lipids
`downstream at the receptor level. Most
`receptor antagonists developed to date
`bind to the receptor with equal or lesser
`efficiency than PAF itself (8), necessitat-
`ing a concentration of the antagonist sev-
`eral-fold higher than the natural ligand
`to reduce cellular effector responses to
`very low levels (i.e., ⬍50%, which may
`not be sufficient to yield a clinical bene-
`fit) or to block them entirely. This may be
`difficult to achieve in a sustained fashion
`in sick patients. In addition, the effective
`concentration of PAF may be very high at
`cellular surfaces where it acts in a juxta-
`crine fashion (Figs. 2 and 3), making
`competition by an antagonist more diffi-
`cult.
`
`OXIDIZED PHOSPHOLIPIDS:
`INFLAMMATORY PAF-LIKE
`SIGNALING MOLECULES THAT
`ARE GENERATED IN AN
`UNREGULATED FASHION
`
`Enzymatic synthesis of PAF is not the
`only mechanism by which cellular acti-
`vation triggered through the PAF recep-
`tor is induced; this feature of the PAF
`signaling system is important when con-
`sidering its role in pathologic states. Ox-
`idized, or oxidatively modified, phospho-
`lipids are generated by oxidant attack on
`hydrogen atoms adjacent to olefinic dou-
`ble bonds in unsaturated fatty acid side-
`chains (7). Oxidation of phosphatidylcho-
`lines, which are structural phospholipids
`in the membranes of all cells, generates a
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`

`large series of phospholipids in which the
`polyunsaturated fatty acid at the sn-2 po-
`sition, which is often arachidonic acid, is
`fragmented to shorter chain lengths.
`Some of these oxidized phospholipids
`have sufficiently short sn-2 residues and
`other structural features that allow them
`to be recognized by the PAF receptor (7).
`Other compounds, including phospholip-
`ids that bind to nuclear peroxisome pro-
`liferator-activated receptors, are also pro-
`duced (7). Analysis of the structural
`features and biological characteristics of
`the subset of oxidized phospholipids with
`PAF-like activity has been accomplished
`using neutrophils, monocytes, and other
`primary cells that bear the PAF receptor,
`heterologous cells expressing the cloned
`PAF receptor, and other strategies (7, 72).
`Together, these studies demonstrate that
`oxidized PAF-like lipids ligate the PAF
`receptor and trigger responses that are
`similar to those induced by PAF itself (G
`Marathe et al., unpublished observa-
`tions), although the binding affinities of
`the oxidatively modified compounds vary
`depending on specific structural features
`of the individual oxidized phospholipid.
`There is considerable evidence that
`oxidized phospholipids with PAF-like ac-
`tivity are produced in inflammatory and
`vascular conditions in vivo (7, 46, 72). A
`key point is that they are generated by
`free radical attacks on membrane phos-
`pholipids, which are unregulated reac-
`tions (7). This contrasts with enzymatic
`synthesis of PAF in endothelial cells and
`other cells in response to receptor-
`mediated agonists, oxidants, and other
`stimuli, which are highly regulated and
`often of short duration (5, 6, 41). In prin-
`ciple, oxidized phospholipids can be gen-
`erated in high local concentrations on an
`ongoing basis if there is a continuous
`source of oxygen radicals and if mem-
`brane phospholipid precursors are not
`depleted. Thus, oxidized phospholipids
`with PAF-like activity may be equally or
`more important compared with PAF itself
`in ischemia-reperfusion and other conse-
`quences of shock and resuscitation and
`also in sustained inflammatory events
`that involve generation of oxygen radi-
`cals. High, local concentrations of these
`PAF-like ligands may be difficult to block
`with receptor antagonists in clinical syn-
`dromes for reasons outlined above. A sec-
`ond important point is that oxidized PAF-
`like phospholipids can be released from
`injured cells in membrane microvesicles
`(46, 73), a mechanism that disrupts spa-
`tially regulated juxtacrine signaling and
`
`S298
`
`may propagate intravascular activation of
`leukocytes and other target cells (4, 5, 41,
`45, 52).
`
`PLASMA PAF AH: A SIGNAL
`TERMINATOR
`
`PAF AHs are enzymes that recognize
`PAF and PAF-like oxidized lipids as sub-
`strates. They cleave the short sn-2 resi-
`dues to yield products that are no longer
`recognized by the PAF receptor when
`they are present in physiologic concen-
`trations (74). The preference for short
`acyl groups at the second position of the
`glycerol backbone prevents these en-
`zymes from attacking “building block”
`phospholipids in cell membranes. A
`group of structurally diverse isoenzymes,
`including both intracellular and extracel-
`lular proteins, have this unusual sub-
`strate selectivity and other distinctive
`properties (74).
`Plasma or Secreted Form of PAF AH.
`The plasma or secreted form of PAF AH
`(74, 75) has been called a signal termina-
`tor (76). The primary structure of plasma
`PAF AH is unique and includes only a
`small region of homology, a GXSXG mo-
`tif, with esterases and other lipases. An
`active site triad, which was determined by
`site-directed mutagenesis, is critical for
`its catalytic properties and is similar to
`that in several neutral lipases (74). The
`plasma enzyme limits the half-life of PAF
`to a few minutes in whole human blood
`(77, 78). Plasma PAF AH cleaves oxida-
`tively modified PAF-like lipids with an
`efficiency similar to that of PAF as a sub-
`strate (74). The secreted form of PAF AH
`is constitutively present in plasma in
`tight association with low- and high-
`density lipoproteins (74, 78).
`Expression of Plasma PAF AH by Mac-
`rophages. Extensive biochemical and bi-
`ological characterization and partial pu-
`rification (74, 79) led to cloning of the
`enzyme (75). The discovery that the gene
`for plasma PAF AH is induced when hu-
`man monocytes differentiate into macro-
`phages in culture was among the critical
`observations of the cloning strategy (75,
`80, 81). Subsequent observations con-
`firmed that plasma PAF AH is produced
`by macrophages (6, 82), and studies of
`patients with allogenic bone marrow
`transplantation indicate that plasma PAF
`AH is largely derived from hematopoietic
`lineage cells (83). Thus, during inflam-
`matory states, plasma PAF AH may be a
`marker for macrophage activation or ex-
`pansion. Analysis of the promoter for the
`
`plasma PAF AH gene demonstrated that it
`contains response elements for inflam-
`matory and myeloid-specific transcrip-
`tion factors and that it is transcriptionally
`regulated during macrophage differenti-
`ation and by mediators of inflammation
`(82). Hormonal stimuli, glucocorticoids,
`and PAF itself modulate expression of
`plasma PAF AH by macrophages (74, 82).
`Plasma PAF AH as a Terminator of
`Inflammation. Many observations indi-
`cate that plasma PAF AH terminates sig-
`nals by PAF and oxidized PAF–like lipids
`and thereby regulates inflammatory re-
`sponses. These include in vitro experi-
`ments and in vivo observations in animal
`models and in humans (5, 6, 56, 84).
`Partially purified and recombinant forms
`of plasma PAF AH block inflammatory
`responses of human leukocytes to PAF (1,
`55, 75, 84, 85) and inhibit responses to
`exogenous PAF in experimental animals
`(75), thus providing important proofs of
`principle. In addition, genetic, develop-
`mental, and acquired deficiency states of
`plasma PAF AH have been identified in
`humans and are correlated with severity
`or negative outcomes in inflammatory
`and thrombotic conditions that include
`asthma and cardiovascular syndromes,
`necrotizing enterocolitis, hemolytic ure-
`mic syndrome, and sepsis (Fig. 4) (5, 56,
`84). Loss-of-function mutations leading
`to hereditary deficiency of plasma PAF
`AH activity occur, and molecular charac-
`terization of these variants has been ac-
`complished (84, 86, 87). These muta-
`tions, together with naturally occurring
`polymorphisms that alter the catalytic
`properties of the enzyme, may contribute
`to the spectrum of severity in inflamma-
`tory syndromes such as asthma, atopy,
`and perhaps, other complex inflamma-
`tory diseases (84).
`Decreased Plasma PAF AH Activity in
`Sepsis. Studies in different populations
`
`Figure 4. Plasma platelet-activating factor (PAF)
`acetylhydrolase is deficient in clinical syndromes
`of
`inflammation, thrombosis, and injury. Ac-
`quired deficiency of plasma PAF acetylhydrolase
`may be important in sepsis and multiple organ
`failure, in addition to genetic and developmental
`deficiency in other