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Ryan M Kramer, Michelle C Archer, Mark T Orr, Natasha Dubois Cauwelaert, Elyse A Beebe, Po-wei D Huang, Quinton M Dowling, Alicia M Schwartz, Dawn M Fedor, Thomas S Vedvick, Christopher B Fox Infectious Disease Research Institute, Seattle, WA, USA Background: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). Materials and methods: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. Results: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. Conclusion: These efforts will aid in the development of a platform formulation for use with other similar vaccines. Keywords: adjuvant, lyophilization, tuberculosis, formulation development, design of experiments, controlled temperature chain, GRAS

This study addresses an important but neglected topic by investigating the social judgment processes that experts (studio executives and producers in Hollywood) use to assess the creative potential of unknown others (relatively unknown screenwriters) during "pitch" meetings in which screenwriters attempt to sell their ideas. The findings suggest a dual-process social judgment model. In one process, person categorization, the experts used behavioral and physical cues to match "pitchers" with seven creative and uncreative prototypes. In another process, relationship categorization, the experts used relational cues and self-perceptions to match pitchers with two relational prototypes.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel.

This research investigates how organizational members respond to events that threaten their perceptions of their organization's identity. Using qualitative, interview, and records data, we describe how menebers from eight "top-20" business schools responded to the 1992 Business Week survey rankings of U.S. business schools. Our analysis suggests that the rankings posed a two-pronged threat to many members' perceptions of their schools' identities by (1) calling into question their perceptions of highly valued, core identity attributes of their schools, and (2) challenging their beliefs about their schools' standing relative to other schools. In response, members made sense of these threats and affirmed positive perceptions of their school's identity by emphasizing and focusing on their school's membership in selective organizational categories that highlighted favorable identity dimensions and interorganizational comparisons not recognized by the rankings. Data suggest that members' use of these categorization tactics depended on the level of identity dissonance they felt following the rankings. We integrate these findings with insights from social identity, self-affirmation, and impression management theories to develop a new framework of organizational identity management.

With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis ( Mtb ). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development., Competing Interests: CBF and RMK are co-inventors on patents claiming priority to WO/2015/103167, “Single Vial Vaccine Formulations,” and CBF is a co-inventor on WO/2013/119856, “Improved Adjuvant Formulations Comprising TLR4 Agonists and Methods of Using the Same.” The remaining authors declare that the research was conducted in the absence of any personal or financial relationships that could be construed as a potential competing interest., (© 2023 The Authors.)

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls-1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection., Competing Interests: MG, NC, MCA, RK, RV, and CF are inventors on a patent application involving spray-dried vaccine adjuvant compositions and methods, and CF is an inventor on a patent involving oil-in-water emulsions with low oil content including GLA-SE. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gomez, Ahmed, Das, McCollum, Mellett, Swanson, Gupta, Carrigy, Wang, Barona, Bachchhav, Gerhardt, Press, Archer, Liang, Seydoux, Kramer, Kuehl, Vehring, Khader and Fox.)

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness., Competing Interests: 7Conflict of Interest CF and RK are co-inventors on patents claiming priority to WO/2015/103167, “Single Vial Vaccines,” and WO/2013/119856, “Improved Adjuvant Formulations Constituting TLR4 Agonists and Methods of Using Same.” TD and ET are/were employed by Lyophilization Technology, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Antimicrobial peptides (AMPs) constitute promising alternatives to classical antibiotics for the treatment of drug-resistant infections, which are a rapidly emerging global health challenge. However, our understanding of the structure-function relationships of AMPs is limited, and we are just beginning to rationally engineer peptides in order to develop them as therapeutics. Here, we leverage a physicochemical-guided peptide design strategy to identify specific functional hotspots in the wasp-derived AMP polybia-CP and turn this toxic peptide into a viable antimicrobial. Helical fraction, hydrophobicity, and hydrophobic moment are identified as key structural and physicochemical determinants of antimicrobial activity, utilized in combination with rational engineering to generate synthetic AMPs with therapeutic activity in a mouse model. We demonstrate that, by tuning these physicochemical parameters, it is possible to design nontoxic synthetic peptides with enhanced sub-micromolar antimicrobial potency in vitro and anti-infective activity in vivo. We present a physicochemical-guided rational design strategy to generate peptide antibiotics., Competing Interests: The authors declare no competing interests.

The Gcn5 histone acetyltransferase (HAT) subunit of the SAGA transcriptional coactivator complex catalyzes acetylation of histone H3 and H2B N-terminal tails, posttranslational modifications associated with gene activation. Binding of the SAGA subunit partner Ada2 to Gcn5 activates Gcn5's intrinsically weak HAT activity on histone proteins, but the mechanism for this activation by the Ada2 SANT domain has remained elusive. We have employed Fab antibody fragments as crystallization chaperones to determine crystal structures of a yeast Ada2/Gcn5 complex. Our structural and biochemical results indicate that the Ada2 SANT domain does not activate Gcn5's activity by directly affecting histone peptide binding as previously proposed. Instead, the Ada2 SANT domain enhances Gcn5 binding of the enzymatic cosubstrate acetyl-CoA. This finding suggests a mechanism for regulating chromatin modification enzyme activity: controlling binding of the modification cosubstrate instead of the histone substrate., Competing Interests: The authors declare no conflict of interest.

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligo-oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore., (Copyright © 2013 The Protein Society.)

The development of vaccines containing adjuvants has the potential to enhance antibody and cellular immune responses, broaden protective immunity against heterogeneous pathogen strains, enable antigen dose sparing, and facilitate efficacy in immunocompromised populations. Nevertheless, the structural interplay between antigen and adjuvant components is often not taken into account in the published literature. Interactions between antigen and adjuvant formulations should be well characterized to enable optimum vaccine stability and efficacy. This review focuses on the importance of characterizing antigen-adjuvant interactions by summarizing findings involving widely used adjuvant formulation platforms, such as aluminum salts, emulsions, lipid vesicles, and polymer-based particles. Emphasis is placed on the physicochemical basis of antigen-adjuvant associations and the appropriate analytical tools for their characterization, as well as discussing the effects of these interactions on vaccine potency.

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.

Will we ever learn? We'd barely recovered from Enron and WorldCom before we faced the subprime mortgage meltdown and more scandals that shook our trust in businesspeople. Which raises the question: Do we trust too much? In this article, Stanford professor and social psychologist Kramer explores the reasons we trust so easily--and, often, so unwisely. He explains that genetics and childhood learning make us predisposed to trust and that it's been a good survival mechanism. That said, our willingness to trust makes us vulnerable. Our sense of trust kicks in on remarkably simple cues, such as when people look like us or are part of our social group. We also rely on third parties to verify the character of others, sometimes to our detriment (as the victims of Bernard Madoff learned). Add in our illusions of invulnerability and our tendencies to see what we want to see and to overestimate our own judgment, and the bottom line is that we're often easily fooled. We need to develop tempered trust. For those who trust too much, that means reading cues better; for the distrustful, it means developing more receptive behaviors. Everyone should start with small acts of trust that encourage reciprocity and build up. Having a hedge against potential abuses also helps. Hollywood scriptwriters, for instance, register their treatments with the Writers Guild of America to prevent their ideas from being stolen by the executives they pitch. To attract the right relationships, people must strongly signal their own honesty, proactively allay concerns, and, if their trust is abused, retaliate. Trusting individuals in certain roles, which essentially means trusting the system that selects and trains them, also works but isn't foolproof. And don't count on due diligence alone for protection; constant vigilance is needed to make sure the landscape hasn't changed.

We examine whether racial/ethnic/language-based variation in measured levels of patients' trust in a physician depends on the survey items used to measure that trust. Survey items include: (1) a direct measure of patients' trust that the doctor will put the patient's medical needs above all other considerations, and (2) three indirect measures of trust asking about expectations for specific physician behaviors, including referring to a specialist, being influenced by insurance rules, and performing unnecessary tests. Using a national survey, we find lower scores on indirect measures of trust in a physician among minority users of health care services than among non-Hispanic white users. In contrast, the direct measure of trust does not differ among non-Hispanic whites and nonwhites once we control for potential confounding factors. The results indicate that racial/ethnic/language-based differences exist primarily in those aspects of patients' trust in a physician that reflect specific physician behaviors.

After Disney's Michael Eisner, Miramax's Harvey Weinstein, and Hewlett-Packard's Carly Fiorina fell from their heights of power, the business media quickly proclaimed thatthe reign of abrasive, intimidating leaders was over. However, it's premature to proclaim their extinction. Many great intimidators have done fine for a long time and continue to thrive. Their modus operandi runs counter to a lot of preconceptions about what it takes to be a good leader. They're rough, loud, and in your face. Their tactics include invading others' personal space, staging tantrums, keeping people guessing, and possessing an indisputable command of facts. But make no mistake--great intimidators are not your typical bullies. They're driven by vision, not by sheer ego or malice. Beneath their tough exteriors and sharp edges are some genuine, deep insights into human motivation and organizational behavior. Indeed, these leaders possess political intelligence, which can make the difference between paralysis and successful--if sometimes wrenching--organizational change. Like socially intelligent leaders, politically intelligent leaders are adept at sizing up others, but they notice different things. Those with social intelligence assess people's strengths and figure out how to leverage them; those with political intelligence exploit people's weaknesses and insecurities. Despite all the obvious drawbacks of working under them, great intimidators often attract the best and brightest. And their appeal goes beyond their ability to inspire high performance. Many accomplished professionals who gravitate toward these leaders want to cultivate a little "inner intimidator" of their own. In the author's research, quite a few individuals reported having positive relationships with intimidating leaders. In fact, some described these relationships as profoundly educational and even transformational. So before we throw out all the great intimidators, the author argues, we should stop to consider what we would lose.

Women carrying BRCA1 and BRCA2 mutations face difficult and confusing reproductive decisions that fall into three categories: issues relating to risk-reducing surgeries, issues relating to use of oral contraceptives/tubal ligation, and issues relating to pregnancy and breastfeeding. Risk-reducing surgeries may confer survival benefits, but they also affect quality of life. Oral contraceptives potentially protect mutation carriers against ovarian cancer but increase the risk of early-onset breast cancer, and evidence for the efficacy of tubal ligation in reducing ovarian cancer risk in BRCA mutation carriers is contradictory. Women with BRCA mutations may increase their risk of breast cancer by becoming pregnant before age 40 years, but breastfeeding may decrease risk of breast cancer in women with BRCA mutations, regardless of age. BRCA mutation carriers desiring to become pregnant must deal with a variety of psychosocial issues, some with significant ethical implications, with minimal guidance from research.

The past decade may well be remembered as the era of the high-flying, aggressive leader. Corner-office titans like Kenneth Lay, Dennis Kozlowski, and Bernard Ebbers graced the covers of business magazines. They captured the public's fascination with their bold business moves and charismatic sound bites. Then scandal set in, and the stars fell to earth. In this article, social psychologist Roderick M. Kramer asks an important question: Why do so many leaders--not just in business, but also in politics, religion, and the media--display remarkable adeptness and ability while courting power, only to engage in even more remarkable bouts of folly once that power has been secured? Kramer, who has spent most of his career researching how leaders get to the top, says there is something about the process of becoming a leader that changes people in profound ways. The systems through which we select our leaders force executives to sacrifice the attitudes and behaviors that are essential to their survival once they have reached the top. Society has learned to consider risk taking and rule breaking as markers of good leadership. As a result, CEOs and other leaders lack the modesty and prudence needed to cope with the rewards and trappings of power. They come to believe that normal limits don't apply to them and that they are entitled to any spoils they can seize. The leaders who do remain grounded--who get to the top and stay there--exhibit five common psychological and behavioral habits: They simplify their lives, remaining humble and "awfully ordinary." They shine a light on their weaknesses instead of trying to cover them up. They float trial balloons to uncover the truth and prepare for the unexpected. They sweat the small stuff. And they reflect more, not less.

On September 11, 2001, in the space of a few horrific minutes, Americans realized the fragility of trust. The country's evident vulnerability to deadly terrorism rocked our faith in the systems we rely on for security. Our trust was shaken again only a few months later with the stunning collapse of Enron, forcing us to question many of the methods and assumptions underpinning the way we work. These two crises are obviously very different, yet both serve as reminders of the perils of trusting too much. The abiding belief that trust is a strength now seems dangerously naive. This new doubtfulness runs contrary to most management literature, which has traditionally touted trust as an organizational asset. It's an easy case to make. When there are high levels of trust, employees can fully commit themselves to the organization because they can be confident that their efforts will be recognized and rewarded. Trust also means that leaders don't have to worry so much about putting the right spin on things. They can act and speak forthrightly and focus on essentials. In short, trust is an organizational superglue. Nevertheless, two decades of research on trust and cooperation in organizations have convinced social psychologist Roderick Kramer that--despite its costs--distrust can be beneficial in the workplace. Kramer has observed that a moderate form of suspicion, which he calls prudent paranoia, can in many cases prove highly beneficial to the distrustful individual or organization. In this article, he describes situations in which prudent paranoia makes sense and shows how, when properly deployed, it can serve as a powerful morale booster--even a competitive weapon--for organizations.

Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A2 (cPLA2). We propose to call these cPLA2beta (114 kDa) and cPLA2gamma (61 kDa), giving the name cPLA2alpha to the well known 85-kDa enzyme. cPLA2beta mRNA is expressed more highly in cerebellum and pancreas and cPLA2gamma more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA2beta on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA2beta is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA2beta has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA2gamma on chromosome 19 near calmodulin. cPLA2gamma lacks the C2 domain, which gives cPLA2alpha its Ca2+ sensitivity, and accordingly cPLA2gamma has no dependence upon calcium, although cPLA2beta does. cPLA2gamma contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA2alpha residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA2alpha, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A2 (cPLA2) in human platelets and HeLa cells. Tryptic digests of 32P-phosphorylated and -immunoprecipitated cPLA2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-alpha or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stress-activated protein kinase (p38(mapk)), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38(mapk) inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA2 expressed in a baculovirus/insect cell system to cPLA2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA2 in different mammalian cells stimulated with multiple agonists.

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

The 85-kDa cytosolic PLA2 (cPLA2) is present in many cells and tissues and its unusual functional properties and catalytic mechanism are being elucidated. Notably, cPLA2 becomes catalytically active in the presence of free Ca2+ concentrations as present in stimulated cells and preferentially cleaves arachidonic acid-containing phospholipids. A variety of agonists, growth factors and cytokines, as well as stressful stimuli activate cPLA2 to hydrolyze cellular phospholipids thereby liberating fatty acids and lysophospholipids and providing the precursor substrates for the biosynthesis of eicosanoids and platelet-activating factor. These products of cPLA2 contribute to inflammatory and degenerative disease states and cPLA2 is therefore an attractive target for the development of novel therapies.

Phosphorylation and activation of cytosolic phospholipase A2 (PLA2) can occur independently of the activation of 42/44-kDa mitogen-activated protein (MAP) kinase in human platelets. We have investigated the hypothesis that the stress-activated p38 MAP kinase plays a role in the regulation of cytosolic PLA2. The specific inhibitor of p38 MAP kinase, SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], completely blocked the collagen-stimulated phosphorylation of cytosolic PLA2 in the presence of a cyclooxygenase blocker, and reduced the release of [3H]arachidonic acid by low concentrations of collagen. Stimulation of platelets with collagen (100 microg/ml) enhanced in vitro PLA2 activity of platelet lysates twofold over basal levels. In vitro PLA2 activity was reduced to basal levels when platelets were stimulated in the presence of SB 203580, but not in the presence of an inhibitor of the kinase that activates p42/p44 MAP kinase. SB 203580 only partially inhibited phosphorylation of cytosolic PLA2 in platelets that had not been treated with a cyclooxygenase blocker indicating that secondary stimulation by thromboxane A2 induces cytosolic PLA2 phosphorylation, by kinase(s) other than p38 MAP kinase. Under these conditions, inhibition of p42/p44 MAP kinase did not result in a reduction of cytosolic PLA2 phosphorylation, which is in agreement with the results obtained in the presence of cyclooxygenase blockers. In contrast to collagen, both p38 MAP kinase and p42/p44 MAP kinase participated in the phosphorylation of cytosolic PLA2 in platelets stimulated by cross-linking of the low-affinity receptor for immune complexes, Fc gammaRIIA. The present results demonstrate an important role for p38 MAP kinase in the regulation of cytosolic PLA2 activity in collagen-stimulated human platelets.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol esterstimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 microM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST-MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

Background and Purpose: Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A2 (cPLA2) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA2 in the rat brain subjected to global ischemia., Methods: Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four- vessel occlusion. PLA2 was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA2 with various glial cell types. cPLA2 levels were also measured by enzymatic assay and Western blot analysis., Results: A marked induction of cPLA2 was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA2. Twenty-four hours after ischemia, numerous cPLA2 immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA2, and a threefold induction in cPLA2 activity was demonstrated by enzymatic assay., Conclusions: These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.

In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca(2+)-dependent cytosolic phospholipase A2 (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J.A. (1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.

Human platelets pretreated with indomethacin release arachidonic acid predominantly through the activity of cytosolic phospholipase A2 (cPLA2), an 85-kDa protein. This enzyme is regulated by an increase in intracellular Ca2+, a necessary condition of for arachidonic acid liberation, and by phosphorylation. Phosphorylation of cPLA2 enhanced the Ca(2+)-induced arachidonic acid release in platelets stimulated by the ionophore A23187 and phorbol ester (phorbol 12,13-dibutyrate (PDBu)). In thrombin-stimulated platelets, however, phosphorylation appeared not to be necessary for arachidonic acid release since the latter response was not impaired in the presence of staurosporine, which inhibited phosphorylation. Collagen, thrombin, and PDBu induced phosphorylation of platelet cPLA2 as well as activation of mitogen-activated protein kinase (MAPK; p42mapk and p44mapk). cPLA2 activation was not dependent on protein kinase C (PKC) in thrombin- and collagen-stimulated platelets, as preincubation with the PKC inhibitor Ro 31-8220 neither interfered with cPLA2 phosphorylation nor reduced arachidonic acid release. However, collagen- and thrombin-induced activation of MAPK was inhibited by Ro 31-8220, indicating that PKC is necessary for MAPK stimulation in platelets. Although MAPK may underlie phosphorylation of cPLA2 in PDBu-activated human platelets, our results provide evidence for PKC- and MAPK-independent phosphorylation of cPLA2 in platelets stimulated by the physiological activators collagen and thrombin.

Inhibition of Na+,K(+)-ATPase activity by hyperglycemia could be an important etiological factor of chronic complications in diabetic patients. The biochemical mechanism underlying hyperglycemia's inhibitory effects has been thought to involve the alteration of the protein kinase C (PKC) pathway since agonists of PKC can normalize hyperglycemia-induced inhibition of Na+,K(+)-ATPase activity. Paradoxically, elevated glucose levels and diabetes have been shown to increase PKC activities in vascular cells. The present study tested the hypothesis that the inhibition of Na+,K(+)-ATPase activity is mediated by the sequential activation of PKC and cytosolic phospholipase A2 (cPLA2). In cultured rat vascular smooth muscle cells (VSMC), increasing glucose levels in the medium from 5.5 to 22 mM elevated cPLA2 activity and increased [3H]arachidonic acid release and PGE2 production by 2.3-, 1.7- and 2-fold, respectively. Similar increases in cPLA2 activity were also induced by elevated glucose levels in human VSMC and rat capillary endothelial cells. The activation of cPLA2 was mediated by PKC since the increases in cPLA2 phosphorylation and enzymatic activity were inhibited by the PKC inhibitor GFX. In contrast, elevation of glucose levels decreased Na+,K(+)-ATPase activity as measured by ouabain-sensitive 86Rb uptake by twofold in rat VSMC. Surprisingly, both PMA, a PKC agonist, and GFX, a PKC inhibitor, were able to prevent glucose-induced decreases in 86Rb uptake. Further, the PLA2 inhibitor AACOCF3 abolished both glucose-induced activation of cPLA2 and the decrease in 86Rb uptake. These data indicated that hyperglycemia is inhibiting Na+,K(+)-ATPase activity by the sequential activation of PKC and cPLA2, resulting in the liberation of arachidonic acid and increased the production of PGE2, which are known inhibitors of Na+,K(+)-ATPase.

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A2 by human platelets. We found that while SFLLRN evokes an initial transient increase in cystolic free calcium concentration ([Ca2+]i) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+]i declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+]i may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the "activating" phosphorylation of cytolic phospholipase A2 (cPLA2). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr669 derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA2 and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+]i and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA2 in the absence of ERK1/2 stimulation suggest that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA2.

In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.

The effect of pancreatic group I phospholipase A2 (PLA2-I) on receptor-mediated expression of arthritic group II phospholipase A2 (PLA2-II) and its correlation with prostaglandin E2 (PGE2) synthesis were examined in cultured rat mesangial cells. Scatchard analysis using 125I-PLA2-I revealed the existence of a single class of specific binding sites for PLA2-I in rat mesangial cells with an equilibrium dissociation constant (Kd) of 1.6 nM and a maximum binding capacity of 10.1 fmol/10(6) cells. The mammalian mature type of PLA2-I specifically recognized this binding site, whereas its inactive zymogen and mammalian PLA2-II showed much lower affinities. PLA2-I markedly increased PLA2-II mRNA levels as well as PLA2-II secretion from the cells in a time- and dose-dependent manner that was closely correlated with PGE2 production. Both PLA2-II expression and PGE2 synthesis were completely suppressed by pretreatment of the cells with actinomycin D, cycloheximide, or dexamethasone. These results strongly suggest that there may be crosstalk between PLA2-I and PLA2-II via the specific PLA2-I receptor that elicits PGE2 synthesis.

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.

Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.

Human monoblast U937 cells contain a soluble phospholipase A2 (PLA2) that is activated over the range of 150-600 nM Ca2+ and is stable only at neutral pH. We have purified this PLA2 over 34,000-fold to near homogeneity using sequential ion exchange, hydrophobic interaction, and gel filtration chromatography steps. The protein has a Mr of approximately 100,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 5.1. Four lines of evidence indicate that this 100-kDa polypeptide represents the PLA2. (i) The intensity of staining of the 100-kDa protein was proportional to the degree of purification of PLA2 activity, (ii) the relative staining intensity of the 100-kDa protein precisely paralleled the elution profile of PLA2 activity during chromatography steps, (iii) the PLA2 activity recovered from a nondenaturing gel (greater than 60% of the total activity applied) coincided exactly with the major high molecular weight protein detected by silver staining, and (iv) monoclonal antibodies against the 100-kDa protein immunoprecipitated the PLA2. We conclude that the cytosolic PLA2 isolated from U937 cells represents a novel, high molecular weight PLA2 responding to physiological (intracellular) changes in Ca2+ concentration and therefore may play a critical role in cellular signal transduction processes and the biosynthesis of lipid mediators.

The cholesterol to phospholipid mole ratio (C/PL) of human platelets was increased 1.3-fold or maintained at a normal value by incubating platelets with sonicated dispersions of cholesterol and phosphatidylcholine (PC) (C/PL = 3 or 1, respectively). Thrombin-induced mobilization of [3H]arachidonic acid from prelabeled phospholipids and subsequent formation of labeled cyclo-oxygenase and lipoxygenase products were increased in cholesterol-enriched platelets as a function of thrombin concentration. Elevated platelet cholesterol content affected thrombin-induced changes in platelet phospholipids: (a) hydrolysis of PC was more sensitive to thrombin and was markedly enhanced over a wide range of thrombin concentrations (0.1-2 units/ml); (b) hydrolysis of phosphatidylinositol (PI) was increased at thrombin concentrations greater than or equal to 0.2 unit/ml. Increased metabolism of [3H]arachidonic acid in stimulated cholesterol-enriched platelets was due to loss of [3H]arachidonate from PC at 0.1 unit/ml of thrombin. At higher thrombin concentrations (0.2-2 units/ml) it reflected enhanced hydrolysis of predominantly PC, but also PI. We conclude that cholesterol, possibly through its effect on platelet lipid organization, influences arachidonic acid metabolism in stimulated plates by promoting enhanced activity of platelet phospholipase(s) for liberation of arachidonic acid.

Platelet membranes contain two distinct transacylase activities catalyzing the synthesis of arachidonoyl phosphatides by acylation of added lysophosphatides with endogenous esterified arachidonate. In the absence of CoA, arachidonate is incorporated only into ethanolamine lysophosphatides with a high preference for the plasmalogen form (Kramer, R. M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811). In the presence of CoA, however, lysophospholipids are acylated in the order 1-acyl-lysophosphatidylserine greater than 1-acyl-lysophosphatidylethanolamine greater than 1-acyl-lysophosphatidylinositol. The CoA-mediated transacylation reaction was characterized with 1-acyl-lysophosphatidylserine as acyl acceptor. It was highly specific for arachidonate and preferentially used phosphatidylcholine as the arachidonoyl donor. This enzymatic pathway may be part of a deacylation-transacylation cycle for remodeling of phospholipids synthesized de novo (according to the Lands pathway) representing a mechanism for enrichment of phospholipids with arachidonic acid.

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.

Human platelets contain an enzyme that catalyzes CoA-independent release of arachidonic acid from phosphatidylcholine with concomitant incorporation into plasmenylethanolamine. Addition of lysoplasmenylethanolamine (10-80 microM) to a crude membrane preparation of prelabeled platelets (0.24 mg of protein/ml) induces transfer of [3H]arachidonate from endogenous phosphatidylcholine to lysoplasmenylethanolamine (0.8 nmol of arachidonic acid/min/mg of protein). The transacylation reaction occurs in the absence of Ca2+, has a broad pH optimum from 7 to 8, is not affected by excess unlabeled arachidonic acid, and is inhibited by N-ethylmaleimide (0.2 mM) and Triton X-100 (0.1 mg/ml). The enzyme shows a high specificity toward the acyl donor (phosphatidylcholine), transfers fatty acids in the order: arachidonic greater than eicosatrienoic greater than oleic, and preferentially acylates lysoplasmenylethanolamine but also other lysophosphatides (lysophosphatidylethanolamine greater than lysophosphatidylserine greater than lysophosphatidylinositol = 0). Platelet acyltransferase, on the other hand, acylates ethanolamine lysophosphatides with free arachidonic acid in the order: lysophosphatidyl-ethanolamine greater than lysoplasmenylethanolamine. These results suggest that a distinct acylation mechanism exists for introduction of arachidonic acid into plasmalogen phosphatides. In stimulated platelets, the transacylase may play an additional role in the controlled release of esterified arachidonic acid for synthesis of the biologically active oxygenated metabolites.

We examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated 2.5 microM-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A2 activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to 29 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonoylglycerol. 1-Stearoyl-2-arachidonoylglycerol at concentrations of 2-5 mol% increased phospholipase A2 activity 1.3-1.8-fold. Exogenously added 1-oleoyl-2-acetylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A2. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca2+-activated phospholipase A2.

The present study demonstrates that inactivation of exogenous 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensitive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl-GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC was provided by phosphatidylcholine. We conclude that the previously described platelet arachidonoyl transacylase (Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811) may play an important role in the metabolism of platelet-activating factor.