External validation of the Revised Cardiac Risk Index and National Surgical Quality Improvement Program Myocardial Infarction and Cardiac Arrest calculator in noncardiac vascular surgery.

BACKGROUND: The National Surgical Quality Improvement Program Myocardial Infarction & Cardiac Arrest (NSQIP MICA) calculator and the Revised Cardiac Risk Index (RCRI) were derived using currently outdated methods of diagnosing perioperative myocardial infarctions. We tested the external validity of these tools in a setting of a systematic perioperative cardiac biomarker measurement. METHODS: Analysis of routinely collected data nested in the Vascular Events In Noncardiac Surgery Patients Cohort Evaluation Study. A consecutive sample of patients ≥45 yr old undergoing in-hospital noncardiac surgery in a single tertiary care centre was enrolled. The predictive performance of the models was tested in terms of the occurrence of major cardiac complications defined as a composite of a nonfatal myocardial infarction, a nonfatal cardiac arrest, or a cardiac death within 30 days after surgery. The plasma concentration of high-sensitivity troponin T was measured before surgery, 6-12 h after operation, and on the first, second, and third days after surgery. Myocardial infarction was diagnosed according to the Third Universal Definition. RESULTS: The median age was 65 (59-72) yr, and 704/870 (80.9%) subjects were male. The primary outcome occurred in 76/870 (8.7%; 95% confidence interval [CI], 6.9-10.8%) patients. The c-statistic was 0.64 (95% CI, 0.57-0.70) for the NSQIP MICA and 0.60 (95% CI, 0.54-0.65) for the RCRI. Predicted risks were systematically underestimated in calibration belts (P<0.001). The RCRI and the NSQIP MICA showed no clinical utility before recalibration. CONCLUSIONS: The NSQIP and RCRI models had limited predictive performance in this at-risk population. The recently updated version of the RCRI was more reliable than the original index.

Altered preoperative coagulation and fibrinolysis are associated with myocardial injury after non-cardiac surgery.

BACKGROUND: Myocardial injury after non-cardiac surgery (MINS), a complication with unclear pathogenesis, occurs within the first 30 days after surgery and worsens prognosis. Hypercoagulability induced by surgery might contribute to plaque rupture, with subsequent thrombosis and myocardial injury. This study assessed haemostatic markers before surgery and evaluated their association with MINS. METHODS: This is a substudy of VISION, a prospective cohort study of perioperative cardiovascular events. Of 475 consecutive vascular surgery patients, 47 (9.9%) developed MINS, defined as postoperative high-sensitivity troponin ≥50 ng litre -1 , with ≥20% elevation from the preoperative concentration. The control group consisted of 84 non-MINS patients matched for patient characteristics and co-morbidities. The following preoperative markers of hypercoagulability and fibrinolysis were measured: antithrombin, factor VIII activity, von Willebrand factor concentration and activity, fibrinogen, D-dimer, plasmin-antiplasmin complex, and tissue plasminogen activator. Moreover, C-reactive protein and CD40L concentrations were measured to assess inflammatory activity. RESULTS: Patients with MINS compared with the non-MINS group had a significantly higher concentration of factor VIII (186 vs 155%, P =0.006), von Willebrand factor activity (223 vs 160%, P <0.001), von Willebrand factor concentration (317 vs 237%, P =0.02), concentrations of fibrinogen (5.6 vs 4.2 g litre -1 , P =0.03), D-dimer (1680.0 vs 1090.0 ng ml -1 , P =0.04), plasmin-antiplasmin complex (747 vs 512 ng ml -1 , P =0.002) and C-reactive protein (10 vs 4.5 mg litre -1 , P =0.02) but not antithrombin (95 vs 94%, P =0.89), tissue plasminogen activator (11 vs 9.7 ng ml -1 , P =0.06) and CD40L (8790 vs 8580 pg ml -1 , P =0.73). CONCLUSIONS: Preoperative elevation of blood markers of hypercoagulability in patients undergoing vascular surgery is associated with a higher risk of MINS. CLINICAL TRIAL REGISTRATION: NCT00512109.

Leukotrienes biosynthesis in vascular surgery patients during perioperative period.

Leukotrienes (LTs), highly bioactive lipid mediators play a major role in inflammation, wound healing and in the development of atherosclerosis. LTs biosynthesis have been suggested to be increased in myocardial infarction (MI) and in surgical patients with abdominal aortic aneurysms. Among LTs, Cysteinyl-LTs have the most potent biological properties and their production is well reflected by LTE4 concentration in urine (uLTE4). Aim of the study was to evaluate perioperative biosynthesis of uLTE4 in noncardiac vascular surgery patients, and its impact on patients' outcomes. Twenty eight consecutive patients aged 61.5 (59.0-72.5) that undergone an elective surgery for abdominal aortic aneurysm (AAA; n=6) or peripheral artery disease (PAD; n=22) were studied. uLTE4 was measured in urine samples using ELISA: before surgery (LT0), 6 hours postoperatively (LT1), and on three following days (LT2-LT4), and the results were adjusted for the urinary creatinine concentration. Patients were followed-up for 30-days for cardio-vascular complications including myocardial infarction (MI) with active post-surgery troponin T screening. One way analysis of variance (ANOVA) for repeated measurements and logistic regression tests were used to analyse the data with P<05 considered significant. Excretion of uLTE4 raised in the first two urine sample (LT1 and LT2) after surgery as compared to preoperative baseline value (LT0) (P=0.008) and returned to normal values on the second day (LT3). Patients that suffered MI during postoperative period had increased uLTE4 levels when compared to the no-MI patients (P=0.006). In conclusion we state that uLTE4 biosynthesis is increased shortly after surgery and returns to the preoperative level on the second day. The increase in uLTE4 biosynthesis is higher in patients that suffer MI after surgery, however this warrants further investigations.

Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding.

Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH2-terminal gamma-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca2+-binding sites: two high affinity (Kd 100 +/- 30 microM), four intermediate affinity (Kd 450 +/- 70 microM), and five to six low affinity (Kd 2 +/- 0.2 mM). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (Kd 140 +/- 20 microM). In contrast, factor X lacking the Gla domain as well as a part of the helical hydrophobic residues (des-44-X) retained only one high affinity Ca2+-binding site (Kd 130 +/- 20 microM). Moreover, a synthetic peptide composed of residues 238-277 (58-97 in chymotrypsinogen numbering) from the protease domain of factor X bound one Ca2+ with high affinity (Kd 150 +/- 20 microM). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the Kd for peptide-Va interaction was calculated to be approximately 10 microM as compared with 30 pM for factor Xa and approximately 1.5 microM for decarboxylated factor Xa. A peptide containing residues 238-262(58-82) bound Ca2+ with reduced affinity (Kd approximately 600 microM) and did not inhibit Xa:Va interaction. In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (Kd approximately 10 microM) but did not bind Ca2+. In additional studies, Ca2+ increased the amidolytic activity of native and des-44-Xa toward a tetrapeptide substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by approximately 1.6-fold. The half-maximal increase was observed at approximately 150 microM Ca2+ and the effect was primarily on the kcat. Ca2+ also significantly protected cleavage at Arg-332-Gln-333(150-151) in the protease domain autolysis loop. Des-44-Xa in which the autolysis loop was cleaved possessed

Edman sequencing as tool for characterization of synthetic peptides.

Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor technique for assessing quantitative aspects and the type and degree of adduct formation that arise from the synthetic chemistry. For these latter considerations, techniques such as mass spectrometry can often give more precise and informative data about the integrity of a synthetic peptide. Thus, sequence analysis is best applied judiciously and then used in combination with other methods. Furthermore, proper interpretation of the results of sequence analysis of synthetic peptides relies on a thorough knowledge of the sequencing process.

Indirect allorecognition of HLA class I peptides by CD4+ cytolytic T lymphocytes.

T-cell responses to alloantigens can occur either by "direct" recognition of donor MHC molecules, or "indirect" recognition of MHC peptides in association with self-MHC. To evaluate human T cells mediating indirect allorecognition, a CD4+ TCL and clones specific for HLA-A1 or HLA-B8 (residues 60-84) were generated from normal PBLs (A2,29 B62,- DR1,4 DQ3). Most clones were A1 specific (16 out of 17 tested), HLA-DR4 restricted (8 out of 8), and lysed targets pulsed with A1 peptide (16 out of 16). An amino acid substitution at position 86 of the DR4 beta chain (G -> V) abrogated the capacity of CD4+ CTLs to lyse target cells. Chloroquine treatment of A1-pulsed targets reduced their susceptibility to lysis, indicating a requirement for peptide processing. The TCL and clones were stimulated to proliferate by cells bearing intact HLA-A1 when autologous APCs were present, indicating that the epitope contained within the A1 60-84 peptide being recognized is produced when APCs process native HLA-A1. Furthermore, the clones and TCL did not recognize HLA-A1 on target cells carrying this allele plus self-HLA-DR4. These studies suggest a much wider role for CD4+ T cells in allograft immunity.

High affinity Ca(2+)-binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity.

Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino acids 1-152) of factor VIIa consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like domains, whereas the heavy chain (amino acids 153-406) contains the serine protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high-affinity (Kd approximately 150 microM) Ca2+ binding sites. The rVIIa also contained approximately 6-7 low-affinity (Kd approximately 1 mM) Ca(2+)-binding sites. By analogy to other serine proteases, one of the two high affinity Ca(2+)-binding sites in factor VIIa may be formed involving Glu-210 and Glu-220 of the protease domain. In support of this, a synthetic peptide composed of residues 206-242 of factor VIIa bound one Ca2+ with Kd approximately 230 microM; however, Ca2+ binding was observed only in Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containing 0.1 M NaCl. In both low or high salt +/- Ca2+, the peptide existed as a monomer as determined by sedimentation equilibrium measurements and had no detectable secondary structure as determined by CD measurements. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high salt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhibited the amidolytic activity of rVIIa toward the synthetic substrate, S-2288. The concentration of the peptide required for half-maximal inhibition was approximately 5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibition assays of active site-blocked 125I-rVIIa binding to TF, the Kd for peptide-TF interaction was calculated to be approximately 15 microM in the high salt and approximately 55 microM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa.TF interaction was approximately 1.5 microM in the absence of Ca2+, and, as inferred from factor X activation studies, it was approximately 10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa.TF interaction approximately 150,000-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a complement receptor 2 (CR2, CD21) ligand binding site for C3. An initial model of ligand interaction with two linked short consensus repeat modules.

Human CR2 (CD21, EBV receptor) is an approximately 145-kDa receptor and a member of the regulators of complement activation gene family. Regulators of complement activation proteins are characterized by the presence of repeating motifs of 60 to 70 amino acids that are designated short consensus repeats (SCR). CR2 serves as a receptor for four distinct ligands. Three of these ligands (complement C3, gp350/220 of EBV, and CD23) interact with the amino terminal 2 of 16 SCR (SCR 1 and 2). Previous studies have determined that at least four sites are important in allowing CR2 to efficiently bind EBV. Two of these sites are also important for binding mAb OKB7, a reagent that blocks both EBV and iC3b/C3dg binding to CR2. We have identified and characterized important sites of iC3b ligand binding by utilizing human-mouse CR2 chimeras, a rat anti-mouse CR2 mAb designated 4E3 that blocks receptor binding to C3, and human CR2-derived peptides. In addition to demonstrating an important role for the same sequence in SCR 1 that is part of the mAb OKB7 and EBV binding site, we have identified a new region within SCR 2 that interacts with C3. These results, when compared with a model of a dual SCR solution structure derived from human factor H SCR, predict that two distinct largely surface-exposed sites on CR2 interact with iC3b. A relative twist of 130 degrees about the long axis of the second SCR in this model would be necessary for these sites to form a single patch for iC3b binding on CR2.

Peptide binding to surface class II molecules is the major pathway of formation of immunogenic class II-peptide complexes for viable antigen presenting cells.

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-class II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules was occurring after endocytosis of peptides as well as by surface binding, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surface complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lymphoblastoid cells (BLCLs). Detection of the bound peptide was made possible by a rabbit anti-serum (alpha HAP) raised against HAP 128-145, which recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significant inhibition in the appearance of these complexes was caused by a decrease in the temperature at which the cells were incubated with peptide. Temperature-specific inhibition was also observed for fixed DRw11-positive APCs and purified DRw11 molecules indicating that the effect of temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such complexes. Moreover, class II molecules have evolved to stably bind peptide optimally at physiologic temperatures, independent of cell metabolism.

Model for the in vivo assembly of nascent Ld class I molecules and for the expression of unfolded Ld molecules at the cell surface.

To characterize the process of class I assembly and maturation, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface Ld molecules can be detected in an alternative conformation designated Ldalt1. Nascent Ldalt molecules are non-peptide ligand associated and are weakly associated with beta 2-microglobulin (beta 2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of Ldalt/beta 2m heterodimer were detected, suggesting that beta 2m association is not necessarily sufficient to induce Ld conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta 2m-associated Ldalt to conformed Ld. Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to Ld. The precursor-product relationship of Ldalt and conformed Ld was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of Ld was studied in the peptide transport-deficient cell line, RMA.S-Ld, and in beta 2m-/- splenocytes. In contrast to wild-type cell lines, both Ldalt and conformed Ld are poorly expressed at the cell surface of RMA.S-Ld and beta 2m-/- splenocytes. Therefore, surface expression of Ldalt is dependent upon the concomitant expression of conformed Ld molecules. To determine whether surface Ldalt molecules can result from melting of conformed Ld molecules, surface Ld molecules were loaded with several different known Ld peptide ligands. Complexes of Ld with different ligands were found to have dramatically disparate surface half-lives. Importantly, the Ld peptide complexes that turned over the most rapidly resulted in the most gain in surface Ldalt, implying that peptide dissociation can induce the accumulation of nonconformed Ld heavy chains at the cell surface.

Specific prolongation of allograft survival by a T-cell-receptor-derived peptide.

Allograft rejection results from the specific recognition by host CD8+ T cells of allogeneic major histocompatibility complex (MHC) molecules on the tissue graft. The specificity of this cellular response is determined by the molecular interaction of the T-cell receptor (TCR) on host T cells with the MHC molecule and its bound ligand on the grafted tissue. To better understand the precise manner by which the TCR interacts with the MHC-peptide complex and how to therapeutically intervene, we have studied the allogeneic response to the mouse class I MHC molecule Ld. In this report, the therapeutic potential of a synthetic peptide derived from the TCR V beta 8 variable region that predominates in responses to Ld was tested. This V beta 8-derived peptide was found to dramatically and specifically block the in vivo and in vitro allogeneic response to Ld. Furthermore, this specific blocking is not dependent upon the presence of V beta 8+ effector cells nor does the V beta 8 peptide bind to the Ld ligand binding cleft. We propose that this peptide functions as an antagonist, competing with the native TCR for recognition of the Ld molecule.

Binding of peptides lacking consensus anchor residue alters H-2Ld serologic recognition.

CTL recognize class I MHC/peptide complexes on the surface of target cells. Crystallographic and serologic data have indicated that peptide ligands can influence the conformation of class I molecules and hence T cell recognition. How the binding of peptides with disparate sequence motifs affects the conformation of distinct regions within a class I molecule remains unknown. A series of site-directed mutants of the murine class I molecule H-2Ld was studied to address this question. These mutants were generated by in vitro mutagenesis and used to map the serologic epitopes recognized by a panel of Ld-reactive mAb. The influence of six different ligands on serologic recognition by these mAb was then examined. Of 12 mAb tested, only one, B22/249, was found to be significantly influenced by the bound peptide. Peptide discrimination by B22/249 was observed at the cell surface and in immunoprecipitates of Ld after incubation with two of the six ligands. The two peptides that caused suboptimal B22/249 recognition of Ld/peptide lack a proline at position 2, which is present in the other four peptides and has previously been defined as an anchor residue for Ld ligands. The epitope on Ld detected by mAb B22/249 includes residues 63 to 70 on the alpha 1 domain helix. Two of these residues are in pocket B, which computer modeling predicts to be in contact with the second residue of Ld-binding peptides. Therefore, these data imply that a mAb to a class I molecule can distinguish peptides with different motifs, possibly reflecting peptide-dependent conformational changes in the class I molecule.

Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin.

Certain class I major histocompatibility complex molecules expressed on live cells have been shown to bind exogenous peptide ligands. However, it remains controversial whether this binding occurs by peptide exchange or to empty surface class I molecules. In this report we compare the surface binding and dissociation of two virus-derived ligands of the Ld class I molecule of the mouse. The peptide ligands were previously identified in immune responses to cytomegalovirus or lymphochoriomeningitis virus as immunodominant, optimally sized, and Ld restricted. Ligand dissociation was monitored on live cells indirectly by measuring the surface turnover of Ld-peptide complexes or directly by using labeled peptides. The cytomegalovirus-derived and lymphochoriomeningitis virus-derived peptides appeared to dissociate relatively rapidly; however, the cytomegalovirus-derived peptide had a more rapid off-rate than the lymphochoriomeningitis-derived peptide. Furthermore, these rates of dissociation appear to span that seen with endogenous Ld-associated peptides expressed by cells at 37 degrees C. Exploiting the extraordinary accessibility of the surface Ld ligand binding site we developed an assay to quantitate peptide ligand exchange. Cells were precoated with saturating amounts of unlabeled peptide by overnight incubation and were then tested for secondary binding of labeled peptides in a 4-h assay. Our results unequivocally demonstrate the potential for surface class I molecules to undergo peptide exchange. Furthermore, peptide exchange was found to be largely independent of exogenous beta 2-microglobulin. This result implies that beta 2-microglobulin association and not beta 2-microglobulin exchange is the critical factor in peptide exchange by surface class I molecules. Because of the exquisite ability of T cells to discriminate different amounts of ligand bound to class I, the binding of exogenous peptides could play a critical role in normal or aberrant immune responses.

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.

The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.

Recognition of the influenza hemagglutinin by class II MHC-restricted T lymphocytes and antibodies. I. Site definition and implications for antigen presentation and T lymphocyte recognition.

We have identified the site encompassing residues 126-145 on the A/Japan/57 influenza hemagglutinin molecule that is recognized in association with HLA-DRw11 by a clonal population of human, influenza specific, CD4+ cytolytic T lymphocytes. The critical core sequence of the T cell determinant spans hemagglutinin residues 129-140 and overlaps a putative antibody binding site. Hemagglutinins of influenza field strains that are not recognized by the T cell clones contain sequence alterations within the 129-140 target site of the CD4+ T cells. Functional analyses, with synthetic peptides, of the contribution of each of the residues within the sequence toward the capacity of the antigenic fragment to associate with both the restriction element and the TCR revealed a continuous linear array of residues necessary for MHC binding and/or Ag receptor engagement. At least one residue, the lysine at position 134, was shown to be critical for both DRw11 association and TCR recognition. The significance of these findings for recognition of glycoproteins by human CD4+ T cells is discussed.

Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex.

The A/Japan/57 influenza hemagglutin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface of living cells without the addition of a non-amino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system.

Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.

The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.

The recognition of a viral antigenic moiety by class I MHC-restricted cytolytic T lymphocytes is limited by the availability of the endogenously processed antigen.

The transmembrane hydrophobic domain of the type A influenza A/JAPAN/305/57 (H2N2) hemagglutinin (HA) contains an immunodominant site encompassing amino acids 523-545 (J523-545) recognized by class I MHC-restricted cytolytic T lymphocytes (CTL). Class I CTL of two fine specificity subsets map to this transmembrane (TM) site. One of these CTL subpopulations is subtype specific. These T lymphocytes recognize the site generated during infection of target cells with A/JAPAN/305/57 virus (H2N2) but not target cells expressing the comparable TM site of the influenza A/PR/8/34 virus (H1N1) hemagglutinin (P527-549) after infection with this virus. The other CTL subpopulation is cross-reactive and recognizes the TM site of the A/JAPAN/305/57 HA and the A/PR/8/34 HA with similar efficiency. Analyses of the critical amino acids in the TM site necessary for CTL recognition with the use of synthetic peptides unexpectedly revealed reactivity for the A/PR/8 HA TM site by subtype-specific CTL. This reactivity was only observed with truncated peptides corresponding to a limited portion of the A/PR/8 HA TM site but also required peptide concentrations greater than 10(-7) M. These results suggested either that the endogenously processed A/PR/8 HA TM site generated during infection was larger than the site defined by the truncated cross-reactive peptides or that the concentration of endogenously processed TM site produced during infection was limiting. To distinguish between these possibilities, we expressed in target cells synthetic minigenes encoding only the portion of the A/PR/8 HA transmembrane sites defined by the synthetic peptides. Unlike the peptides, the "preprocessed" endogenous minigene products were not recognized by subtype-specific CTL. These data suggest that the level of available endogenously processed Ag rather than selectivity in the site of fragmentation of newly synthesized Ag may play a critical role in determining whether the complex of the antigenic moiety and class I MHC is efficiently presented to and recognized by class I CTL.