Activated protein C-protein C inhibitor complex, activation peptide of carboxypeptidase B and C-reactive protein as predictors of severe acute pancreatitis.

INTRODUCTION: The concentration of carboxypeptidase B activation peptide (CAPAP) is proposed to be a predictor of severe acute pancreatitis. The activated protein C (APC)-protein C inhibitor (PCI; APC-PCI) complex in plasma could be useful in detecting the hypercoagulative condition in severe acute pancreatitis. METHOD: In this prospective study, mild (n = 50) and severe (n = 9) cases of acute pancreatitis were compared with respect to levels of CAPAP and APC-PCI, and sorted in time intervals from onset of symptoms to sampling. The peak values of the C-reactive protein (CRP) within the 1st week were also compared. RESULTS: CRP detected the severe cases with a sensitivity of 0.89 and a specificity of 0.74 (cut-off level 200 mg/l). In the interval 0-72 h, CAPAP could predict the severity of the disease in serum and urine (sensitivity 0.52/0.29, specificity 0.73/0.93, cut-off 2 nM/60 nM). The level of APC-PCI in plasma could predict the severe condition in the interval 0-24 h after the onset of symptoms (sensitivity 0.6, specificity 0.66, cut-off level 0.54 microg/l). CONCLUSION: Of the parameters explored, CRP is still the best biochemical marker to distinguish between severe and mild acute pancreatitis. CAPAP could be useful in combination with other tests, but the APC-PCI complex's diagnostic time interval is too short to be used in the clinical routine.

Explorative investigation of biomarkers of brain damage and coagulation system activation in clinical stroke differentiation.

INTRODUCTION: A simple and accurate method of differentiating ischemic stroke and intracerebral hemorrhage (ICH) is potentially useful to facilitate acute therapeutic management. Blood measurements of biomarkers of brain damage and activation of the coagulation system may potentially serve as novel diagnostic tools for stroke subtypes. METHODS: Ninety-seven stroke patients were prospectively investigated in a multicenter design with blood levels of brain biomarkers S100B, neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) as well as a coagulation biomarker, activated protein C-protein C inhibitor complex (APC-PCI), within 24 hours of symptom onset. RESULTS: Eighty-three patients (86%) had ischemic stroke and fourteen patients (14%) had ICH. There were no differences in S100B (p=0.13) and NSE (p=0.67) levels between patients with ischemic stroke or ICH. However, GFAP levels were significantly higher in ICH patients (p=0.0057). APC-PCI levels were higher in larger ischemic strokes (p=0.020). The combination of GFAP and APC-PCI levels, in patients with NIHSS score more than 3, had a sensitivity and negative predictive value of 100% for ICH in our material (p=0.0052). CONCLUSION: This exploratory study indicated that blood levels of biomarkers GFAP and APC-PCI, prior to neuroimaging, may rule out ICH in a mixed stroke population.

Activated protein C-protein C inhibitor complex in patients with abdominal aortic aneurysms: is it associated with diameter and growth rate?

Increased thrombin activation was documented in patients with abdominal aortic aneurysm (AAA). Activated protein C-protein C inhibitor (APC-PCI) complex, a new biological marker of thrombin generation, was measured in a population of 232 patients with AAA and a control group, and the association between aneurysm size, growth rate, and APC-PCI was studied. The patients were divided into cohorts according to AAA diameter and compared with a control group. APC-PCI was significantly higher in all AAA cohorts (n = 232; median, 0.36 microg/L; 10th to 90th percentile, 0.18-1.01) compared with the control group (n = 41; median, 0.19 microg/L; 10th to 90th percentile, 0.11-0.31; P < or = .001). APC-PCI correlated with AAA diameter (r = .22; P = .001), body mass index (r = -.19; P = .004), and age (r = .19; P = .004). APC-PCI did not correlate with AAA growth rate (r = .11; P = .14).

Venomous auger snail Hastula (Impages) hectica (Linnaeus, 1758): molecular phylogeny, foregut anatomy and comparative toxinology.

The >10,000 living venomous marine snail species [superfamily Conoidea (Fleming, 1822)] include cone snails (Conus), the overwhelming focus of research. Hastula hectica (Linnaeus, 1758), a venomous snail in the family Terebridae (Mörch, 1852) was comprehensively investigated. The Terebridae comprise a major monophyletic group within Conoidea. H. hectica has a striking radular tooth to inject venom that looks like a perforated spear; in Conus, the tooth looks like a hypodermic needle. H. hectica venom contains a large complement of small disulfide-rich peptides, but with no apparent overlap with Conus in gene superfamilies expressed. Although Conus peptide toxins are densely post-translationally modified, no post-translationally modified amino acids were found in any Hastula venom peptide. The results suggest that different major lineages of venomous molluscs have strikingly divergent toxinological and venom-delivery strategies.

Structure of native protein C inhibitor provides insight into its multiple functions.

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into beta-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.

The APC-PCI complex concentration predicts outcome of aortic surgery.

INTRODUCTION: Coagulation activation may be related to complications during surgery for abdominal aortic aneurysm. The complex formed between activated protein C (APC) and the serpin, protein C inhibitor (PCI), is a sensitive indicator of the activation of blood coagulation. The purpose of the study was to establish whether the APC-PCI complex can provide information useful for the assessment of outcome after aortic surgery. MATERIALS AND METHODS: In 38 patients, the APC-PCI complex was initially determined every 6 h and daily from day three. Protein C, antithrombin, global haemostatic tests, and clinical scores were investigated. Length of stay at the intensive care unit (ICU) and hospital, and vital status up to two years were recorded. RESULTS: The median APC-PCI complex concentration in samples drawn 0-6 h after surgery was more than 20-fold higher than the upper limit of the reference interval. The level then declined rapidly, but remained elevated during the first two days. In patients with higher initial APC-PCI complex concentrations, Sequential Organ Failure Assessment (SOFA) scores were higher, the ICU stay was longer, and survival up to two years was lower. Patients who did not survive the ICU care had higher APC-PCI complex levels at 6-12 h and 12-18 h. CONCLUSIONS: High concentrations of the APC-PCI complex within 6-18 h after the aortic surgery predict a sinister outcome. The results suggest that the APC-PCI complex is indicative of the severity of the disease.

A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

Binding of calcium to anticoagulant protein S: role of the fourth EGF module.

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile.

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.

Activated protein C-protein C inhibitor complex: a new biological marker for aortic aneurysms.

OBJECTIVE: The concentration of the complex between activated protein C (APC) and protein C inhibitor (PCI) is a measure of thrombin generation. We studied whether it can provide information useful for the diagnosis and treatment of arterial vascular disease. METHODS: Blood was obtained from 429 vascular patients admitted consecutively during September 2004 to March 2005. The APC-PCI complex was measured by using a sandwich immunofluorometric method. The patients were divided into cohorts according to the planned treatment and compared with a control group of healthy individuals. RESULTS: The APC-PCI complex concentration varied from 0.08 to 2.50 microg/L. In the cohort of patients with aortic aneurysms (n = 78), the median APC-PCI value was 0.45 (10th to 90th percentile, 0.24-1.47), and values were clearly increased compared with all other cohorts (P < .0001). Patients with carotid disease (n = 73) yielded a median of 0.22 (10th to 90th percentile, 0.15-0.48). The median for claudicants (n = 74) was 0.26 microg/L (10th to 90th percentile, 0.15-0.75), which was higher than in those (n = 97) with critical ischemia (0.20; 10th to 90th percentile, 0.13-0.36; P < .0023). The cohort with other forms of atherosclerotic disease (n = 40) had a median of 0.23 (10th to 90th percentile, 0.14-0.42), whereas the value for a cohort of 21 patients with venous disease was 0.19 (10th to 90th percentile, 0.10-0.34). The median was 0.15 (10th to 90th percentile, 0.10-0.23) for the control group (n = 121). CONCLUSIONS: Patients with atherosclerosis had an increased APC-PCI concentration that corresponded to increased generation of thrombin. Patients with aortic aneurysm had a threefold higher median concentration than the control group. We suggest that this remarkable increase is caused by the local activation of coagulation, and we surmise that APC-PCI measurements can be used as a screening tool to identify patients with aortic aneurysms.

Early identification of acute myocardial infarction by activated protein C--protein C inhibitor complex.

INTRODUCTION: Increased coagulation activity due to coronary thrombosis in a ruptured plaque should result in activation of the protein C anticoagulant system with formation of complexes between activated protein C (APC) and the protein C inhibitor (PCI), which reflects coagulation activity. We hypothesized that elevated APC-PCI concentration might allow earlier detection of ongoing myocardial infarction than traditional biochemical markers. We have evaluated a newly devised immunofluorimetric assay for measuring plasma concentration of APC-PCI complexes among patients with suspected acute coronary syndrome. MATERIALS AND METHODS: Blood samples were taken from 340 patients (median 71 years, range 31-97) with suspected acute coronary syndrome at first presentation in the emergency department. Electrocardiogram was recorded and APC-PCI, Troponin I and Creatine kinase-MB concentrations were repeatedly measured 3 times at 6 h interval. RESULTS: The 74 patients who were eventually diagnosed with myocardial infarction had a higher median level of APC-PCI complex than those without myocardial damage; 0.27 vs. 0.20 microg/L (p = 0.001). In a multivariate regression model, APC-PCI level in the fourth quartile (>0.32 microg/L) independently predicted myocardial infarction with an odds ratio of 3.7 (95% CI 1.4-9.6, p < 0.01). CONCLUSION: Early APC-PCI elevation can be detected among patients with a normal first Troponin I and non-ST-elevation myocardial infarction and provides additional risk assessment in acute coronary syndrome.

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3.

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.

Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-carboxylation.

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

No effect of the prothrombin G20210A mutation on protein C activation in a large kindred with type I protein C deficiency.

Previously, we observed a positive association of prothrombin concentrations with thrombin generation (fragment 1 + 2) and thrombin activity (fibrinopeptide A), but no association with protein C activation peptide levels. We further evaluated a potential beneficial effect of increased prothrombin concentrations on activated protein C generation by assessing the plasma concentration of activated protein C in complex with protein C inhibitor (APC-PCI). Blood samples were used from 195 family members of a large French-Canadian kindred with type I protein C deficiency due to a 3363C insertion in the protein C gene. We utilized a new and highly sensitive assay for measuring the concentration of APC-PCI complex as a measure of the level of activation of protein C. Means of the plasma concentrations of the APC-PCI complex were compared among carriers and non-carriers of the prothrombin G20210A mutation. Protein C activity levels were positively associated with APC-PCI complex plasma concentrations; however, APC-PCI complex levels were not different for carriers of the prothrombin G20210A mutation than for non-carriers. Thus, carriers of the prothrombin G20210A mutation do not have increased protein C activation despite the increased thrombin generation resulting from the higher prothrombin concentrations associated with the G20210A mutation.

N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules.

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern.

One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel gamma-carboxyglutamic acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five gamma-carboxyglutamic acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two gamma-carboxyglutamic acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal acid.

The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus.

Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

The metal-free and calcium-bound structures of a gamma-carboxyglutamic acid-containing contryphan from Conus marmoreus, glacontryphan-M.

Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca(2+) channels expressed in mouse pancreatic beta-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453-32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a D-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two gamma-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional (1)H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys(11)-Cys(5)-Pro(6) region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla(2) and Ser(3), moves away from the His(8) and Trp(10) aromatic rings and the alignment of the D-Trp(7) and His(8) aromatic rings with respect to the Trp(10) rings is altered. The blockage of L-type voltage-gated Ca(2+) channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone C alpha conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.

Probing plasma clearance of the thrombin-antithrombin complex with a monoclonal antibody against the putative serpin-enzyme complex receptor-binding site.

A high-affinity monoclonal antibody (M27), raised against the human thrombin-antithrombin complex, has been identified and characterized. The epitope recognized by M27 was located to the linear sequence FIREVP (residues 411-416), located in the C-terminal cleavage peptide of antithrombin. This region overlaps, by two residues, the putative binding site of antithrombin for the serpin-enzyme complex receptor. Studies in rats and with HepG2 cells in culture indicated that the Fab fragment of M27 does not block binding and uptake of the thrombin-antithrombin complex, suggesting that this region does not play a major role in the recognition and clearance of the thrombin-antithrombin complex. M27 blocked the ability of antithrombin to inhibit thrombin as well as antithrombin cleavage, both in the presence and absence of heparin.