Fbg αC 389-402 Enhances Factor XIII Cross-Linking in the Fibrinogen αC Region Via Electrostatic and Hydrophobic Interactions.

Coagulation Factor XIII (FXIII) stabilizes blood clots by cross-linking glutamines and lysines in fibrin and other proteins. FXIII activity in the fibrinogen αC region (Fbg αC 221-610) is critical for clot stability and growth. Fbg αC 389-402 is a binding site for thrombin-activated FXIII, (FXIII-A*), with αC E396 promoting FXIII-A* binding and activity in αC. The current study aimed to discover additional residues within Fbg αC 389-402 that accelerate transglutaminase activity toward αC. Electrostatic αC residues (E395, E396, and D390), hydrophobic αC residues (W391 and F394), and residues αC 328-425 were studied by mutations to recombinant Fbg αC 233-425. FXIII activity was monitored through MS-based glycine ethyl ester (GEE) cross-linking and gel-based fluorescence monodansylcadaverine (MDC) cross-linking assays. Truncation mutations 403 Stop (Fbg αC 233-402), 389 Stop (Fbg αC 233-388), and 328 Stop (Fbg αC 233-327) reduced Q237-GEE and MDC cross-linking compared to wild-type (WT). Comparable cross-linking between 389 Stop and 328 Stop showed that FXIII is mainly affected by the loss of Fbg αC 389-402. Substitution mutations E396A, D390A, W391A, and F394A decreased cross-linking relative to WT, whereas E395A, E395S, E395K, and E396D had no effect. Similar FXIII-A* activities were observed for double mutants (D390A, E396A) and (W391A, E396A), relative to D390A and W391A, respectively. In contrast, cross-linking was reduced in (F394A, E396A), relative to F394A. In conclusion, Fbg αC 389-402 boosts FXIII activity in Fbg αC, with D390, W391, and F394 identified as key contributors in enhancing αC cross-linking.

Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates.

Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+. Cellular FXIII is activated nonproteolytically (FXIII-A°) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A° that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A° toward Q residues within Fbg αC (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α2-antiplasmin (α2AP), fibronectin, and Fbg αC (233-388, missing FXIII-binding site αC 389-402). Furthermore, the N-terminal α2AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233-425 WT, αC E396A, and truncated αC (233-388) further documented that FXIII-A* exerts greater benefit from the αC 389-402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII-binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are achieved.

Economic and survival burden of dysphagia among inpatients in the United States.

The inpatient burden of dysphagia has primarily been evaluated in patients with stroke. It is unclear whether dysphagia, irrespective of cause, is associated with worse clinical outcomes and higher costs compared to inpatients with similar demographic, hospital, and clinical characteristics without dysphagia. The aim of this study is to assess how a dysphagia diagnosis affects length of hospital stay (LOS), costs, discharge disposition, and in-hospital mortality among adult US inpatients. Annual and overall dysphagia prevalence, LOS, hospital charges, inpatient care costs, discharge disposition, and in-hospital mortality were measured using the AHRQ Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (2009-2013). Patients aged 45 years or older with ≤180 days of stay in hospital with and without dysphagia were included. Multivariable survey regression methods with propensity weighting were used to assess associations between dysphagia and different outcomes. Overall, 2.7 of 88 million (3.0%) adult US inpatients had a dysphagia diagnosis (50.2% male, 72.4% white, 74.6% age 65-90 years) and prevalence increased from 408,035 (2.5% of admissions) in 2009 to 656,655 (3.3%) in 2013. After inverse probability of treatment weighting adjustment, mean hospital LOS in patients with dysphagia was 8.8 days (95% CI 8.66-8.90) compared to 5.0 days (95% CI 4.97-5.05) in the non-dysphagia group (P < 0.001). Total inpatient costs were a mean $6,243 higher among those with dysphagia diagnoses ($19,244 vs. 13,001, P < 0.001). Patients with dysphagia were 33.2% more likely to be transferred to post-acute care facility (71.9% vs. 38.7%, P < 0.001) with an adjusted OR of 2.8 (95% CI 2.73-2.81, P < 0.001). Compared to non-cases, adult patients with dysphagia were 1.7 times more likely to die in the hospital (95% CI 1.67-1.74). Dysphagia affects 3.0% of all adult US inpatients (aged 45-90 years) and is associated with a significantly longer hospital length of stay, higher inpatient costs, a higher likelihood of discharge to post-acute care facility, and inpatient mortality when compared to those with similar patient, hospital size, and clinical characteristics without dysphagia. Dysphagia has a substantial health and cost burden on the US healthcare system.

Charge Distribution Fine-Tunes the Translocation of α-Helical Amphipathic Peptides across Membranes.

Hundreds of cationic antimicrobial and cell-penetrating peptides (CPPs) form amphipathic α-helices when bound to lipid membranes. Here, we test two hypotheses for the differences in the ability of these peptides to translocate across membranes. The first, which we now call the hydrophobicity hypothesis, is that peptide translocation is determined by the Gibbs energy of insertion into the bilayer from the membrane interface. The second, which we call the charge-distribution hypothesis, is that translocation is determined by whether the distribution of cationic residues in the peptide can transiently stabilize a high-energy inserted intermediate by forming salt bridges to the phosphates of lipid headgroups. To test these hypotheses, we measured translocation of two series of peptide variants. The first series was based on TP10W, a peptide derived from the amphipathic CPP transportan 10; the second was based on DL1a, a synthetic peptide derived from staphylococcal δ-lysin. The peptides in those two series had small sequence changes relative to TP10W and DL1a: either single-residue substitutions or two-residue switches, which were designed to increase or decrease translocation differently according to the two hypotheses. We found that with regard to the changes introduced in the sequences, five out of six peptide variants translocated in agreement with the charge-distribution hypothesis, whereas none showed agreement with the hydrophobicity hypothesis. We conclude that large effects on translocation are probably determined by hydrophobicity, but the fine tuning appears to arise from the distribution of cationic residues along the peptide sequence.

Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles.

The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. However, if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energies of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period, but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 min. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. However, some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle.