Variant Gamma Research Articles

Assessing the impact of the Gamma variant on COVID-19 patient admissions in a southern Brazilian tertiary hospital-A comparison of dual pandemic phases.

Since the first case of COVID-19, Brazil has undergone infection waves with distinct characteristics. The description of new variants has alerted the emergence of more contagious or virulent viruses. The variant of concern Gamma emerged in Brazil and caused an epidemic wave, but its spread outside the country was limited. We report the clinical-epidemiological profile of hospitalized patients with COVID-19 by comparing two periods. A retrospective cohort study was performed. The primary outcome was to assess individuals with COVID-19 admitted in wards and intensive care units at the academic hospital of the Federal University of Parana (CHC-UFPR) between March 2020 and July 2021, correlating demographic, clinical-epidemiologic, and survival data with the most prevalent viral variant found in each period. We used Kaplan-Meier analysis to estimate the probability of survival and ROC curves to evaluate laboratory tests to find a cutoff point for poor outcomes. Data from 2,887 individuals were analyzed, 1,495 and 1,392 from the first and second periods, respectively. Hospitalization predominated among males in both periods, and the median age was significantly lower in the second one. The frequency of comorbidities was similar. Various demographic factors, clinical assessments, and laboratory tests were examined in relation to greater severity. When comparing the two periods, we observed predominance of the Wild virus during the first wave and the Gamma variant during the second, with no significant difference in outcomes. The findings suggest that despite the association of many factors with increased severity, the temporal variation between the two periods did not result in a notable divergence in the measured outcomes. The COVID-19 pandemic has lasted for a long time, with periods marked by peaks of cases, often caused by the emergence of viral variants, resulting in higher infection rates and rapid dissemination but, for variant Gamma, no apparent greater virulence.

Infection with SARS-CoV-2 variant Gamma (P.1) in Chile increased ICU admission risk three to five-fold.

The 2021 wave of SARS-CoV-2 infection in Chile was characterized by an explosive increase in ICU admissions, which disproportionately affected individuals younger than 60 years. This second wave was also accompanied by an explosive increase in Gamma (P.1) variant detections and the massive vaccine rollout. We unveil the role the Gamma variant played in stressing the use of critical care, by developing and calibrating a queueing model that uses data on new onset cases and actual ICU occupancy, symptom's onset to ICU admission interval, ICU length-of-stay, genomic surveillance, and vaccine effectiveness. Our model shows that infection with the Gamma (P.1) variant led to a 3.5-4.7-fold increase in ICU admission for people younger than 60 years. This situation occurred on top of the already reported higher infection rate of the Gamma variant. Importantly, our results also strongly suggest that the vaccines used in Chile (inactivated mostly, but also an mRNA), were able to curb Gamma variant ICU admission over infections.

Abstract 11051: A Common Variant Near the Fibrinogen Gamma Chain Gene is an Independent Risk Factor for Thrombosis After Congenital Heart Surgery

Introduction: Postoperative thrombosis is a common complication after congenital heart disease (CHD) surgery in children, and is associated with increased mortality and morbidity. Identification of risk factors may allow preventive therapy and improved outcomes. Hypothesis: We tested the hypothesis that genetic variants are associated with postoperative thrombosis after CHD surgery in children. Methods: Children with CHD were prospectively enrolled in an observational cohort study at the time of initial surgical repair/palliation. Clinically detected thrombosis within 30 days of surgery while in hospital was the primary outcome. Patients were genotyped on the Axiom Precision Medicine Research Array. Clinical and genetic (8 common variants in candidate genes) factors were assessed for association with the primary outcome with univariate and multivariate analysis. A two-tailed P-value of < 0.05 was considered statistically significant. Results: A total of 2,398 unique patients (median age 6 months, median weight 6.7 kg) had complete clinical and genetic data for analysis. Postoperative thrombosis occurred in 242 (10.1%). Patients with thrombosis were younger (P<0.001), with longer bypass time (P<0.001) and higher initial postoperative lactate (P<0.001). Thrombosis was also associated with longer hospital stay (50 ± 59 days vs 16 ± 31days, P<0.001) and mortality (15.3% vs 2.9%, P<0.001). One common variant near fibrinogen gamma ( FGG , rs7659024, NC_000004.12:g.154599778G>A) was associated with postoperative thrombosis, with a gene-dose effect (thrombosis observed in 8.9% GG, n=1370, 11% AG, n=869, 15.1% AA, n=159, P=0.025). In multivariate analysis age (per year older OR 0.7, 95% CI 0.61-0.79), bypass time (per hour OR 1.24, 95% CI 1.09-1.39), lactate (per mmol/L OR 1.15, 95% CI 1.19-1.32) and FGG genotype (GG reference, AG OR 1.51, 95% CI 1.09-2.08; AA OR 2.35, 95% CI 1.39-3.97) were each independently associated with postoperative thrombosis. Conclusions: A common variant near FGG previously associated with fibrinogen γ′ levels is an independent risk factor for postoperative thrombosis after CHD surgery. Preoperative genotyping with thromboprophylaxis for patients at sufficient risk may be considered.

Gamma Prime Fibrinogen and Childhood Obesity: Ready for Prime Time?

Gamma Prime Fibrinogen and Childhood Obesity: Ready for Prime Time?

Optimal time scheduling for carrying out minor maintenance on a steam turbine

Almost all the major equipment of ships or industry today undergo regular maintenance on its components and sub-components. In most of the cases it is observed that there are generally two kinds of maintenance in terms of times to repair and its effect on the availability of the main equipment; minor maintenance and a major maintenance or overhaul. The minor maintenance are generally carried at a component or a sub-component level, either at fixed intervals or based on the component condition. The minor maintenance however, requires that the equipment is brought down from operation for a shorter duration and put back in operation immediately after completion of the minor maintenance. The major maintenance on the other hand is carried out preferably in a workshop, where the equipment is completely stripped down to undertake maintenance which otherwise would not have been possible in its original location (for e.g. inside a ship). The time required for carrying out major maintenance is therefore substantial when compared to the minor maintenance period. The paper puts forward a method for scheduling minor maintenance of a major equipment—a steam turbine of a ship, within the time frame of the major maintenance interval based upon the deterioration of the components. The wear processes of the important components of the turbine are assumed to be time variant gamma processes and allow for continuous monitoring of the wear. Since turbines are a complex mix of components, sub-components and other ancillary systems, most of which are easily replaceable in miniscule of time periods, the paper considers only the most important wearing components which play a direct part in deterioration of performance of the turbine. All other random failures, which based on the past experience are only a very few in numbers and have very little impact on the availability of the turbine, have been ignored. MLE and a Gibbs sampling method has been used to estimate the parameters of distribution of gamma wear processes for the components of turbine.

Adhesion of Nanoparticles

We have developed a new model of nanoparticle adhesion which explicitly takes into account the change in the nanoparticle surface energy. Using combination of the molecular dynamics simulations and theoretical calculations we have showed that the deformation of the adsorbed nanoparticles is a function of the dimensionless parameter beta proportional, variant gamma(p)(GR(p))(-2/3)W(-1/3), where G is the particle shear modulus, R(p) is the initial particle radius, gamma(p) is the polymer interfacial energy, and W is the particle work of adhesion. In the case of small values of the parameter beta < 0.1, which is usually the case for strongly cross-linked large nanoparticles, the particle deformation can be described in the framework of the classical Johnson, Kendall, and Roberts (JKR) theory. However, we observed a significant deviation from the classical JKR theory in the case of the weakly cross-linked nanoparticles that experience large shape deformations upon particle adhesion. In this case the interfacial energy of the nanoparticle plays an important role controlling nanoparticle deformation. Our model of the nanoparticle adhesion is in a very good agreement with the simulation results and provides a new universal scaling relationship for nanoparticle deformation as a function of the system parameters.

Fibrinogen and fibrin: scaffold proteins in hemostasis

Elevated fibrinogen is a cardiovascular risk factor. Recent work provides a rationale for this risk, as abnormal fibrin clot structure, strength and stability correlates with coronary artery disease. This review describes in-vitro experiments whose intent is to define the molecular mechanisms that control clot architecture and function in vivo. Biochemical and structural data continue to define the interactions between monomer units that assemble into a fibrin clot. In particular, 'A: a' interactions dominate the first step in fiber formation, while the analogous 'B: b' interactions have a minor role. Studies show the N-terminus of Bbeta, the C-terminus of Aalpha, and the splice variant gamma' modulate fibrin clot structure. Measurement of the mechanical properties of fibrinogen and fibrin show fibrin fibers are among the strongest in nature. Studies have identified fibrinogen-binding proteins that influence clot structure and function. These findings defined mechanisms that control fibrin clot structure, strength and stability. This basic information provides direction for clinical studies to examine clot properties in pathologic thrombosis and pharmaceutical studies to develop therapeutic interventions to prevent or control cardiovascular disease. These studies also establish novel techniques to examine individual bonds, molecules and fibers.

Novel fibrinogen mutation (γ 313 Ser→Asn) associated with hypofibrinogenemia in two unrelated families

Congenital hypofibrinogenemia is a rare disorder caused by a number of different mutations in the fibrinogen genes. The aim of the study was the elucidation of molecular defects in two unrelated families with hypofibrinogenemia. DNA samples from the patients were screened for mutations in the fibrinogen genes by direct sequencing of polymerase chain reaction-amplified gene segments. Isolated plasma fibrinogen was studied by sodium dodecyl sulfate electrophoresis and electrospray ionization mass spectrometry in order to detect variant polypeptides. Fibrin polymerization was analyzed both in plasma and using purified fibrinogen samples. A novel mutation in the FGG gene (G7590A) was found in all patients from the two families with hypofibrinogenemia. This mutation causes the amino acid exchange 313 Ser-->Asn in the gamma chain. When plasma fibrinogen from a heterozygous individual was analyzed for the presence of variant gamma chains by reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry, only normal gamma chains could be detected. The molecular defect affecting an evolutionary highly conserved amino acid residue in human fibrinogen interferes with plasma expression of the variant molecules and is causative for the observed hypofibrinogenemic phenotype.

Pathophysiologic roles of the fibrinogen gamma chain.

Fibrinogen binds through its gamma chains to cell surface receptors, growth factors, and coagulation factors to perform its key roles in fibrin clot formation, platelet aggregation, and wound healing. However, these binding interactions can also contribute to pathophysiologic processes, including inflammation and thrombosis. This review summarizes the latest findings on the role of the fibrinogen gamma chain in these processes, and illustrates the potential for therapeutic intervention. Novel gamma chain epitopes that bind platelet integrin alpha IIbbeta3 and leukocyte integrin alphaMbeta2 have been characterized, leading to the revision of former dogma regarding the processes of platelet aggregation, clot retraction, inflammation, and thrombosis. A series of studies has shown that the gamma chain serves as a depot for fibroblast growth factor-2 (FGF-2), which is likely to play an important role in wound healing. Inhibition of gamma chain function with the monoclonal antibody 7E9 has been shown to interfere with multiple fibrinogen activities, including factor XIIIa crosslinking, platelet adhesion, and platelet-mediated clot retraction. The role of the enigmatic variant fibrinogen gamma chain has also become clearer. Studies have shown that gamma chain binding to thrombin and factor XIII results in clots that are mechanically stiffer and resistant to fibrinolysis, which may explain the association between gammaA/gamma' fibrinogen levels and cardiovascular disease. The identification of new interactions with gamma chains has revealed novel targets for the treatment of inflammation and thrombosis. In addition, several exciting studies have shown new functions for the variant gamma chain that may contribute to cardiovascular disease.

Effect of particle inertia on turbulence in a suspension.

We propose a one-fluid analytical model for a turbulently flowing dilute suspension, based on a modified Navier-Stokes equation with a k-dependent effective density of suspension rho(eff)(k) and an additional damping term proportional, variant gamma(p)(k), representing the fluid-particle friction (described by Stokes law). The statistical description of turbulence within the model is simplified by a modification of the usual closure procedure based on the Richardson-Kolmogorov picture of turbulence with a differential approximation for the energy transfer term. The resulting ordinary differential equation for the energy budget is solved analytically for various important limiting cases and numerically in the general case. In the inertial interval of scales, we describe analytically two competing effects: the energy suppression due to the fluid-particle friction and the energy enhancement during the cascade process due to decrease of the effective density of the small-scale motions. An additional suppression or enhancement of the energy density may occur in the viscous subrange, caused by the variation of the extent of the inertial interval due to the combined effect of the fluid-particle friction and the decrease of the kinematic viscosity of the suspensions. The analytical description of the complicated interplay of these effects supported by numerical calculations is presented. Our findings allow one to rationalize the qualitative picture of the isotropic homogeneous turbulence of dilute suspensions as observed in direct numerical simulations.

Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein

To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411. The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen. Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical. We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids.

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

The structure and biological features of fibrinogen and fibrin.

Fibrinogen and fibrin play important, overlapping roles in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, and neoplasia. These events are regulated to a large extent by fibrin formation itself and by complementary interactions between specific binding sites on fibrin(ogen) and extrinsic molecules including proenzymes, clotting factors, enzyme inhibitors, and cell receptors. Fibrinogen is comprised of two sets of three polypeptide chains termed A alpha, B beta, and gamma, that are joined by disulfide bridging within the N-terminal E domain. The molecules are elongated 45-nm structures consisting of two outer D domains, each connected to a central E domain by a coiled-coil segment. These domains contain constitutive binding sites that participate in fibrinogen conversion to fibrin, fibrin assembly, crosslinking, and platelet interactions (e.g., thrombin substrate, Da, Db, gamma XL, D:D, alpha C, gamma A chain platelet receptor) as well as sites that are available after fibrinopeptide cleavage (e.g., E domain low affinity non-substrate thrombin binding site); or that become exposed as a consequence of the polymerization process (e.g., tPA-dependent plasminogen activation). A constitutive plasma factor XIII binding site and a high affinity non-substrate thrombin binding site are located on variant gamma' chains that comprise a minor proportion of the gamma chain population. Initiation of fibrin assembly by thrombin-mediated cleavage of fibrinopeptide A from A alpha chains exposes two EA polymerization sites, and subsequent fibrinopeptide B cleavage exposes two EB polymerization sites that can also interact with platelets, fibroblasts, and endothelial cells. Fibrin generation leads to end-to-middle intermolecular Da to EA associations, resulting in linear double-stranded fibrils and equilaterally branched trimolecular fibril junctions. Side-to-side fibril convergence results in bilateral network branches and multistranded thick fiber cables. Concomitantly, factor XIII or thrombin-activated factor XIIIa introduce intermolecular covalent epsilon-(gamma glutamyl)lysine bonds into these polymers, first creating gamma dimers between properly aligned C-terminal gamma XL sites, which are positioned transversely between the two strands of each fibrin fibril. Later, crosslinks form mainly between complementary sites on alpha chains (forming alpha-polymers), and even more slowly among gamma dimers to create higher order crosslinked gamma trimers and tetramers, to complete the mature network structure.

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.

Normal Binding of Calcium to Five Fibrinogen Variants with Mutations in the Carboxy Terminal Part of the γ-Chain

Calcium ions are known to accelerate polymerization of fibrin monomers. Each of the two carboxy terminal domains of normal fibrinogen contains one high-affinity calcium binding site that seems to be situated close to the polymerization site in the gamma-chain. Most hitherto described functionally defective fibrinogen variants showed impaired clot formation. Since the tightly bound calcium ions may influence the conformation of the polymerization site, the question arises whether the abnormal clotting of a dysfibrinogen might be due to defective calcium binding. We investigated binding of calcium to fibrinogen and the effect of calcium on the clotting properties of five heterozygous fibrinogen variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinogens has one single amino acid substitution in the carboxy-terminal part of the gamma-chain: fibrinogen Claro (gamma 275 Arg-->His), Milano V (gamma 275 Arg-->Cys), Milano I (gamma 330 Asp-->Val), Bern I (gamma 337 Asn-->Lys), and Milano VII (gamma 358 Ser-->Cys). The shortest thrombin clotting time and the earliest onset of turbidity increase were observed in fibrinogen gamma 358 Ser-->Cys; both parameters were little affected by calcium concentration. In the variant gamma 337 Asn-->Lys, the thrombin time was abnormally prolonged at 0.01 mM Ca2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen gamma 330 Asp-->Val was barely improved at increasing calcium concentrations. Both variants with the substitution of gamma 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and the lowest turbidity of fibrin clots at any Ca2+ concentration used. High affinity calcium binding was found to be normal in all five fibrinogen variants studied, suggesting that their abnormal clotting was not due to defective binding of calcium. The gamma-chain in the fragment D1 derived from the variant gamma 337 Asn-->Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas fragments D1 from the other four gamma-chain variants as well as from normal fibrinogen were protected against plasmic degradation in the presence of 1 mM Ca2+.

Binding of recombinant fibrinogen mutants to platelets.

Platelet aggregation is mediated by the interaction of fibrinogen with platelet membrane glycoprotein IIb-IIIa, a member of the integrin family (integrin alpha IIb beta 3). Three different binding sites on fibrinogen for IIb-IIIa have been proposed, two RGD-containing sequences in the alpha chain and one dodecapeptide sequence at the carboxyl terminus of the gamma chain. However, recent evidence shows that mutations in either of the alpha chain sequences have no effect on platelet aggregation, whereas the substitution of a variant gamma chain (gamma') for the gamma chain results in a major reduction in platelet aggregation activity. The present investigation demonstrates that the gamma' chain shows decreased binding to IIb-IIIa as measured by direct binding experiments. In addition, adhesion studies indicate that the binding of both stimulated and unstimulated platelets to immobilized fibrinogens is mediated primarily through the gamma chain carboxyl terminus. Furthermore, a peptide corresponding to the carboxyl terminus of the gamma chain inhibits fibrinogen binding and platelet adhesion, whereas a peptide corresponding to the carboxyl terminus of the gamma' chain is significantly less inhibitory. These data show that the defective platelet aggregation activity of the fibrinogen gamma' chain is due to decreased binding to platelet glycoprotein IIb-IIIa.

Purification and Functional Characterization of Homodimeric γB-γB Fibrinogen From Rat Plasma

Heterogeneity in the human and rat plasma fibrinogen (FBG) gamma chains is due to differential splicing of the primary gamma chain mRNA transcript. The subunit composition of FBG containing the predominant form of the gamma chain is homodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 50; rat: A alpha 2, B beta 2, gamma A-gamma A). The subunit composition of FBG containing the longer, minor form of the gamma chain is heterodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 57.5; rat: A alpha 2, B beta 2, gamma A-gamma B). The variant gamma chain-FBGs comprise about 10% of the total plasma FBG in the human and 30% in the rat. Although the presence of homodimeric gamma B-gamma B FBG has been speculated, proof of its existence has been difficult to obtain experimentally. We now show that 5% to 6% of rat plasma FBG is found as homodimeric FBG with a subunit composition of A alpha 2, B beta 2, gamma B-gamma B. Commercially purified rat FBG was further purified by diethylaminoethyl-Sephacel chromatography with a combined pH and ionic strength gradient. The enriched gamma B-gamma B FBG fraction eluted at the lowest pH (6.3) and highest ionic strength (4.5 mmho) due to its increased net negative charge. To further purify gamma B-gamma B FBG, a Mono Q column with an NaCl gradient was used. The subunit composition of the purified gamma B-gamma B FBG was confirmed by its electrophoretic mobility under reducing and denaturing conditions both as FBG, and clotted and cross-linked fibrin. Homodimeric gamma B-gamma B FBG was unable to support adenosine diphosphate (ADP)-induced platelet aggregation, whereas heterodimeric gamma A-gamma B FBG was able to support ADP-induced platelet aggregation at 40% of that achieved with homodimeric gamma A-gamma A FBG, similar to previous observations with human FBGs. Taken together, these results support the hypothesis that the A alpha-RGD sites alone are not sufficient for dimeric FBG support of platelet aggregation. Furthermore, the data suggest that at least one intact platelet recognition site at the carboxyterminus of the gamma A or gamma 50 chain is required.

Purification and functional characterization of homodimeric gamma B- gamma B fibrinogen from rat plasma

Heterogeneity in the human and rat plasma fibrinogen (FBG) gamma chains is due to differential splicing of the primary gamma chain mRNA transcript. The subunit composition of FBG containing the predominant form of the gamma chain is homodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 50; rat: A alpha 2, B beta 2, gamma A-gamma A). The subunit composition of FBG containing the longer, minor form of the gamma chain is heterodimeric (human: A alpha 2, B beta 2, gamma 50- gamma 57.5; rat: A alpha 2, B beta 2, gamma A-gamma B). The variant gamma chain-FBGs comprise about 10% of the total plasma FBG in the human and 30% in the rat. Although the presence of homodimeric gamma B- gamma B FBG has been speculated, proof of its existence has been difficult to obtain experimentally. We now show that 5% to 6% of rat plasma FBG is found as homodimeric FBG with a subunit composition of A alpha 2, B beta 2, gamma B-gamma B. Commercially purified rat FBG was further purified by diethylaminoethyl-Sephacel chromatography with a combined pH and ionic strength gradient. The enriched gamma B-gamma B FBG fraction eluted at the lowest pH (6.3) and highest ionic strength (4.5 mmho) due to its increased net negative charge. To further purify gamma B-gamma B FBG, a Mono Q column with an NaCl gradient was used. The subunit composition of the purified gamma B-gamma B FBG was confirmed by its electrophoretic mobility under reducing and denaturing conditions both as FBG, and clotted and cross-linked fibrin. Homodimeric gamma B-gamma B FBG was unable to support adenosine diphosphate (ADP)-induced platelet aggregation, whereas heterodimeric gamma A-gamma B FBG was able to support ADP-induced platelet aggregation at 40% of that achieved with homodimeric gamma A-gamma A FBG, similar to previous observations with human FBGs. Taken together, these results support the hypothesis that the A alpha-RGD sites alone are not sufficient for dimeric FBG support of platelet aggregation. Furthermore, the data suggest that at least one intact platelet recognition site at the carboxyterminus of the gamma A or gamma 50 chain is required.

The Detection of Hemoglobin Variants by Isoelectrofocusing Using EDTA-Collected and Filter Paper-Dried Cord Blood Specimens

A comparative study was conducted aimed at the detection of abnormal hemoglobin conditions (mainly AS, SS, AC, CC, SC, AE, AD, Hb Bart's or gamma 4) by isoelectrofocusing of cord blood samples stored as liquid blood and as dried hemoglobin on filter paper. Analyses were made within four to six days after the collection of the samples; storage conditions mimicked those of testing programs using liquid blood samples (as in Georgia) or dried blood filter paper samples (as in several other states). During analysis of hemoglobin solutions extracted from dried blood samples, considerable difficulties were encountered in detecting significant hemoglobinopathies such as SS and SC, whereas even simple abnormalities such as AS, AC, AD, and AE were also often not diagnosed. Detection of the fast-moving variant Hb Bart's or gamma 4 was not possible. These results again cause doubt regarding the general use of dried blood filter paper samples in newborn hemoglobin testing programs. Perhaps special precautions--such as speed in analyzing the samples, storage at -20 degrees C (or perhaps 4 degrees C) instead of at room temperature, and removal of unstable hemoglobin from the filter paper extract by centrifugation--might eliminate some of the problems that were observed.

The C-terminal sequences of the gamma 57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin

The gamma chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two gamma chain mRNAs encoding gamma 50 and gamma 57.5 polypeptides, while fibrinogen gamma 55 is produced by post- translational modification of the gamma 57.5 chain. The composition of purified variant gamma chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (A alpha, B beta, gamma 50/gamma 55 or A alpha, B beta, gamma 50/gamma 57.5), whereas the composition of purified fibrinogen with the major form of the gamma chain is homodimeric (A alpha, B beta, gamma 50/gamma 50). These gamma chain variations interrupt sequences that mediate platelet- fibrinogen interactions. Therefore, the structure and function of gamma 57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included gamma 57.5 chain residues 409–412 (Arg-Pro-Glu- His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen gamma 57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide gamma 57.5 405– 416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. Binding of fibrinogen gamma 50 (IC50 = 780 mumol/L) or gamma 57.5 (IC50 = 650 mumol/L) to ADP- stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen gamma 57.5 binding. Peptide gamma 57.5 408–416 failed to dissociate platelet-bound fibrinogens. These data indicate that the gamma 408–416 sequence of fibrinogen gamma 55 or gamma 57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein llb-llla (GPllb-llla), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPllb-llla binding site peptide sequences [Arg-Gly-Asp- Ser (RGDS) or gamma 50 400–411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with gamma 57.5 408–416. This epitope is available for binding MoAb L2B in gamma 55 or gamma 57.5 chain dimers and binds to all gamma 57.5 408–416 epitopes equally in non-crosslinked and factor Xllla crosslinked fibrin clots.(ABSTRACT TRUNCATED AT 400 WORDS).