Fibrinogen Peak Research Articles

Palifermin reduces infection rate and hyperfibrinogenemia in patients treated with high-dose chemotherapy based on beam or BU-thiothepa.

We performed a retrospective study in patients who underwent high-dose chemotherapy and auto-SCT because of haematological malignancies. Forty patients were treated with palifermin while 80 were controls selected after being matched for diagnosis and length of neutropenia. Patients treated with BEAM or BU-CY or THIO-CY (BEAM/BUS) displayed, after palifermin, a lower rate of severe oral mucositis (P=0.03). This beneficial effect of palifermin was not evident in the stratum of patients treated with high-dose melphalan (HD-PAM). After palifermin, we observed in the whole treated population a reduced rate of 'fever of unknown origin' (FUO, P=0.02) and of severe infections not related to Gram-positive bacteria (FUO, Gram-negative bacteremia or pneumonia) (P=0.003). This effect of palifermin on infections not related to Gram-positive bacteria was evident only in patients receiving BEAM/BUS (P=0.01) and not in patients treated with HD-PAM (P=0.11). Fibrinogen peak in plasma was found to be reduced after palifermin in the whole population (P=0.01) and in the stratum who received BEAM/BUS (P=0.02) but not in the stratum of HD-PAM. In conclusion, anti-infectious beneficial effects of palifermin are more evident in BEAM/BUS-treated patients and toward some types of infections. Reduction of fibrinogen level after palifermin suggests that this agent reduces not only the rate of infections but also their severity.

Cellulose acetate electrophoresis of canine plasma after fibrinogen precipitation by ethanol

In routine canine medicine, anticoagulated blood is often the only sample sent to laboratories for diagnostic purposes. This hampers the interpretation of protein electrophoretic tracings because plasma contains fibrinogen, which migrates in the beta-gamma region. In human medicine, fibrinogen can be precipitated from plasma using ethanol. The purpose of this study was to assess ethanol precipitation as a method for removing fibrinogen from canine plasma so as to facilitate the interpretation of electorphoresis results. Blood samples collected from 40 dogs were divided into plain tubes and tubes containing EDTA (n=20) or lithium-heparin (n=20). An aliquot of plasma from each sample was incubated with ethanol at a final concentration of 100 mL/L. Cellulose acetate electrophoresis was then performed on serum, plasma, and plasma treated with ethanol. To verify the efficiency of ethanol treatment, fibrinogen was added to 5 canine serum samples at final concentrations of 2.5, 5.0, and 10.0 g/L, and electrophoresis was performed before and after ethanol treatment. Visual analysis of electrophoretograms from ethanol-treated samples confirmed the disappearance of the fibrinogen peak from the beta(2)-globulin region. Treatment with ethanol caused a significant decrease in the percentage of beta(2)-globulins and a significant increase in the percentage of alpha(2)-globulins. Absolute values of most electrophoretic fractions were significantly decreased in ethanol-treated plasma compared with serum. Ethanol treatment successfully removed fibrinogen from canine plasma and normalized electrophoretic profiles, but probably also precipitated proteins other than fibrinogen. Ethanol treatment is recommended to facilitate visual identification of abnormal monoclonal peaks, but not for determining absolute protein concentrations in electrophoretic fractions.

Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains.

The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.

Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation

Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.

Temperature dependent dissociation of soluble fibrin monomer complexes demonstrated by agarose gel filtration

The temperature dependent dissociation behaviour of soluble fibrin monomer complexes (SFMC) in plasma of post surgical patients was studied using gel filtration chromatography of β-Ala precipitated plasma samples. A temperature dependency in the investigated range between 12° and 37°C was demonstrated with a linear regression y = 5.56−0.07 x and a correlation coefficient r = 0.83. Additional experiments using 125 I-des- A fibrin injected into heparinized rabbits revealed that after gel filtration of whole plasma samples at 20°C 68% of the activity was eluted in front of the fibrinogen elution volume and 32 % with the fibrinogen volume. At 37°C 24 % of the activity was eluted in front of fibrinogen, while 76 % eluted with the fibrinogen peak. The experiments indicate that SFMC are subject to a temperature dependent dissociation behaviour which is also influenced by the type of the complexes.

Occurrence of Soluble Very High Molecular Weight Fibrinogen Complexes in Hypercoagulable States. An in vitro and in vivo Study on their Features.

The gel-chromatography of the beta-alanine precipitate of human plasma and of purified fibrinogen shows the presence of different classes of fibrinogen complexes. A first class of complexes is detectable in correspondence with the ascending branch of the fibrinogen peak ("shoulder" complexes or high molecular weight fibrinogen complexes, HMWFC). A second class of complexes at very high molecular weight is eluted at the void volume (peak 1 complexes or VHMWFC). Treatment of plasma or fibrinogen in vitro with thrombin induces the formation of both classes of complexes. Also thrombin infusion in rabbits induces an increase of both VHMWFC and HMWFC; pretreatment with heparin prevents these changes. In the detection of VHMWF 4% agarose and beta-alanine precipitation seem to be necessary for a good resolution of the chromatographic profile. VHMWFC are devoid of albumin, transferrin and immunoglobulins on radial immunodiffusion and their elution volume suggests a molecular weight over 1 million. They show a procoagulant activity; namely they accelerate the fibrinogen polymerization and increase the resistance of normal plasma to heparin. They also induce the formation of platelet aggregates, reversible in EDTA, when incubated without stirring with control PRP, but in Born apparatus in presence of ADP they show a slight antiaggregating activity. In normal healthy young volunteers (control group A) the average values were respectively 7.81 ± 3.53 mg% for VHMWFC and 12.19 ± 4.74 mg% for HMWFC. Both VHMWFC and HMWFC are increased in patients with hypercoagulable states (patients with history of myocardial infarction, MIP, with chronic cerebrovascular disorders, CVP, with acute thrombophlebitis and with subacute intravascular coagulation, cancer patients). The increase of VHMWFC and HMWFC is however different in the various groups of patients. An increase predominantly of VHMWFC (sometimes with normal HMWFC) is detectable in MIP and CVP, whereas in subjects with acute thrombophlebitis and with cancer a predominant increase of HMWFC associated with increased levels of FDP is detectable. The detection of VHMWFC represents a useful tool for the evaluation of hypercoagulable state, and their occurrence can cooperate to enhance hypercoagulability as VHMWFC possess procoagulant activity.

Formation and dissociation of soluble fibrin complexes in plasma at 20°C and at 37°C

Small amounts of thrombin were added to human plasma containing 125I-fibrinogen to generate fibrin. Thereafter, thrombin was quenched with hirudin and 131I-fibrinogen added for internal calibration. Gel filtration of this mixture on sepharose CL-6B columns equilibrated with plasma revealed two peaks, one (peak A) eluting with the void volume, the other (peak B) with the volume corresponding to the fibrinogen peak. At 20°C, a small amount of 131I-fibrinogen (4.5%) was eluted together with the thrombin-treated 125I-fibrinogen in peak A, whereas at 37°C, no 131I-fibrinogen was recovered in peak A. If isolated fractions of peak A eluted at 20°C were rechromatographed at 20°C, they were eluted again in the void volume. At 37°C, however, this material was mainly eluted in peak B corresponding to the fibrinogen peak. If isolated fractions of peak B at 37°C were rechromatographed at 37°C, they were eluted with the same volume as in the original chromatography. At 20°C, however, the material was eluted in peak A as well as in peak B. These data show that at 20°C fibrin aggregates generated in a plasma medium from monomeric fibrin without incorporating fibrinogen. From these experiments, we conclude that so-called fibrin-fibrinogen complexes not stabilized by factor XIIIa generate only in vitro at 20°C, but do not exist at 37°C.

Soluble Fibrin Complexes: Separation as a Function of pH and Characterization

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH. 2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aalpha, Bbeta and gamma chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aalpha, alpha, Bbeta and gamma chains were found. In the peak containing the high polymers, only the presence of alpha, Bbeta and gamma chains was demonstrated. 3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

Complex formation of crosslinked fibrin oligomers with agarose-coupled fibrinogen and fibrin.

Soluble oligomers of fibrin were produced by limited incubation of fibrinogen with thrombin in the presence of calcium2 positive earth, cysteine and factor XIII. They were separated from fibrinogen by gel filtration. Gamma-gamma dimers were demonstrated in fractions from the void volume and the shoulder prior to the fibrinogen peak. These fractions were subjected to affinity chromatography on agarose-coupled fibrinogen and fibrin. Cross-linked oligomers of fibrin were adsorbed to agarose-coupled fibrin, whereas no adsorption occured during affinity chromatography on agarose-coupled fibrinogen. This indicates that crosslinked fibrin oligomers have no affinity to fibrinogen, yet readily form complexes with fibrin.

Semiquantitative Determination of Soluble Fibrin Monomer Complexes by Chromatography and Serial-dilution Protamine Sulfate Test

Plasmas were prepared from blood obtained from rabbits and from healthy individuals and patients with thrombotic disorders. Gel filtration (Sepharose-4B) chromatography and the serial-dilution protamine sulfate (SDPS) test were performed on each sample. For healthy individuals and rabbits, a narrow fibrinogen peak was obtained on chromatogram and was associted with a negative SDPS test. After administration of thrombin or endotoxin to rabbits and in plasmas from patients with intravascular coagulation, major shifts to earlier effluent volumes occurred. These higher-molecular-weight derivatives corresponded to soluble fibrin monomer complexes (FM). Positive SDPS tests were found in these plasmas. There was a highly significant correlation between percentage area occupied by FM on chromatogram and SDPS test titer. It is concluded that the SDPS test is a reliable semiquantitative measure of FM that can be used clinically as well as in experimental animal models.

Soluble Fibrin Monomer Complexes Demonstrated by Agarose Gel Filtration and by Adsorption on Insolubilized Fibrinogen

Incubation of fibrinogen with small amounts of thrombin resulted in the occurrence of soluble fibrin monomer complexes. These complexes consisted predominantly of a derivative with a higher molecular weight than that of fibrinogen. It was characterized by its relative electrophoretic mobility in 5% PAA gel (0.28 X 10(-5) cm2/V X sec) and its elution position prior to the fibrinogen peak following gel filtration. Using adsorption chromatography on insolubilized fibrinogen the derivative dissociated at a ratio of almost 1 : 1 into one part which was adsorbed and into fibrinogen which was not adsorbed. The part which was adsorbed seemed to be the thrombin mediated fibrin monomer. This study confirms the concept that dissociable dimeric fibrinogen-fibrin monomer complexes occur after limited action of thrombin on fibrinogen.

Letter: Oral contraception and increased formation of high molecular weight derivatives of fibrinogen.

Experiments conducted in the writers' laboratory on oral contraception and increased formation of high molecular weight derivatives of fibrinogen confirm results of a report by L.O. Pilgeram and others on an increased formation of soluble fibrin in patients taking oral contraceptives. 7 healthy women 17-35 years old were examined before and during a 4-12 week period of treatment with an oral contraceptive containing norgestrel .25 mg and ethinyl estradio. .05 mg. On Days 24 and 26 of the anovulatory menstrual cycles, citrated plasma was chromatographed on a Biogel-A-5m-column. Fibrinogen-related material in the eluates were titrated manually with the staphylococcal clumping test, and the mean concentrations before and during contraception were calculated for each fraction. The results indicated 1) an unchanged elution position of the quantitatively increased fibrinogen, 2) increased amounts of high molecular weight derivatives in the early fractions, and 3) the elution position of the main fraction of high molecular weight derivatives in the position calculated for the fibrinogen-fibrin-dimer. Mathematical processing of the data suggests that the polymers represent 5.6% of the total amount of the fibrinogen peak, which would correspond to a monomer concentration of 2%-3%, assuming that a 1:1 complex of fibrinogen and fibrin monomers is formed.

Incorporation of fibrinogen into soluble fibrin complexes

Fibrinogen was incubated with traces of thrombin, and the reaction stopped with hirudin prior to visible gelation. When I 125-labelled fibrinogen was added to the mixture before chromatography on Sepharose 4B, labelled fibrinogen eluted not only in the main fibrinogen peak, but also in the earlier fractions containing high molecular weight complexes. This implies that fibrinogen not acted upon by thrombin, may form complexes with soluble fibrin. The complexes detected by gel chromatography could not be demonstrated by SDS-urea polyacrylamide gel electrophoresis, indicating that non-covalent bonds are involved.

Formula omitted] formation of soluble fibrin monomer complexes in human plasma

Plasma samples from asphyctic newborns as well as maternal plasma samples during delivery contain soluble fibrin monomer complexes (SFMC) as evidenced by the elution of FR-antigen material in a shoulder prior to the fibrinogen peak after agarose gel filtration. PAA gel electrophoresis of fractions from the shoulder revealed a component with the relative electrophoretic mobility of 0.28 × 10 −5 cm 2/V × sec (fibrinogen 0.43 × 10 −5 cm 2/V × sec). This component consisted of FR-antigen material only and no other plasma proteins were associated to it. Elution behaviour, migration pattern and immunological properties of the 0.28 derivative are in agreement with the assumption of a dimeric structure, which may occur either as a fibrinogen fibrin monomer complex or as a covalently linked dimer.

Formation of soluble fibrin monomer complexes in human plasma

Small amounts of thrombin were added in vitro to plasma for a short time interval. Following β-alanine precipitation PAA-gel-electrophoresis revealed a component with a relative electrophoretic mobility of 0.28 × 10 −5 cm 2 /V × sec. (fibrinogen 0.43 × 10 −5 cm 2 /V × sec.). Following agarose gel filtration the 0.28 derivative was eluted in the fractions of the shoulder prior to the fibrinogen peak. According to the quantitative relation of the 0.28 derivative to higher forms of monomer units it was demonstrated that short exposure of plasma to small amounts of thrombin resulted predominantly in the occurrence of the 0.28 derivative.

Experiments with factor 8 separated from fibrinogen by electrophoresis in free buffer film.

Approaches to an understanding of the chemistry of Factor VIII (antihaemophilic factor) have been few and necessarily indirect since Factor VIII occurs in plasma in only trace amounts, possibly less than 1 mg./l., is very labile, and the biological assay methods are subject to large errors. There have been repeated suggestions that the actual active Factor‐VIII molecule might be rather small and that in plasma it is associated with another protein (Thelin and Wagner, 1961; Surgenor, 1964). Methods of purification of Factor VIII are essentially modifications of methods applicable to fibrinogen. The highest purification of Factor VIII is probably that of Michael and Tunnah (1963) but the protein of even their best preparation is at least 50 per cent fibrinogen. Methods directed towards removing fibrinogen specifically by conversion to fibrin or heating result in a loss of Factor‐VIII activity. It seemed possible, therefore, that if there is an association between Factor VIII and another protein, the partner might be fibrinogen.Cross‐linked gels provide the possibility of separating molecules according to their size. The degree of cross linking determines the size of molecules which cannot enter the lattice of the gel particles, and so travel fastest down a column of gel. According to the manufacturers (Pharmacia Ltd., Uppsala, Sweden) Sephadex G‐200 has a structure which should totally exclude molecules having a molecular weight above 200,000, so that fibrinogen (molecular weight about 340,000) should be separable under very mild conditions from any substances having molecular weights much below 200,000. Thelin and Wagner (1961) produced evidence that in high salt concentration (0.4 M‐NaCl) Factor VIII dissociated to a size comparable to that of albumin (molecular weight about 70,000). Molecules of this size should be readily and completely separable from fibrinogen on a Sephadex G‐200 column unless the two had some definite bond.In experiments reported below we tried to separate bovine Factor VIII from fibrinogen in commercial bovine antihaemophilic globulin using columns of Sephadex G‐200, but found the Factor‐VIII activity at the front of the fibrinogen peak even when the gel filtration took place in M‐NaCl or M‐urea in an effort to minimize protein‐protein interaction. We confirmed the observation of Lewis (1964) that Factor VIII in whole human plasma appears to be totally excluded from Sephadex G‐200. Although correlation with molecular weight would not be precise if the molecule were markedly asymmetrical this probably indicates that the Factor‐VIII molecule or complex has a molecular weight in excess of 200,000. These experiments do not distinguish between the possibilities that Factor VIII is itself a large molecule, or is a small molecule in association with fibrinogen. It seemed that discussion about the possible association between Factor VIII and fibrinogen could only be settled by repeating this type of molecular sieve experiment with Factor VIII prepared free from fibrinogen by some other method. Even the best preparations of Factor VIII made by methods depending on salt fractionation and chromatography still contained a large proportion of fibrinogen (Michael and Tunnah, 1963). Only electrophoretic methods seemed to offer the possibility of a complete separation of these two factors.