Fibrinogen Derivatives Research Articles

Structure and refractive index of fibrin protofibril aggregates according to laser phase microscopy accompanied by DLS and AFM.

The structures, sizes, and refractive indices (RI) of protein aggregates formed in a fibrinogen-thrombin system are examined using laser phase microscopy (LPM) accompanied by dynamic light scattering (DLS) and atomic force microscopy (AFM) measurements. Fibrin aggregates found in pure fibrinogen and fibrinogen with thrombin solutions by the DLS method, after drying the sample, form complex structures of different shapes and sizes on a glass surface. The LPM reveals submicron-sized dimeric structures in the pure fibrinogen solution, elongated micron-length structures, and rectangular structures in the fibrinogen-thrombin sample. AFM measurements show that the elongated structures form branched fibers, which in turn assembly into rectangular structures. All sizes obtained by LPM and AFM are consistent with DLS measurements. The refractive indices of all the structures, estimated by optical thickness, vary from 1.53 to 1.62, which indicates that they are fibrinogen derivatives. Effective visualization of the structure and determination of the optical properties for fibrin gel indicate that laser phase microscopy is capable of tissue imaging and characterization.

Platelet collagen receptor Glycoprotein VI‐dimer recognizes fibrinogen and fibrin through their D‐domains, contributing to platelet adhesion and activation during thrombus formation

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi.GPVI‐dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D‐domains.Collagen, D‐fragment and D‐dimer may share a common or proximate binding site(s) on GPVI‐dimer.GPVI‐dimer–fibrin interaction supports spreading, activation and adhesion involving αIIbβ3. SummaryBackgroundPlatelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin.ObjectivesTo determine if GPVI‐dimer, GPVI‐monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site.MethodsRecombinant GPVI monomeric extracellular domain (GPVI ex) or dimeric Fc‐fusion protein (GPVI‐Fc2) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti‐GPVI‐dimer or anti‐αIIbβ3.ResultsUnder static conditions, GPVI ex did not bind to any fibrinogen substrate. GPVI‐Fc2 exhibited specific, saturable binding to both D‐fragment and D‐dimer, which was inhibited by mFab‐F (anti‐GPVI‐dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI‐Fc2 binding to D‐fragment or D‐dimer was abrogated by collagen type III, Horm collagen or CRP‐XL (crosslinked collagen‐related peptide), suggesting proximity between the D‐domain and collagen binding sites on GPVI‐dimer. Under low shear, adhesion of normal platelets to D‐fragment, D‐dimer, fibrinogen and fibrin was inhibited by mFab‐F (inhibitor of GPVI‐dimer) and abolished by Eptifibatide (inhibitor of αIIbβ3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbβ3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab‐F, supporting some independent GPVI‐dimer involvement in this interaction.ConclusionOnly dimeric GPVI interacts with fibrinogen D‐domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

Antioxidant and Anticoagulant Status Were Improved by Personalized Dietary Intervention Based on Biochemical and Clinical Parameters in Cancer Patients

We investigated whether personalized dietary intervention could improve clinical measurements such as immune cell-mediated cytotoxicity, serum albumin, derivatives of reactive oxygen metabolites (D-ROMS), D-dimer, and fibrinogen. Cancer patients received either a treatment support diet (TD, for those with chemotherapy), or a remission support diet (RD; for those in remission) for at least 3 wk (21–61 days). Both diets were low glycemic, low fat, and high plant protein diets; the diet for the TD group contained an additional 0.5 servings of protein. Based on clinical values, additional amounts of garlic, onion, tomato, shiitake, rice bran, kale, blueberry, pineapples, and/or turmeric powder were provided in regular meals. Estimated daily intake of protein, plant fat, garlic, onion, allicin, and quercetin was greater in the TD compared to the RD. An increased intake of vitamin A, vitamin C, vitamin E and selenium and a reduction in D-dimer were noted compared to baseline diets in both groups. A decrease in D-ROMS in the RD and an increase in albumin and an increased tendency in cytotoxicity in the TD were observed. In conclusion, personalized diets with supplemented functional ingredients improved antioxidant status and/or anticoagulant activity in cancer patients undergoing chemotherapy and in remission.

D-dimer: A novel predictive marker for cardiovascular disease

The D-dimer, a high-weight molecule of the fibrinogen derivative produced by the division of cross-linked fibrin, reflects both thrombin production and the activation of fibrinolysis. High D-dimer levels occur in many diseases in which the coagulation system is activated, such as acute venous thromboembolism, ischemic heart disease, and cancer.

The Assembly of Nonadhesive Fibrinogen Matrices Depends on the αC Regions of the Fibrinogen Molecule

Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7-8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia.

The antileukemic activity of modified fibrinogen–methotrexate conjugate

BackgroundThe search for new, innovative methods to treat all types of diseases, especially cancer-related ones, is a challenge taken by pharmaceutical companies and academic institutions. The use of conjugates containing widely-known and widely-used bioactive substances is one of the ways to solve this problem. Research into drug binding with macromolecular carrier systems has joined the search for new therapeutic strategies. MethodsThe main goal of this paper is the potential offered by the use of fibrinogen derivatives as an antileukemic drug carrier. Physicochemical properties of the obtained conjugate were analyzed, characterizing alterations in relation to the starting carrier and analyzing biological implications. The intraperitoneally (i.p.) inoculated P388 mouse leukemia model for in vivo studies was used. Results and conclusionsConjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug were developed. Carrier preparation and a conjugate synthesis in aqueous solution were formulated, as well as purification of the conjugate was performed. The study showed that the survival of leukemia mice treated with FH–MTX conjugate was indeed significantly longer than survival in both untreated animals (control) and mice treated with unbound MTX. A significant increase in the antileukemic activity of MTX conjugated with hydrolysed fibrinogen was observed as compared with the unconjugated drug. Reported data suggest that hydrolysed fibrinogen can serve as a carrier molecule for the MTX drug with the aim of enhancing its antileukemic activity. General significanceConjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug seem to be a promising anticancer drug.

Radiolabeled and near-infrared fluorescent fibrinogen derivatives create a system for the identification and repair of obscure gastrointestinal bleeding

Gastrointesintal hemorrhage is often difficult to localize. A test that does not depend on active bleeding might prove clinically useful. We tested 2 novel fibrinogen (FBG)-based contrast agents for their ability to localize gastrointestinal (GI) hemorrhage after bleeding stopped. 125I-FBG permits gamma ray-based preoperative or intraoperative scanning, and near-infrared (NIR) flourescent FBG (FBG800) permits real-time intraoperative visualization of active clot. Bovine FBG was radiolabeled with 125I or conjugated to the NIR fluorophore CW800. Sites of bleeding were created by gastrotomy, mucosal resection of the stomach, or laceration of a mesenteric vessel; then 1.7 mg/kg FBG800 or 15 microCi/kg 125I-FBG was injected intravenously into mice, rabbits, or pigs 30 minutes before or after injury. Sites of active clot were quantified by using gamma counting and were also imaged by using invisible NIR light intraoperatively, for up to 3 hours postinjection. After an injection of either 125I-FBG or FBG800, sites of prior bleeding could be identified in the absence of active bleeding. Blood clearance was such that a signal-to-background ratio of 2.0 or greater could be achieved within 20 minutes after injection. A similarly labeled human serum albumin did not accumulate at any site, with an SBR of 1.0 or less. Both radiolabeled (preoperative gamma scanning) and NIR fluorescent (intraoperative real-time imaging) FBG can be used in experimental situations to identify the location of prior bleeding in the absence of active bleeding. Taken together, these contrast agents create a system for the identification and control of obscure GI bleeding.

Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach

Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein α-1 subunit [CapZα-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZα-1. As a result, frequencies of autoantibodies to non-citrullinated CapZα-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZα-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZα-1 is relevant to RA. The antibody titers to the citrullinated CapZα-1 were significantly higher than those to the non-citrullinated CapZα-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZα-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZα-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.

Latex Immunoturbidimetric Assay for Soluble Fibrin Complex

Soluble fibrin complex (SFC), composed of fibrin monomer and fibrinogen derivatives, is known to exist in the circulating blood in patients with thrombosis. Its detection and quantification are useful for obtaining information about the condition and degree of intravascular coagulation in early-stage thrombosis, but there is no rapid method to measure SFC in plasma for clinical use. We obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay (LIA) using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. The method had an analytical range of 3-300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (CVs <3.0% and <2.0%, respectively). The assay detection limit was <0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10%), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470 000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases [mean (SD), 48.9 (57.6) mg/L; n = 160) were significantly higher than those in plasma from healthy individuals [1.8 (2.1) mg/L; P <0.001; n = 304]. In terms of linearity, precision, and sensitivity, the LIA, performed on a Hitachi 911 automated analyzer, may be useful for measurement of SFC in plasma.

Direct chemiluminescent immunodetection of proteins in agarose gels.

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.

The Use of Soluble Fibrin in Evaluating the Acute and Chronic Hypercoagulable State

Soluble fibrin detected in clinical plasma samples includes a variety of complexes consisting of fibrin monomer units, fibrinogen, and various proteolytic derivatives of fibrinogen and fibrin. The advantage of measuring soluble fibrin over fibrinopeptide A to detect thrombin action on fibrinogen is the considerably longer half-life of soluble fibrin in the circulation. Soluble fibrin can be detected by a variety of methods, including paracoagulation and precipitation assays, adsorption of fibrin monomer to insolubilized fibrinogen, functional assays based on the cofactor activity of some soluble fibrin compounds in t-PA-induced plasminogen activation, and by using fibrin-specific antibodies. Fibrin-specific antibodies may react with epitopes generated directly by fibrinopeptide A or B release or with epitopes generated by fibrin polymerization. Epitopes dependent on fibrinopeptide A release are often not accessible in native fibrin complexes and require the disaggregation of fibrin compounds to be reactive, whereas epitopes dependent on fibrinopeptide B release or fibrin polymerization are accessible to the monoclonal antibodies in nondenatured fibrin. Since soluble fibrin assays detect different structural or functional properties of soluble fibrin, no common calibrator for all soluble fibrin assays and, often, little correlation between different assay systems in clinical evaluations exist. Clinical applications of soluble fibrin assays include diagnosis and treatment monitoring of intravascular coagulation processes and prethrombotic states, monitoring anticoagulant treatment, and biocompatibility investigations. Some soluble fibrin assays have been demonstrated to be extremely sensitive indicators of acute fibrin formation. For the exclusion of venous thrombosis, D-dimer assays appear to be more sensitive than current soluble fibrin assays, since D-dimer assays detect freshly formed fibrin and proteolytic fragments of particulate clots. Further clinical studies are needed to establish the clinical utility of specific, soluble fibrin assays. The development of rapid, quantitative soluble fibrin assays for clinical routine use should be encouraged.

49. Catheter-directed intra-arterial thrombolysis

The aim of this study was to investigate the proteolytic degradation of fibrin(ogen), with special emphasis on the size of soluble fibrin(ogen) derivatives identified before, during and after intra-arterial catheter-directed thrombolysis with alteplase. Arteriography was performed before thrombolysis and after 0.5, 3, 10 and 24 h in six patients with peripheral native artery or bypass occlusions. Samples collected simultaneously intra-arterially from the thrombus and from venous blood were studied by immunoblotting patterns obtained after polyacrylamide and agarose gel electrophoresis. Supernatant from centrifuged material aspirated from the thrombus before thrombolysis contained soluble fibrin(ogen) derivatives with molecular weights of several thousand kDa. Two types of soluble fibrin(ogen)-related material were visualized during treatment: high molecular weight species (500-1000 kDa) displaying an almost continuous spectrum of molecular weights, suggesting gradual proteolytic degradation of cross-linked fibrin into X-oligomeric material; and X, Y, DD, D and E fragments. The amount and distribution of fragments strongly indicated that preferential fibrinolysis had taken place. The finding of a sustained level of fragments in post-thrombolytic plasma might indicate that insoluble fragments embolize peripherally and subsequently lyse. A close association between angiographical and molecular findings during both successful and failing thrombolysis was demonstrated.

The Derivatives of Human Fibrinogen with Special Reference to Soluble Fibrin and the D-dimer. Their Generation and Status in the Circulating Blood

The Derivatives of Human Fibrinogen with Special Reference to Soluble Fibrin and the D-dimer. Their Generation and Status in the Circulating Blood

The Derivatives of Human Fibrinogen with Special Reference to Soluble Fibrin and the D-dimer. Their Generation and Status in the Circulating Blood

The Derivatives of Human Fibrinogen with Special Reference to Soluble Fibrin and the D-dimer. Their Generation and Status in the Circulating Blood

The relationship between the fibrinogen D domain self-association/cross-linking site (gammaXL) and the fibrinogen Dusart abnormality (Aalpha R554C-albumin): clues to thrombophilia in the "Dusart syndrome".

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gammaXL"). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 > I-9 > 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alphaC" region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alphaC" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome."

71. A specific test for polymorphonuclear leukocyte-mediated fibrinogenolysis in plasma

In response to inflammatory mediators, polymorphonuclear leukocytes (PMN) are activated and release potent serine proteases, i.e. elastase, cathepsin G and proteinase 3. The aim of this study was to develop an assay to determine PMNmediated fibrinogenolysis in plasma, as a biochemical marker for activated PMN in vivo. On the basis of a special monoclonal antibody, generated against a neo-antigenic site in elastase degraded fibrinogen (EOF), we developed a highly specific plasma test for PMN-generated fibrin(ogen) derivatives. Intact fibrin(ogen) did not cross react in the assay, nor did plasmin-generated fibrin(ogen) derivatives. With this assay we showed, amongst others, that smoking did not affect the values of EDF-like degradation products in plasma of normal healthy volunteers (overall 8.2±1.6 ng/ml; n= 18) and that EDF-values are increased in patients with an ccl-proteinase inhibitor deficiency (18.6±5.3 ng/ml; n=12) and with sepsis (365.7±198.4 ng/ml; n=16). The assay was also applied to assess PMN-mediated fibrinogenolysis in synovial tissue extracts of patients with rheumatoid arthritis; these contained very high levels of EDF (98.2±59.3 ng/ml; n=15), in contrast to synovial tissue extracts of patients with osteoarthritis (6.3±2.7 ng/ml; n=12). The assay may be useful to identify patients with activated PMN, and to monitor drug-efficacy during therapy. This study was partly supported by grants from the Dutch Asthma Foundation (NAF 91.39 and 93.73) and the Praeventiefonds (002819180). © Pearson Professional Ltd 1996.

The detection of d-dimer in plasma by enzyme immunoassay

Most commercial D-dimer enzyme immunoassays employ two antibodies, a fibrin specific capture monoclonal and a less specific antibody cross-reacting with epitopes present on fibrinogen. Two different ELISA systems were compared to test whether this cross-reaction can lead to overestimation of the true levels of cross-linked fibrin derivatives. Both assays used the same D-dimer specific antibody for capture, DD-3B6/22, but utilized signalling antibodies which differed in fibrinogen reactivity. The assays gave low results for 210 normal samples (conventional ELISA, 39 +/- 45 ng/ml; non-fibrinogen reactive ELISA, 23 +/- 20 ng/ml) with a high degree of elevation for 53 patients with active thrombosis (conventional ELISA, 901 +/- 649 ng/ml; non-fibrinogen reactive ELISA, 1,906 +/- 1,725 ng/ml). However, a dramatic difference between results was seen when fibrinogenolysis was induced by treatment of 20 normal plasmas with high levels of t-PA and plasminogen. The D-dimer levels estimated with the conventional fibrinogen-reactive signal antibody rose 25-fold (normal, 36 +/- 27 ng/ml; treated, 833 +/- 272 ng/ml), whereas no increase was obtained with the non-fibrinogen reactive ELISA (normal, 24 +/- 21 ng/ml; treated, 27 +/- 22 ng/ml). These results suggest that a more accurate estimation of D-dimer levels in samples containing high levels of fibrinogen derivatives is achieved in assays incorporating non-fibrinogen reactive antibodies.

Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer

Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B). The results obtained show that paramagnetic particles coated with mAb S4 catch intact D-dimer as well as a variety of cross-linked fibrin molecules of high-molecular-weight (HMW) in plasma. The existence of HMW derivatives in fibrinaemic serum indicates that some of the HMW fibrin related material in such plasma is not clottable. Fibrinogen/fibrin monomers and some of the fibrinogen/fibrin related derivatives found in the eluates were probably non-covalently bound to the complexes caught by mAb S4 coated particles. The present technique combines the selective concentrating power of immunoparticles and the sensitivity of immunovisualization and allows rapid and direct identification of minute amounts of circulating immunoreactive fibrinogen/fibrin derivatives.

GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally, but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer. Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47 degrees C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassie blue. Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.

GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer, Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47°C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassic® blue, Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.