Fibrinogen Molecule Research Articles

Hypofibrinogenaemia associated with a novel heterozygous γ289 Ala→Val substitution (fibrinogen Dorfen)

The molecular basis of hypofibrinogenaemia was investigated in a 34-year-old woman and her 10-year-old daughter. DNA sequencing revealed a single heterozygous GCC-->GTC transition in exon 8 of the fibrinogen gamma ?gene in both subjects, predicting a novel gamma289 Ala-->Val substitution. Examination of fibrinogen gamma ?chains by electrospray ionization mass spectrometry failed to detect the variant chain in plasma fibrinogen. Further evidence for its non-expression came from tryptic peptide mapping. The mutation predicts a mass increase of 28 Da in peptide T32, but only the normal (M + 2H) ion was detected at 1418 m/z in the proposita. Our finding that gamma289 is an important determinant of plasma fibrinogen levels highlights the role of mutational analysis in defining structurally important regions of the fibrinogen molecule. This case suggests that the highly conserved Ala(289) is important in maintaining structure of the "a" polymerization site via hydrogen bonding to Thr(371).

Congenital afibrinogenemia: identification and expression of a missense mutation inFGBimpairing fibrinogen secretion

AbstractCongenital afibrinogenemia is a rare autosomal recessive disorder characterized by complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease: a homozygous deletion of approximately 11 kb of the fibrinogen α-chain gene (FGA). Subsequent studies revealed that the great majority of afibrinogenemia mutations are localized inFGA, but mutations were also found inFGGandFGB. Apart from 3 missense mutations identified in the C-terminal portion ofFGB, all fibrinogen gene mutations responsible for afibrinogenemia are null. In this study, a young boy with afibrinogenemia was found to be a compound heterozygote for 2 mutations inFGB: an N-terminal nonsense mutation W47X (exon 2) and a missense mutation (G444S, exon 8). Coexpression of theFGBG444S mutant cDNA in combination with wild-typeFGAandFGGcDNAs demonstrated that fibrinogen molecules containing the mutant β chain are able to assemble but are not secreted into the media, confirming the pathogenic nature of the identified mutation. (Blood. 2003;102:4413-4415)

A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles

We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gamma-chain of mouse fibrinogen, inhibits factor (F)XIIIa-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction; in addition, it facilitates thrombolysis. To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25-fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan delta; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab')2, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.

Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bβ chain gene

Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bbeta chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL(-1) as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS-PAGE analysis. After sequencing all exons and exon-intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T-->G at position 3356 of the patient fibrinogen Bbeta chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bbeta 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bbeta chain gene caused a truncated fibrinogen Bbeta chain, which may not be assembled in the fibrinogen molecule.

Conformational Changes in the Plasma Protein Fibrinogen upon Adsorption to Graphite and Mica Investigated by Atomic Force Microscopy

Atomic force microscopy has been used to investigate the adsorption of the plasma protein fibrinogen on graphite and mica substrates. These substrates serve as model hydrophobic and hydrophilic surfaces, respectively. The overall structure of submonolayer coverage films is dramatically different on the mica and graphite substrates when imaged under ambient conditions after being dried under a nitrogen stream. The molecules show a tendency to aggregate on the graphite substrate but adsorb as isolated single molecules in the case of mica. On the mica substrate, individual fibrinogen molecules appear globular in structure whereas, on graphite, the trinodular structure is most commonly observed. The average height of the fibrinogen molecules as measured by tapping mode AFM in air is 1.71 ± 0.65 nm, and the average height on the graphite substrate is 1.05 ± 0.13 nm. The average lengths and widths of the molecules on these two substrates vary as well, the average length being 31 ± 7 nm on mica and 63 ± 10 nm on graphite. These differences are consistent with a change in conformation of the protein upon adsorption of these two surfaces due to the differences in surface chemistry of the substrates, which suggests a change in mechanism of adsorption between the two substrates.

Metabolic syndrome: epidemiology and more extensive phenotypic description. Cross-sectional data from the Bruneck Study.

The present study aimed at evaluating the prevalence of the Metabolic Syndrome and at identifying its additional clinical features. Within a prospective population-based survey examining 888 subjects aged 40-79 y, subjects were identified fulfilling the WHO and the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATPIII) criteria for diagnosing the Metabolic Syndrome. In these subjects and in the rest of the sample (controls), several metabolic and nonmetabolic biochemical parameters were compared. The prevalence of the Metabolic Syndrome by WHO criteria was 34.1% (95% CI 31.0-37.2) and by NCEP-ATPIII criteria 17.8% (15.5-20.3). The prevalence was significantly higher in older subjects and in those less physically active. Subjects with the Metabolic Syndrome either by WHO or by NCEP-ATPIII criteria showed higher levels of oxidized low-density lipoprotein, apolipoprotein B, urate, leptin, fibrinogen, leukocytes, erythrocyte sedimentation rate, GOT, gamma-GT and soluble endothelial adhesion molecules (E-selectin, vascular adhesion molecule-1 and intercellular adhesion molecule-1) and lower apolipoprotein A concentrations. Insulin resistance, as assessed by the Homeostasis Model Assessment, increased with the increase in the number of traits composing the syndrome found within the single individual. Subjects with insulin resistance had more pronounced abnormalities in several parameters, including the additional features of the syndrome (eg fibrinogen and soluble adhesion molecules). The Metabolic Syndrome occurs very frequently in the general population aged 40-79 y, and is associated with several additional metabolic and nonmetabolic abnormalities that likely contribute to an increased cardiovascular risk. Insulin resistance seems to play a major role in classic and additional abnormalities featuring the Metabolic Syndrome.

The role of inflammation markers in triggering acute coronary events

Studies have shown disparate results in relation to the role of plasma concentrations of inflammation markers such as fibrinogen, cytokines, and cell adhesion molecules in acute coronary syndromes. The differentiation of primary versus secondary alterations of these markers in response to acute coronary syndromes is not clear. The aim of this study was to investigate the effect of soluble cell adhesion molecules and some inflammatory markers on coronary plaque instability. The prospective study consisted of 15 patients with stable angina pectoris (SAP), 16 with unstable angina pectoris (UAP), and 16 who had undergone percutaneous transluminal coronary angioplasty (PTCA). Blood samples were obtained from the SAP group on admission, from the UAP group at the early stage of pain onset within 6 h of pain, and again after 12 h of pain. Samples from the PTCA group were collected before, 2, 14 h after the procedure. Soluble vascular cell adhesion molecule-1 (VCAM-1), endothelial selectin, interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2), and C-reactive protein (CRP) were analyzed by enzyme-linked immunosorbent assay. CRP serum levels gradually increased although IL-2 gradually decreased in patients with UAP and PTCA. In addition, VCAM-1 levels were sharply decreased after the PTCA procedure. However, this value returned back to the preprocedure levels 14 h after PTCA. Both CRP and IL-2 are directly involved in the triggering mechanisms of acute coronary events.

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution

AbstractMyosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 μM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 × 102 M–1 · s–1 and a dissociation rate constant of 3.07 × 10–4 s–1 are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosinfibrin clot is relatively resistant to plasmin.

Dynamic Changes of Fibrin Architecture during Fibrin Formation and Intrinsic Fibrinolysis of Fibrin-rich Clots

Clotting and fibrinolysis are initiated simultaneously in vivo, and fibrinolysis usually occurs without any individualized lysis front (intrinsic fibrinolysis). We have developed a novel model to assess whether morphological changes resulting from intrinsic fibrinolysis are similar to those previously reported at the lysis front using externally applied lytic agents. Fibrin assembly and fibrinolysis were followed in real-time by confocal microscopy using gold-labeled fibrinogen molecules. An increase in fiber absorbance (30%, p < 0.01) and a decrease in fiber diameter (60%, p < 0.01) due to the ongoing accumulation and packing of fibrin molecules were the most significant detectable features occurring during fibrin assembly. Similar features with a similar magnitude were observed during fibrin dissolution, but in the reverse order and with a 3-fold increase in duration. Then, lysing fibers were progressively transected laterally, and thinner fibers were cleaved at a 2.5-fold faster rate than thicker fibers (p < 0.001). Frayed lysing fibers were seen to interact progressively with adjoining fibers (agglomeration), leading to a 76 and 88% increase in the network pore diameter (p < 0.05) and fiber diameter (p < 0.01), respectively. At the maximum decrease in fiber absorbance (46%, p < 0.05), the network suddenly collapsed with the release of large fragments that gradually vanished. Morphological changes of fibrin that occur during intrinsic fibrinolysis are similar as those observed next to the lysis front, although they are not restricted spatially to the clot/surrounding milieu interface but are observed through the entire clot.

Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies.

The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.

Effect of atorvastatin on lipid parameters, LDL subtype distribution, hemorrheological parameters and adhesion molecule concentrations in patients with hypertriglyceridemia

Background and Aim: Hypertriglyceridemia is a risk factor for atherosclerosis that is typically associated with high concentrations of adhesion molecules, impaired hemorrheology and an unfavourable low-density lipoprotein (LDL) subtype distribution. We hypothesised that some of these risk markers might be beneficially influenced by lipid-lowering therapy with atorvastatin in hypertriglyceridemic patients. Methods and Results: Nineteen patients with primary hypertriglyceridemia were given 10 mg of atorvastatin per day for four weeks. Their cholesterol, triglyceride, LDL and high-density lipoprotein cholesterol (HDL-C) levels, LDL subtype profile, hemorrheological parameters and E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 concentrations were measured before and at the end of atorvastatin therapy. The levels of total and LDL cholesterol respectively decreased by 25% and 24% (both p<0.001). Furthermore, cholesterol was reduced by 8–29% in all seven LDL subfractions (density range: 1.020–1.066 g/mL) ( p<0.05). The reduction in triglyceride concentrations was of marginal significance (9%, p=0.1), but its degree positively correlated with the reduction of small-dense LDL (r=0.5, p<0.025). Plasma viscosity and blood viscosity at low shear rates were respectively reduced by 2% and 16% (both p<0.05). The effect of the treatment on the concentrations of HDL-C, fibrinogen and adhesion molecules was not significant. Conclusions: Atorvastatin (10 mg/day) not only reduced the plasma concentrations of atherogenic lipoproteins but also improved the LDL-subtype profile and reduced plasma and blood viscosity in patients with hypertriglyceridemia; however, it failed to significantly lower triglyceride concentrations.

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

New molecular defects in the γ subdomain of fibrinogen D-domain in four cases of (hypo)dysfibrinogenemia: fibrinogen variants Hannover VI, Homburg VII, Stuttgart and Suhl

Four new molecular abnormalities in the gamma subdomain of the D domain elucidated in three unrelated thrombophilic patients and in one asymptomatic case of hypofibrinogenemia are reported: fibrinogen Suhl, gamma 326, Cys-->Tyr, fibrinogen Hannover VI, gamma 336 Met-->Ile, fibrinogen Stuttgart, gamma 345, Asn-->Asp and fibrinogen Homburg VII, gamma 354,Tyr-->Cys. In all cases, fibrin polymerization in plasma is impaired. In the case of fibrinogen Suhl, there was a normalization of fibrin polymerization in plasma at higher Ca(2+) concentration. The protective effect of Ca(2+) on plasmic degradation of fibrinogen was incomplete with all three variants. The fibrinogen molecules in variants Homburg VII and Suhl contain covalently bound albumin. Fibrin clot structure was abnormal in case of variant Homburg VII, with finer and more branched fibers forming a less porous clot. Experimental data indicate possible effects of the molecular abnormalities on Ca(2+)-binding, D-E interaction and lateral association of protofibrils.

Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation

Thrombin substrate binding is mediated through fibrinogen recognition "exosite 1" in thrombin, resulting in fibrinopeptide cleavage to form fibrin. In addition, thrombin exhibits "non-substrate" binding to fibrin, an activity termed "Antithrombin I". Antithrombin I (AT-I) is characterized by two classes of thrombin binding sites, the first of "low affinity" in the fibrin E domain, and the other of high affinity, that is situated between C-terminal residues 414 and 427 of a variant gamma chain termed gamma'(1-427L), Plasma fibrinogen molecules containing gamma' chains ("fibrinogen 2") are virtually all heterodimers containing one gamma(A) chain (platelet-binding) and one gamma' chain. The remaining fibrinogen (approximately 85%) is homodimeric, lacks high affinity thrombin-binding potential, and is termed " fibrinogen 1" (gamma(A)/gamma(A)). Thrombin generation in recalcified fibrinogen-depleted or congenital afibrinogenemic plasma is increased. Repletion with fibrinogen 1 has a modest effect in normalizing thrombin generation, whereas repletion with fibrinogen 2 (gamma(A)/gamma') has a more marked effect. A post-translational gamma' chain derivative, gamma'(1-423P), accounts for 3%-34% of the gamma' chain population, lacks thrombin binding potential, and arises by proteolytic processing at the expense of gamma' (1-427L) chains. Little is known about its effect on plasma AT-I activity under normal or pathological circumstances. In summary, fibrin formation (Antithrombin I) inhibits thrombin generation in clotting blood by sequestering thrombin, and "high-affinity" thrombin-binding (i.e., via gamma' chains) plays a dominant role in this process. AT-1 should be considered when assessing the pathogenesis of thromboembolic disease.

Fibrinogen storage disease without hypofibrinogenaemia associated with acute infection

The presence of ground glass hepatocytes in a liver biopsy may be related to different conditions, including fibrinogen storage disease. Three types of fibrinogen storage disease have been described, namely types I, II and III. Type I is an hereditary hypofibrinogenaemia genetically characterized by a mutant variant of the fibrinogen molecule designated as fibrinogen Brescia, consistent with a gamma284 Gly-->Arg mutation. Only rare cases of types II and III fibrinogen storage disease have been described. The purpose of the present paper is to describe two cases of fibrinogen storage disease without associated hypofibrinogenaemia, which appeared during acute infectious diseases. Both patients were female, aged 77 and 73 years, who developed high transaminases during an infectious disease. In each case blood coagulation tests were within the normal range, and despite clinical and laboratory investigations no possible cause for liver disease could be found. Liver biopsies were performed; in both cases weakly eosinophilic cytoplasmic inclusions were observed. Using immunohistochemistry the inclusions were found to be due to fibrinogen accumulation. At ultrastructural level features corresponding to type II inclusions were observed. Molecular studies, performed in case 2, excluded the mutation typical of type I fibrinogen storage disease. Both patients also presented features of chronic hepatitis. In case 1, giant cell granulomas were additionally present. No close relatives of the patients presented any clinical or laboratory features of liver disease. In both patients altered liver function test values gradually, spontaneously, returned to within normal ranges after infectious disease was resolved. These cases suggest that, on rare occasions, hepatocytes may accumulate fibrinogen during an infectious disease.

Structural basis of the fibrinogen–fibrin transformation: contributions from X-ray crystallography

During the past several years, a number of crystal structures have been determined of fragments from fibrinogen and fibrin and, most recently, a structure of a native fibrinogen. One feature of the fibrinogen molecule that has emerged from these studies has to do with its “loose ends,” segments of the molecule that are extremely mobile and not discernable by X-ray crystallography. Some, if not all, of this flexibility is functionally important. Small synthetic peptides based on mobile parts of fibrinogen exposed by the action of thrombin have contributed significantly to these studies and may yet prove useful therapeutically. In the end, although crystal structures have added greatly to our understanding of fibrin formation, much still needs to be unraveled about how clots form.

Induced removal of dipalmitoyl phosphatidylcholine by the exclusion of fibrinogen from compressed monolayers at air/liquid interfaces

The induced removal of dipalmitoyl phosphatidylcholine (DPPC) by the exclusion of fibrinogen from mixed DPPC/fibrinogen monolayers at compressed air/liquid interfaces was analyzed. The surface pressure–area hysteresis curves of the monolayers at interfaces were obtained by a Langmuir trough. The hysteresis curves of equilibrium fibrinogen adsorption layers suggest that fibrinogen desorption during the area compression stage became significant at a higher bulk concentration of 1000 ppm. For mixed monolayers of DPPC with fibrinogen, the fibrinogen molecules were expelled from the interface upon compression due to the presence of insoluble DPPC molecules. The squeeze-out of fibrinogen molecules evidently removed a significant number of DPPC molecules from the interface, with the extent depending on fibrinogen surface concentration. During the subsequent area expansion stage, fibrinogen molecules entered the interface and participated in the rise of surface pressure. The induced loss of free DPPC molecules at the interface by the expelled fibrinogen molecules during the area compression stage was then evaluated from the hysteresis curves.

Multi-Bead-and-Spring Model to Interpret Protein Detachment Studied by AFM Force Spectroscopy

This article deals with the detachment of molecules (fibrinogen) from a surface studied experimentally with an atomic force microscope. The detachment (or rupture) forces are measured as a function of the retraction velocity and exhibit a clear dependence on this parameter, even though the interaction between the molecules and the surface are nonspecific. To interpret these data, a mechanical multi-bead-and-spring model is developed. It consists of one to several parallel, “molecular” springs connected to an extra spring representing the cantilever that is moved at constant velocity. The free end of each molecular spring terminates with a particle that interacts with the surface through a Lennard-Jones potential. This Brownian dynamics model is used to analyze the experimental findings. In the framework of this model, it appears that the fibrinogen molecule must be ascribed a stiffness much smaller than that of the cantilever. In addition, several bonds between the molecule and the surface must be taken into account for the range of the molecule-surface interaction not to be unrealistically small. In future work, this model will be extended to more complex mechanisms such as the detachment of cells from a surface.

Enhanced exudation of fibrinogen into the perivascular space in acute inflammation triggered by neutrophil migration.

The present experiments were performed to ascertain whether or not all plasma components are extravasated when vascular permeability is increased. Male Sprague-Dawley strain rats (specific pathogen-free) 8 weeks old (for histamine exudation) or 9-10 weeks old (for carrageenin pleurisy) were used. Histamine or A-carrageenin was injected into the rat right pleural cavity to induce rat pleurisy. Protein components in the inflammatory exudate and plasma were separated by high performance liquid chromatography. Coagulation time was assessed, and the fibrinogen levels in the pleural exudate were determined by thrombin time. The fibrinogen levels were also visualized by immunoblot analysis. Tumor necrosis factor-alpha (TNF-alpha, 0.4 microg/rat, intrapleurally), anti-rat CD18 monoclonal antibody (anti-CD18 antibody, 1 mg/kg, i. v.) and granulocyte-colony stimulating factor (G-CSF, 100 microg/kg, s.c. twice daily for 4 days) were used. In the histamine-induced extravasation, the level of plasma protein components with large molecules over 900 kD in the exudate was 62% of that in the rat's own plasma. The amount of fibrinogen in the pleural exudate was 1/8 of that in the plasma and was faintly detected in immunoblot analysis, but it was clearly detected after the treatment of rats with TNF-alpha. In rat carrageenin pleurisy, fibrinogen was hardly detected in immunoblot analysis in the exudate collected 0.5 h after carrageenin, when neutrophils did not migrate into the exudate. However, it was clearly present after neutrophil migration started 2 h later The increase in the neutrophil counts in the exudate caused by G-CSF enhanced the fibrinogen level in the exudate, whereas intravenous injection of anti-CD18 antibody suppressed the fibrinogen level in immunoblot analysis. Venular permeability increase in the rat histamine exudation induced minimal extravasation of plasma proteins with large molecules, such as fibrinogen, while fibrinogen molecule was detected in rat carrageenin-injected pleurisy, when neutrophil diapedesis occurred. Thus, only when neutrophils started to migrate into the perivascular space was fibrinogen clearly detected in the exudate.

Structural alterations in hereditary dysfibrinogens.

Dysfibrinogens can be grossly divided in two groups: (1) defective thrombin-catalyzed conversion of fibrinogen molecules to fibrin monomers, and (2) defective fibrin polymerization due to structural alterations in polymerization sites, that include "A" and "a" sites, end-to-end D:D abutment surfaces, and lateral association sites involving the carboxyl terminal region of the fibrin alpha-chain. Recently, a number of mutations in the fibrinogen genes have been identified, and many of these encode changes that occur in regions of fibrinogen that have been elucidated by high-resolution structural studies. Here we focus on the structure-function relationships of fibrinogen that can be inferred from studies involving these abnormal molecules.