The Fibrinogen Concentration in Blood of Dairy Cows and its Influence on the Interpretation of the Glutaraldehyde and Formol-Gel Test Reactions

It has earlier been shown that the formol-gel test on serum and glutaraldehyde test on whole blood are simple and rapid methods for evaluation or the immunoglobulin status in the cow. Both tests function as coagulation tests in which aldehyde groups oross-link basic blood globulins at their NH2-groups, forming polymerisates. The glutaraldehyde has in whole blood the capacity to polymerize not only immunoglobulins but also fibrinogen. This investigation was made in order to study whether the fibrinogen level may influence the result of the glutaraldehyde test, so revealing any differences between the results of that and the formol-gel test carried out on serum. In 92 cows with a variety of clinical disorders (most of them with inflammatory processes) the total protein, albumin, total globulin concentration and albumin/globulin ratio in serum and fibrinogen concentration in plasma were recorded. The material was grouped according to glutaraldehyde and formol-gel test reactions. It is shown that increases in the fibrinogen level have an effect on the results of the glutaraldehyde test. A positive glutaraldehyde test in more acute processes is ascribed to a heavy rise of plasma fibrinogen in its capacity of acute-phase protein. A positive glutaraldehyde test in chronic diseases may be viewed as a result of interaction between high immunoglobulin concentrations and elevated fibrinogen concentration. In conclusion the fibrinogen and immunoglobulin status of blood is important to assess in many diseases of cattle. The semiquantitative tests described for field use can separately, or especially in parallel use, provide valuable information about the character and development of a disease and may be regarded as good substitutes for the sedimentation rate (SR), which is not demonstrable in cattle. kw|Keywords|k]bovine fibrinogen; k]bovine serum proteins; k]formol-gel reaction; k]glutaraldehyde test; k]acute and chronic inflammations     

Coagulation Defects in Hypoxic Full-term Newborn Infants

Blood clotting was examined in 24 consecutive full-term newborn infants with hypoxia and 23 normal control infants. There was evidence of a gross alteration in the clotting process in the hypoxic infants. The degree of disturbance in clotting seemed to be dependent on the severity of the hypoxia, suggesting a causal relationship. The mechanism involved seemed to be intravascular coagulation. The consumption of clotting factors involved led to the appearance of a haemorrhagic diathesis. This effect seemed to be of short duration, suggesting that direct liver damage plays at most a minor part.Though there was no evidence of damage in the infants who survived, it is possible that intravascular coagulation induced by hypoxia, by leading to deposition of fibrin, may cause damage to vital organs. Further studies will be required to examine this possibility.     

New strategies in the determination of fibrin and fibrin(ogen) derivatives by monoclonal antibodies

Until recently only tests with a limited specificity were available for the assessment of the products of activated coagulation and/or fibrinolysis. Those assays were based on polyclonal antibodies, which crossreact with fibrinogen, and as a consequence they were performed on serum samples i.e. after removal of fibrinogen by clotting. Serum preparation, however, is a notorious source of artefactually high or low levels of fibrin(ogen) degradation products, and is not suitable for the determination of coagulation products. Recently, highly specific monoclonal antibodies (MoAb's) have been developed, the majority of which do not crossreact with fibrinogen. This has enabled new strategies to be developed, i.e. assays using these MoAb's on plasma samples. Furthermore, the new assays can discriminate between (individual) fibrin and fibrinogen degradation products, and coagulation products can be assessed in the same plasma samples.     

Functional impact of oxidative posttranslational modifications on fibrinogen and fibrin clots

Fibrinogen is a circulating multifunctional plasma protein vital for hemostasis. Activation of the coagulation cascade converts soluble fibrinogen to insoluble polymerized fibrin, which, along with platelets, forms the hemostatic clot. However, inappropriate formation of fibrin clots may result in arterial and venous thrombotic disorders that may progress to life-threatening adverse events. Often thrombotic disorders are associated with inflammation and the production of oxidants. Fibrinogen represents a potential target for oxidants, and several oxidative posttranslational modifications that influence fibrinogen structure and function have been associated with disease pathogenesis. Here, we review various oxidative modifications of fibrinogen and the consequences of these modifications on protein structure and the ability to form fibrin and how the resulting alterations affect fibrin architecture and viscoelastic and biochemical properties that may contribute to disease.     

可溶性血纤蛋白促进细胞伸展及其作用机制

可溶性血纤蛋白（ｓｏｌｕｂｌｅｆｉｂｒｉｎ,ＳＦ）为血纤蛋白单体和血纤蛋白原１∶２的复合物．现已知在血液凝固系统被激活的病理状态下,存在于循环血液中,然而它的生理作用仍然不明．首次发现细胞能在固定的ＳＦ上伸展,并能被外源性ＳＦ及精氨酸－甘氨酸－天冬氨酸（ＲＧＤ）合成肽所抑制,但不能被血纤蛋白原和血纤蛋白单体所抑制,提示ＳＦ形成后其结构变化是引起细胞伸展的关键．片段Ｘ（缺乏ＲＧＤ２序列的血纤蛋白原片段）与血纤蛋白单体形成的复合物,使细胞伸展活性明显减低,提示在ＳＦ结构中,血纤蛋白原的ＲＧＤ２序列在细胞伸展中起重要作用．同时发现ＤＩＣ患者血浆中的ＳＦ也具有细胞伸展活性．ＳＦ作为一个粘附分子在体内血栓形成过程中起重要调节作用     

Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection

Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders.     

Glycolaldehyde induces fibrinogen post-translational modification, delay in clotting and resistance to enzymatic digestion

Glycolaldehyde (GA) is a highly reactive aldehyde that can be generated during inflammation and hyperglycemia. It can react with arginine and lysine residues impairing protein function. As inflammation and diabetes present haemostatic dysfunction, we hypothesized that GA could participate in this process. The aim of this study was to investigate if plasma incubated in the presence of GA presents alteration in the coagulation process. We also aimed to evaluate the role of fibrinogen in GA-induced haemostatic dysfunction. For this purpose, plasma and fibrinogen were each incubated separately, either in the presence or absence of 1 mM GA for 8 and 4 h, respectively. After that, plasma coagulation and fibrin polymerization kinetics were recorded, as well as the kinetic of plasma clot digestion and fibrinolysis protein carbonylation was quantified. An SDS-PAGE was run to check the presence of cross-linking between fibrinogen chains. GA induced a delay in plasma coagulation and in fibrin polymerization. Maximum absorbance decreased after GA treatment, indicating the generation of thinner fibers. Fibrin generated after complete coagulation showed resistance to enzymatic digestion, which could be related to the generation of thinner fibers. Protein carbonylation also increased after GA treatment. All parameters could be reversed with AMG (a carbonyl trap) co-treatment. The data presented herein indicate that GA causes post-translational modification of lysine and arginine residues, which are central to many events involving fibrinogen to fibrin conversion, as well as to fibrinolysis. These modifications lead to the generation of persistent clots and may contribute to mortality seen in pathologies such diabetes and sepsis.     

Affinity purification of fibrinogen using an Affimer column

BackgroundFibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen.MethodsFibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis.ResultsAffimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII.ConclusionsThe Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations.General significanceOur methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.     

Coagulation disorders seen through the window of molecular biology

Coagulation disorders have been traditionally worked up by their clinical phenotypes and coagulation factor assays which are dependent on APTT- and PT-based techniques. Development of chromogenic substrates in the late seventies and early eighties allowed coagulation factors to be measured like enzymes. There was still a major lacuna in the understanding of the biology of different coagulation disorders. Modern molecular biology - which developed as an unique synthesis of biochemistry, immunology, cell biology, and genetics - allowed us to have a more comprehensive understanding of the pathobiology of many of these coagulation disorders. This overview presents several examples which show how we have enriched our understanding about the varied clinical phenotypes of different coagulation disorders.     

Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria

Aeromonas sobria infection often advances to sepsis, in which interaction of bacterial components with plasma proteins possibly causes various disorders. This bacterium releases a serine protease (ASP), a putative virulence factor, and binds to fibrinogen. To study the ASP effect on fibrinogen, we incubated fibrinogen or plasma with ASP and investigated their clotting elicited by thrombin, which converts fibrinogen to a fibrin clot. Enzymatically active ASP retarded plasma clotting in a dose-dependent manner starting at an ASP concentration of 10 nM. ASP also retarded fibrinogen clotting at 3 nM and above, which appeared to correspond to ASP cleavage of fibrinogen at the A alpha-chain. Consistent with containing serine protease activity for an ASP-specific substrate, the culture supernatant of an ASP gene-introduced strain retarded plasma and fibrinogen clotting more than that of the wild-type strain. The culture supernatant of an ASP gene-disrupted strain that releases negligible serine protease activity for the ASP-specific substrate did not affect plasma clotting. These results indicate that ASP is the main fibrinogenolytic protease released from A. sobria. Impaired plasma clottability induction through fibrinogen degradation is a new virulence activity of ASP and may contribute to hemorrhagic tendencies in sepsis caused by infection with this bacterium.     

Iron modulates the alpha chain of fibrinogen

Iron-bound fibrinogen has been noted to accelerate plasmatic coagulation in patients with divergent conditions involving upregulation of heme oxygenase activity, including hemodialysis, Alzheimer’s disease, sickle cell anemia, and chronic migraine. Our goal was to determine if a site of iron-fibrinogen interaction was on the alpha chain. Using thrombelastography, we compared the coagulation kinetic profiles of plasma exposed to 0–10 µM ferric chloride after activation of coagulation with thrombin generated by contact activation of plasma with the plastic sample cup or by exposure to 1 µg/ml of Calloselasma rhodostoma venom (rich in ancrod activity), which causes coagulation via polymerization of alpha chain monomers. Venom mediated coagulation always occurred before thrombin activated thrombus formation, and ferric chloride always diminished the time of onset of coagulation and increased the velocity of clot growth. Iron enhances plasmatic coagulation kinetics by modulating the alpha chain of fibrinogen.     

Coagulation abnormalities and cardiovascular disease.

In patients with excessive venous thrombosis, genetic defects predisposing to thrombosis can be found in 60-80%. Increased plasma levels of coagulation proteins such as fibrinogen and plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of myocardial infarction. However, despite the presence of polymorphisms that regulate plasma levels of factor VIII, PAI-1, and fibrinogen the association between common polymorphisms of these coagulation protein and ischemic cardiac disease remains ambiguous. Up to 10% of the population have defects that predispose them to excessive venous thrombosis. In spite of the essential role of thrombosis in coronary ischemic syndrome, no convincing evidence has implicated the two most common venous hypercoagulable states in ischemic heart disease. Pathogenic polymorphisms in the platelet fibrinogen and collagen receptors remains an area of intense research interest. Finally, it has been shown that lipoproteins can act as mediators of coagulation processes.     

Interaction between fibrinogen and insulin-like growth factor-binding protein-1 in human plasma under physiological conditions

Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.     

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.     

Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.     

Coagulation factor levels in non-metastatic colorectal cancer patients

There is evidence that high plasma levels of factor (F) VIII, FIX, FXI and fibrinogen are independent risk factors for venous thromboembolism. AIM: To determine the plasma concentrations of several coagulation factors and C4b-binding protein (C4BP) in a group of patients with non-metastatic colorectal cancer in order to investigate some aspects of cancer-acquired thrombophilia. METHODS: Plasma fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI and FXII activity levels and C4BP concentrations were determined in 73 patients with non-metastatic colorectal cancer (48 colon and 25 rectum) and in 67 matched control subjects. No one in either group had had previous thrombotic events. RESULTS: Mean plasma concentrations of fibrinogen (functional and antigen), FVIII, FIX, FV and C4BP were significantly higher in colorectal cancer patients than in control subjects, while FVII and FXII levels were significantly decreased. Several correlations were found between the increased coagulation factors and C4BP concentrations, while FVII was highly correlated with FXII. CONCLUSIONS: In colorectal cancer patients high plasma fibrinogen, FVIII and FIX levels might represent further risk factors for venous thrombotic complications in the immediate post-surgery period, while decreased FVII and FXII concentrations may be an index of intravascular coagulation activation, still in a subclinical phase.     

Anti-heparin activity of plasma with various low density lipoprotein content]

Anti-heparin activity correlated with LDL concentration in the plasma. Blood plasma of women in labour is characterized by the high antiheparin activity and low LDL levels. Anti-heparin activity is low and LDL levels are low in blood plasma in childhood. An effect of other factors neutralizing heparin (e.g. fibrinogen, platelet factor 4, acid alpha 1-glycoprotein, globulins, basic proteins) and differences of anti-thrombin III on plasma anti-heparin activity has been excluded. Neutralization of heparin anticoagulation activity by LDL is of clinical value. Blood LDL level should be considered, while heparin therapeutical doses are under scrutiny.     

Ingestion of colostrum from specific cows induces Bovine Neonatal Pancytopenia (BNP) in some calves

Background  

Since 2006, cases of haemorrhagic diathesis in young calves have been observed with a much higher incidence than previously known. The syndrome, now uniformly called Bovine Neonatal Pancytopenia (BNP), is characterized by multiple (external and internal) haemorrhages, thrombocytopenia, leukocytopenia, and bone marrow depletion. Although various infectious and toxicological causes of bleeding disorders in calves have been ruled out, the aetiology of BNP remains unknown. However, field observations have led to the hypothesis that the aetiological principle may be transmitted to calves via colostrum.     

Fibrinogen inhibits red cell adhesion to glass

Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated. The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.     

Reactive carbonyl compounds (RCCs) cause aggregation and dysfunction of fibrinogen

Fibrinogen is a key protein involved in coagulation and its deposition on blood vessel walls plays an important role in the pathology of atherosclerosis. Although the causes of fibrinogen (fibrin) deposition have been studied in depth, little is known about the relationship between fibrinogen deposition and reactive carbonyl compounds (RCCs), compounds which are produced and released into the blood and react with plasma protein especially under conditions of oxidative stress and inflammation. Here, we investigated the effect of glycolaldehyde on the activity and deposit ion of fibrinogen compared with the common RCCs acrolein, methylglyoxal, glyoxal and malondialdehyde. At the same concentration (1 mmol/L), glycolaldehyde and acrolein had a stronger suppressive effect on fibrinogen activation than the other three RCCs. Fibrinogen aggregated when it was respectively incubated with glycolaldehyde and the other RCCs, as demonstrated by SDS-PAGE, electron microscopy and intrinsic fluorescence intensity measurements. Staining with Congo Red showed that glycolaldehyde- and acroleinfibrinogen distinctly formed amyloid-like aggregations. Furthermore, the five RCCs, particularly glycolaldehyde and acrolein, delayed human plasma coagulation. Only glycolaldehyde showed a markedly suppressive effect on fibrinogenesis, none did the other four RCCs when their physiological blood concentrations were employyed, respectively. Taken together, it is glycolaldehyde that suppresses fibrinogenesis and induces protein aggregation most effectively, suggesting a putative pathological process for fibrinogen (fibrin) deposition in the blood.