Inverse inkjet printed gold micro electrodes for the structured deposition of epithelial cells and fibrin

The micro structured deposition of vital cells is an important challenge in tissue engineering, biosensor technology, and in all research dealing with cell-cell and cell-substrate contacts. Hence, an inkjet printing technology has been developed to manufacture Au-based micro electrodes by sputter coating inversely printed polyester-foils. These electrodes feature minimal structure sizes of 35 microm and consist of an anode and a cathode part. They were used with fibrinogenic epithelial cell suspensions to deposit human keratinocytes (HaCaT), mouse fibroblasts (L-929) and the protein fibrin by applying DC voltage. Subsequently cells were electrophoretically attracted to the anode, following exactly its shape, while the insoluble fibrin was simultaneously precipitated due to the electrically mediated polymerization of the soluble fibrinogen molecule. Furthermore, it was demonstrated that this technique is suitable to co-deposit both cell types in a layered fashion. The lower voltage boundary for successful deposition was set at approximately 0.8 V needed for the conversion of fibrinogen into fibrin, while the upper voltage boundary was set at approximately 1.85 V, when commencing electrolysis inhibited the deposition of vital cells. Subsequent to the anodic cell-fibrin deposition, cells were cultivated for up to 4 days and then characterized by FDA+EB staining, methyl violet staining, MNF staining and SEM. The conversion from fibrinogen into fibrin was studied using ATR/FTIR.     

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.     

STUDIES ON BLOOD COAGULATION : I. THE R?LE OF PROTHROMBIN AND OF PLATELETS IN THE FORMATION OF THROMBIN

1. A method is described for the preparation and titration of prothrombin and thrombin. 2. Confirming the views of Morawitz, Howell (1916–17, 1925), and Bordet, thrombin cannot be regarded as an artificial by-product of coagulation (Wooldridge, Nolf (both quoted from Morawitz)). Calcium, a platelet factor, and a plasma factor (prothrombin) interact to form thrombin, and this then acts upon fibrinogen to form fibrin. The amount and rate of thrombin formation in the first reaction are independent of the presence or absence of fibrinogen. After a variable latent period, thrombin suddenly appears in large quantities, coincident with or immediately preceding the deposition of fibrin if fibrinogen is present. 3. The amount of thrombin formed in a mixture of prothrombin, Ca and platelets is independent of the platelet or Ca concentration, and depends primarily upon the amount of prothrombin used. The platelets (or cephalin) enormously accelerate the transformation of prothrombin to thrombin, and this acceleration seems to be their physiological rôle in the coagulation process. 4. Contrary to previous reports, platelets have not been demonstrated to contain significant quantities of prothrombin. 5. The available data do not allow any definite decision as to whether the platelet factor actually combines with prothrombin to form thrombin, or merely catalyzes the transformation. The very slow formation of thrombin in the complete absence of platelets may be due to dissolved traces of platelet material released during the physical manipulation of the plasma (centrifuging, Berkefeld filtration). 6. There was no evidence for a species-specific activity of platelets in the transformation of prothrombin to thrombin.     

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.     

Colon cancer metastasis in mouse liver is not affected by hypercoagulability due to Factor V Leiden mutation

Clinical trials have shown life-prolonging effects of antithrombotics in cancer patients, but the molecular mechanisms remain unknown due to the multitude of their effects. We investigated in a mouse model whether one of the targets of antithrombotic therapy, fibrin deposition, stimulates tumour development. Fibrin may provide either protection of cancer cells in the circulation against mechanical stress and the immune system, or form a matrix for tumours and/or angiogenesis in tumours to develop. Mice homozygous for Factor V Leiden (FVL), a mutation in one of the coagulation factors that facilitates fibrin formation, were used to investigate whether hypercoagulability affects tumour development in an experimental metastasis model. Liver metastases of colon cancer were induced in mice with the FVL mutation and wild-type littermates. At day 21, number and size of tumours at the liver surface, fibrin/fibrinogen distribution, vessel density and the presence of newly formed vessels in tumours were analysed. Number and size of tumours did not differ between mice with and without the FVL mutation. Fibrin/fibrinogen was found in the cytoplasm of hepatocytes and cancer cells, in blood vessels in liver and tumour tissue and diffusely distributed outside vessels in tumours, indicating leaky vessels. Vessel density and angiogenesis varied widely between tumours, but a pre-dominance for vessel-rich or vessel-poor tumours or vessel formation could not be found in either genotype. In conclusion, the FVL mutation has no effect on the development of secondary tumours of colon cancer in livers of mice. Fibrin deposition and thus inhibition of fibrin formation by anticoagulants do not seem to affect tumour development in this model.     

Viscoelasticity of fibrinogen solution and of blood during coagulation studied by a new damped oscillation rheometer

The behavior of a newly developed damped oscillation type rheometer was analyzed for fibrinogen solution and blood during coagulation. This rheometer consists of a cylindrical tube suspended from a torsion wire, that is filled with liquid to be tested. The logarithmic damping factor (LDF) during coagulation for blood and fibrinogen solution was obtained by this rheometer, which was closely related to the changes of viscosity and/or viscoelasticity of the blood sample. The slight increase of LDF prior to the rapid decrease was observed for blood. The increase of LDF would be reflected in the formation of the aggregation structure of red blood cells (rouleaux network) prior to the formation of fibrin network. The value of LDF for fibrinogen solution sharply increased and then decreased through a maximum value with the progress of coagulation, although the change of LDF was remarkably dependent on the fibrinogen concentration. The initial increase in LDF for fibrinogen solution was considered to be due to the formation of small clots in the solution. The decrease in LDF after attaining a maximum value is ascribed to the formation of fully developed fibrin network. The maximum value of LDF during coagulation for fibrinogen solution is higher than that for blood. The behavior was compared with that for non-biological fluids such as viscosity standard liquids and polyvinyl alcohol solution. From those data, it was concluded that the higher value of LDF than that for Newtonian liquids was due to the formation of aggregation structure or inhomogeneous fine clots in the liquid, which was accompanied with the appearance of the elasticity.     

Detection of soluble fibrin monomer complexes. Comparison of a haemagglutination assay with the ethanol gelation test

Hypercoagulability and disseminated intravascular coagulation (DIC) are characterized by the presence of circulating fibrin monomer complexes in plasma. In 342 patients with possible DIC fibrin monomers, fibrinogen, Reptilase Time, antithrombin III and other coagulation parameters were determined at frequent intervals. Testing of soluble fibrin monomer complexes was performed using a sensitive and reliable hemagglutation assay with red cells sensitized by fibrin monomers (FM-Test) and the ethanol gelation test (EGT). Method comparison regarding the influence of fibrinogen levels and fibrin degradation products shows that high fibrinogen levels lead to false-positive results with EGT. The same effect is observed for fibrin degradation products and EGT whereas no influence of fibrinogen level and fibrin degradation products on the FM-Test occurs. It is well-known that during DIC AT III level decreases caused by proteolytic activity. In this study it could be shown that fibrin monomer increases parallel to the decrease of AT III. The same effect does not occur due to fibrin degradation products.     

The thrombomodulin-protein C system is essential for the maintenance of pregnancy

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

STUDIES ON BLOOD COAGULATION : II. THE FORMATION OF FIBRIN FROM THROMBIN AND FIBRINOGEN

Although calcium is essential for the formation of thrombin, it can be recovered quantitatively from formed horse thrombin without affecting its coagulating activity. Citrate also has no significant effect. As stated in the text, this does not exclude the possibility that thrombin is actually a calcium compound present in minute concentration; but confirming the results of Hammarsten, it does show that fibrin cannot be a calcium-protein compound unless one assumes molecular weights for fibrinogen greater than 1,000,000. Although the available experimental data concerning the properties of thrombin, the kinetics of its reaction with fibrinogen, and the quantitative relationships between the two do not allow a definite decision as to whether thrombin is an enzyme analogous to rennin, or whether it combines with fibrinogen to form an insoluble compound, fibrin, the weight of evidence does favor the enzyme theory. A given quantity of thrombin can form at least 200 times its weight of fibrin, and in view of the crudeness of the preparation this ratio is probably many times greater. There is no apparent stoichiometric relationship between thrombin and fibrinogen, and thrombin does not disappear from a mixture of the two until the moment of coagulation; the quantity which then disappears is many times the minimal quantity necessary to form the amount of fibrin produced.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP)

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bβ chain (Bβ1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.     

Babesia bovis (argentina): observations of coagulation parameters, fibrinogen catabolism and fibrinolysis in intact and splenectomized cattle

The results of this paper indicate that cattle infected with B. bovis (argentina) have a markedly altered and activated coagulation system. A degree of thrombin activation occurs due partly to release of thromboplastin-like substances from lysed erythrocytes but due primarily to activation of kallikrein by babesial proteases. This produces a hyperfibrinogenaemia, particularly in intact cattle, with soluble fibrin complexes constituting up to one-third of the total fibrinogen concentration. High molecular weight non-coagulable fibrinogen-like proteins are detected terminally but more so in splenectomized cattle. Plasminogen concentration decreases in splenectomized but not intact cattle while low molecular weight fibrinogen degradation products are not easily detected. It is suggested that a hypercoagulable intermediate state with little or no fibrin deposition occurs rather than terminal disseminated intravascular coagulation.     

Force spectroscopy of the fibrin(ogen)-fibrinogen interaction

Fibrin aggregation is of vital importance in many physiological and pathological processes, such as blood coagulation, wound healing, and thrombosis. In the present study, we investigated the forces involved in the initial steps of the fibrinogen fibrin aggregation by force spectroscopy using the atomic force microscope. Our data confirm the existence of strong specific interactions between fibrin and fibrin(ogen), with unbinding forces ranging from 290 to 375 pN and a logarithmic dependence on the loading rate between 0.8 and 23 nN/s.     

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.     

Citrullinated fibrinogen inhibits thrombin-catalysed fibrin polymerization

Citrullination is the post-translational modification of arginine residues by peptidylarginine deiminases (PADIs). Fibrinogen is one substrate of PADIs under physiological conditions. Fibrinogen is an important factor for blood coagulation and inducing inflammation. The citrullinated form of fibrinogen appears in rheumatoid arthritis synovial tissue together with the production of autoantibodies that target self-peptides containing citrulline. However, whether the function of fibrinogen changes after citrullination remains unclear. We found that citrullinated fibrinogen markedly impairs the function of thrombin-catalysed fibrin polymerization and also inhibits fibrin formation. Increased citrullinated fibrinogen might thus affect the balance between coagulation and fibrinolysis and alter antigenicity under physiological conditions. These data suggest that citrullination of proteins could physiologically change functions and subsequently generate pro-inflammatory conditions and autoimmune reactions.     

Early postischaemic renal fibrin deposition and reduction of glomerular filtration rate in the rat: effect of the defibrinating agent Arwin

The correlation between the extent of intrarenal fibrin deposition induced by 15, 20 and 30-min bilateral occlusion of the renal arteries and the reduction of glomerular filtration rate (GFR) has been studied. The tissue level of fibrin was estimated by 125I-labelled human fibrinogen. There was a significant negative correlation between cortical and medullary fibrin content and GFR. In rats pretreatment with the defibrinating agent Arwin failed to prevent postischaemic coagulation in the kidney and the reduction of GFR.     

Multidomain structure of fibrinogen and its transformations

Generalized concepts of some structural peculiarities of fibrinogen, its transformation into fibrin and assembly have been considered on the basis of author's and published data. The role of local conformational changes in different areas of fibrinogen molecule and of separate reaction centers in formation of single- and double-stranded rod-like equilibrium fibrin oligomers and flexible branched copolymers of fibrinogen with fibrin E fragment has been considered. The mechanism of compactization has been discussed.     

Novel aspects of fibrin(ogen) fragments during inflammation

Coagulation is fundamental for the confinement of infection and/or the inflammatory response to a limited area. Under pathological inflammatory conditions such as arthritis, multiple sclerosis or sepsis, an uncontrolled activation of the coagulation system contributes to inflammation, microvascular failure and organ dysfunction. Coagulation is initiated by the activation of thrombin, which, in turn, triggers fibrin formation by the release of fibrinopeptides. Fibrin is cleaved by plasmin, resulting in clot lysis and an accompanied generation of fibrin fragments such as D and E fragments. Various coagulation factors, including fibrinogen and/or fibrin [fibrin(ogen)] and also fibrin degradation products, modulate the inflammatory response by affecting leukocyte migration and cytokine production. Fibrin fragments are mostly proinflammatory, however, Bβ15-42 in particular possesses potential antiinflammatory effects. Bβ15-42 inhibits Rho-kinase activation by dissociating Fyn from Rho and, hence prevents stress-induced loss of endothelial barrier function and also leukocyte migration. This article summarizes the state-of-the-art in inflammatory modulation by fibrin(ogen) and fibrin fragments. However, further research is required to gain better understanding of the entire role fibrin fragments play during inflammation and, possibly, disease development.     

The impact of delayed fibrinopeptide-A release on fibrin structure. Studies of an abnormal fibrinogen

The impact of delayed fibrinopeptide-A release on polymerization and structure of fibrin gels was studied utilizing a heterozygously transmitted variant fibrinogen. An arginine to histidine substitution at position 16 of the alpha chain of the abnormal fibrinogen delayed release of an abnormal fibrinopeptide-A (A) by thrombin and completely blocked release of A by reptilase. When clotted with thrombin, patient fibrin formed more slowly than normal fibrin, but clottability was normal and gel fiber mass/length ratios were decreased less than 10%. Gels formed with reptilase clotted slowly, demonstrated reduced clottability, but had normal fiber mass/length ratios. Reptilase clotted the normal but not the variant component of the patient fibrinogen. Thrombin-induced cleavage of fibrinopeptide-B prior to A occurred in these experiments, but polymerization of this species beyond trimers has been reported to be minimal under the conditions used. With time, A is removed by thrombin resulting in the slow production of normal fibrin monomer from the abnormal component. These monomers subsequently polymerize. The minimal change in gel fiber size caused by slow A release implies that fibrin fiber size is primarily a function of ionic environment and not of the sequence of peptide release.