Characterization of the amino acids of bovine fibrinogen involved in the fibrinogen-thrombin interaction of the blood clotting process. Comparison with the milk clotting process

Summary Bovine fibrinogen and the A and B chains of bovine fibrinogen have been subjected to chemical modification by a number of reagents and the effects of these procedures on the susceptibility of the proteins to thrombin hydrolysis is described. The reagents used were rose bengal (for photo-oxidation), 2-hydroxy-5-nitrobenzyl bromide, N-acetylimidazole, iodoacetic acid and diethyl pyrocarbonate. Evidence is presented which indicates that the tryptophan and tyrosine residues of fibrinogen are not involved to any great extent in the interaction of this protein with thrombin. Modification with iodoacetic acid suggests that methionine residues play a major role in such interactions, but the fibrinogen chains on which the important residues reside remain uncertain. The use of diethyl pyrocarbonate indicates the participation also of histidine in fibrinogen-thrombin interactions and that, whereas the histidine residues of the B chain are involved to a great extent, it appears that those of the Aa chain are not. The similarities which exist between the fibrinogen-thrombin and the -casein-chymosin systems are discussed.Abbreviations used DEP diethyl pyrocarbonate (ethoxyformic anhydride) - HNBB 2-hydroxy-5-nitrobenzyl bromide - N-Acl N-acetylimidazole - PTC phenylthiocarbamyl - PTH 3-phenyl-2-thiohydantoin.     

The binding of calcium to fibrinogen: influence on the clotting process.

The influence of Ca2+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2+-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermeidate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin monomer devoid in only the peptide A, (b) Ca2+ enhances the proteolytic step as illustrated by the measure of the kinetics of H+ release at pH 8.6. On the basis of these observations it is proposed that Ca2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer.     

Thromboelastographic assays of the clotting process in situations of obesity and caloric restriction

A study on blood clotting has been carried out in a number of obese individuals and compared to a group of non-obese persons, in order to assess if the former can be considered to be in "high risk" regarding the onset of a thromboembolic process. The technique of thromboelastography was chosen. The results point out that in obese people a series of alterations take place, both in the time of clot formation, which is enlarged, as in the organization of its nets, which appear strongly structured, favoured by the hyperfibrinogenemia and thrombocytosis detected in these subjects. Likewise, the effect of a hypocaloric diet on clotting in obese persons has been evaluated and compared with the former groups. Clotting in treated obese individuals is modified in the same way as in the untreated group when compared to the non-obese population; nevertheless, when both groups of obese people are compared, no significant difference is observed in the different parameters studied, even though constants determined in citrated whole blood are closer to normality in the subjects undergoing caloric restriction.     

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC–MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.     

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.     

Phenomenological analysis of the clotting curve

A model-independent (phenomenological) characterization of the clotting curve is proposed. Three parameters are used to encapsulate the main features of the increase in absorbance observed at 350 nm due to the reaction of thrombin with fibrinogen that leads to clot formation: (1) the maximum increase in absorbance per unit time, A _m , at the inflection point of the clotting curve; (2) the time needed to reach the maximum increase in absorbance,t _m ; and (3) the clotting time,t _c , obtained from extrapolation of the slope att _m to the zero absorbance baseline. Clotting curves at low fibrinogen concentrations (0.125 ÷ 0.250 µM), well below the K_m, where thrombin amidase activity is rate-limiting with respect to the subsequent aggregation process, have been measured under a wide variety of experimental conditions, (i.e., as a function of thrombin concentration,pH and temperature) in order to explore the basic response of each parameter to changes in solution conditions. Under all conditions examined in this study we have observed thatt _m andt _c are linked through a linear relationship that appears to be an important invariant property of the clotting curve, regardless of experimental conditions. No such clear relationship exists between A _m andt _c , witht _c being associated with several possible values of A _m and vice versa, depending upon solution conditions. It is proposed thatt _c is strictly dependent on thrombin amidase activity, while A _m reflects properties of the aggregation process leading to clot formation. The clotting time shows apH and temperature dependence that closely resembles that of K_m/V_m for synthetic amide substrates. Futhermore,t _c changes linearly with either the inverse thrombin concentration and the concentration of competitive inhibitors of fibrinogen binding to thrombin, as expected for the ratio K_m/V_m. We show how the analysis of clotting curves obtained at different thrombin and inhibitor concentrations yields a quantitative measure of K_I that is in excellent agreement with the value determined independently from steady-state measurements of thrombin amidase activity.     

Cross induced coagulations between human and crustacean clotting factors. Considerations on the clotting processes

1. Cross induced coagulations show that human factor XIII and crustacean coagulin are to some extent functionally equivalent and may be substituted for each other. 2. In crustacea, fibrinogen and coagulin appear as in situ activated products since they are both able to react with non-activated human clotting factors. 3. The coagulin catalyzed transamidation which stabilizes the clot and renders it insoluble in 1-5% monochloroacetic acid solutions seems to be the basic reaction of the clotting process in the animals in which coagulation occurs. 4. The possibility of a two step clotting in crustacea is discussed.     

Blood clotting in space

We describe herein a novel in vitro approach that can be used effectively to obtain valuable insights into the role of platelets, various coagulation proteins as well as proteins of the subendothelial extracellular matrix involved in the hemostatic and thrombotic processes occurring under microgravity. At difference with other experimental approaches proposed in the past our device operates in a closed system and under different shear forces, which better mimics flow conditions occurring in vessels. Furthermore our device by allowing real time monitoring of the thrombotic process and its underlying mechanisms can be regarded as a reliable system for the precise assessment of platelet function.     

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.