Birger Blombäck

Human fibrinopeptides. Isolation, characterization and structure.

Abstract 1. 1. Four different fibrinopeptides, A, AP, Y and B, have been isolated from human fibrinogen by means of chromatography and precipitation procedures. 2. 2. The amino acid sequence has been determined with the phenylisothiocyanate degradation method and by means of fragmentation with proteolytic enzymes and partial acid hydrolysis. The A-, AP- and Y-peptides are similar in structure, the AP-peptide being an A-peptide phosphorylated at the serine residue in Position 3 from the N-terminal end and the Y-peptide being one amino acid residue shorter than the A-peptide from the N-terminal end. The B-peptide has been found to have pyroglutamic acid as N-terminal residue. 3. 3. The phosphorus in human fibrinogen is partly bound to an AP-peptide and partly to other structures of the fibrinogen molecule. 4. 4. On basis of the results the structure of human fibrinogen and the specificity of thrombin is discussed.

Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes

Abstract New, synthetic substrates for trypsin-like enzymes are described. These substrates consist in general of arginine p-nitroanilides in which the NH2-group of arginine is acylated with hydrophobic amino acids or peptides. The susceptibility of the peptide substrates is enhanced by benzoylation of the free NH2-terminal group. One of the most reactive substrates is Nα-benzoyl-phenylalanyl-valyl-arginine p -nitroanilide (Bz-Phe-Val-Arg-pNA). Some kinetic parameters for the substrates with trypsin, thrombin and Reptilase have been studied. Trypsin has a much higher affinity for Bz-Phe-Val-Arg-pNA than for benzoyl-DL-arginine p-nitroanilide (BAPNA). Bz-Phe-Val-Arg-pNA also has a high susceptibility toward thrombin and Reptilase. The narrow substrate specificity of thrombin as compared with trypsin is reflected by the fact that Bz-L-Phe-L-Val-L-Arg-pNA is rapidly hydrolyzed by thrombin while the rate of hydrolysis of Bz-D-Phe-L-Val-L-Arg-pNA is very slow. On the other hand the action of trypsin on the D-isomer is only slightly reduced. The synthetic peptide substrates are useful for determination of enzymes of the trypsin type, and they can also be used in biological fluids. Small amounts of trypsin, thrombin and Reptilase can be determined.

Fibrin in human plasma: Gel architectures governed by rate and nature of fibrinogen activation

The porosity, fiber dimension and architecture of fibrin gels formed in recalcified plasma on addition of thrombin are, within a certain range of thrombin concentrations, determined by the initial rate of fibrinogen activation. Furthermore, the initial network formed in this range creates the scaffold into which subsequently activated fibrinogen molecules are deposited. Change in thrombin concentration that occurs during gelation, as a result of indigenous thrombin generation in plasma, does not qualitatively alter this scaffold. The formation of the networks obeys a more complex rule when low amounts of thrombin are added or with recalcified plasma without added thrombin. These networks are tighter than would be expected from the initial rate of fibrinogen activation. In this case an extremely porous network is probably formed initially, followed by formation of a secondary, superimposed network of a less porous architectural quality. The latter structure appears to be governed by the rate of indigenous generation in plasma of thrombin-like enzymes in combination with the particular type of fibrinmonomers being produced. In addition our findings establish the rules for proper determination of gel structures in clinical plasma samples. The sequelae of a variety of clot structures that may be formed in vivo are discussed.

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.

Fibrin gel structure and clotting time

Liquid permeation studies of fibrin gels, which were formed in the presence of thrombin (Fibrin II) and in the presence of Batroxobin (Fibrin I), showed that those gels have distinctly different flow properties. For both gels the permeability coefficient (Ks) and average pore size varied depending on the conditions for gel formation. Low pH and ionic strength favour high Ks and large pore sizes, whereas high pH and ionic strength produce gels with low Ks and small pore sizes. Parallel turbidity studies showed correlations between the optical properties of the gels and permeation data. Of particular importance was the finding that the clotting time (Ct) is directly related to Ks of the final gels. Thus events preceding gel formation determine the gel structure. It is proposed that the average lengths of polymers formed prior to gelatin varies directly with Ct. Shorter polymers give infinite net works (gels) which are tight and longer polymers provide for formation of infinite net works which are more porous. At the same Ct, the net work formed with thrombin is, over a wide range of Ct, tighter than that formed with Batroxobin. It was also found that Ks is inversely related to the fibrin concentration (C) in the gels. The ratio Ct/C (permeability index) is thus an important determinant for the gel structure. The applicability of the permeability index to whole blood as a predictive test for thromboembolic and bleeding conditions is discussed.

Prothrombin activation induced by ecarin - A prothrombin converting enzyme from echis carinatus venom

Abstract Purified prothrombin was activated by a physiological activator of a thromboplastin type (AC) and by Ecarin, a procoagulant enzyme derived from Echis carinatus venom. Intermediate products obtained during 2 min. to 2 hours of activation were analyzed by SDS poly-acrylamide gel electrophoresis and NH2-terminal analysis. Thrombin activity was measured on fibrinogen as substrate. It was found that Ecarin cleaves prothrombin at a bond, which results in release of the NH2 -terminal portion of the molecule (A-fragment; intermediate 3). A second cleavage site appears to be at the ArgIle bond linking the A and B chain in thrombin precursors. The latter cleavage was considered to be responsible for the appearance of the biological activity. The resulting Ecarin thrombin in constrast to thrombin produced by physiological activation appears to consist of a B-chain and an extended A-chain (A-chain + intermediate 4). This is believed to be the reason for the lower specific activity of Ecarin thrombin on fibrinogen. Our results do not exclude the possibility that prothrombin is cleaved by Ecarin as well as by physiological activator at other bonds than already identified.

Molecular weight analysis of fibrinogen and fibrin chains by an improved sodium dodecyl sulfate gel electrophoresis method

Abstract Modification of the procedure for sodium dodecyl sulfate polyacrylamide electrophoresis to use dithiothreitol as reducing agent gave reproducible molecular weights in good agreement with other methods and was easier and faster to perform. It was found that the migration of some proteins in sodium dodecyl sulfate-containing gels was significantly affected by both reduction and alkylation and that accurate estimates of molecular weight could be obtained only when both reference and sample proteins were treated in the same manner. It was also found that the sodium dodecyl sulfate method does not yield accurate molecular weights for proteins with polypeptides held together by interchain peptide bonds. The sodium dodecyl sulfate method was used to estimate the molecular weights of the chains of fibrinogen, crosslinked, and non-crosslinked fibrin. Average molecular weight values obtained for the carboxymethylated derivatives were: α(A), 64 000; β(B), 57 000; γ, 48 000; α1, 61 000; α2, 63 000; β, 55 000, and γ-γ dimer, 79 000. Summation of the values for the chains of fibrinogen, (α(A), β(B), γ)2), gave a molecular weight of 338 000. The value for the γ-γ dimer of crosslinked fibrin was less than twice the molecular weight of free γ-chain because it is held together by peptide bonds and does not have the effective surface area of two free γ-chains.

Purification of the factor VIII complex

Two high purity factor VIII concentrates, type I and type II were developed for clinical trials in patients with hemophilia A and von Willebrands disease. Fresh frozen plasma containing 1% polyethylene glycol 4000 was thawed to form cryoprecipitate, which was subsequently dissolved in citrate buffer. By addition of glycine buffer to a final concentration of 2.0 M at 26 degrees C, the bulk of fibrinogen was precipitated while factor VIII remained in solution. Factor VIII was precipitated from the glycine supernatant by addition of solid sodium chloride. The recovery of factor VIII procoagulant activity (VIII:C) per kg plasma was 271 +/- 23 units (n = 4) and 386 +/- 47 units (n = 7) for the type I and the type II preparations, respectively, while the recovery of von Willebrand factor related activity (ristocetin cofactor, VIIIR:RC) was 518 +/- 75 units and 718 +/- 90 units per kg plasma, respectively. The specific activity (units per mg protein) of VIII:C in the type I and type II preparations were 2.53 +/- 1.02 and 7.56 +/- 1.33, respectively. The specific activity (units per mg protein) of VIIIR:RC for the type I and type II preparations were 4.86 +/- 2.32 and 13.6 +/- 3.7, respectively. VIIIR:Ag was present as multimers in both preparations, and the multimeric pattern was similar to that of normal plasma. The preparations have the ability to correct the prolonged bleeding time in severe von Willebrands disease. The factor VIII complex in the type II preparations was further purified by gel filtration on Sephacryl S-1000. This preparation was free of fibrinogen and fibronectin. Its specific activity in terms of VIII:C was 47 u/mg protein and 104 u/mg protein in terms of VIIIR:RC. The subunit of reduced factor VIIIR:Ag had Mr of 210 Kd on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Measurement in human blood of fibrinogen/fibrin fragments containing the Bβ 15–42 sequence

This paper describes a radioimmunoassay for the B beta 15-42 peptide derived from human fibrinogen or fibrin. Iodinated B beta 15-42 is bound by specific antiserum and binding can be completely inhibited by excess of the non-iodinated B beta 15-42, B beta 1-42 or B beta 1-118. These peptides cannot be distinguished in this assay. Furthermore, fibrinogen can also completely inhibit binding of iodinated B beta 15-42 peptide. Due to the cross-reaction with fibrinogen, clinical blood samples require a processing step prior to their use in this radioimmunoassay. Ethanol precipitation allows for fibrinogen removal and a near quantitative recovery of both added B beta 15-42 peptide as well as of the endogenous blood peptide(s) containing the B beta 15-42 sequence. The mean level of B beta 15-42 immunoreactive material in normal individuals was found to be 0.41 pmol/ml while that in one group of patients was 20-40 times this value.

The assay of antithrombin using a synthetic chromogenic substrate for thrombin

Abstract A rapid method for the assay of antithrombin activity is described in which a chromogenic synthetic tripeptide, benzoylPheValArgp.nitroanilide, is used as substrate for thrombin. During the reaction p-nitroaniline is released. The optimal conditions of the thrombin-antithrombin interaction with respect to pH, ionic strength and temperature have been determined. The results obtained with this method compare well with antithrombin assays using coagulation or immunological procedures.