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.

The binding of calcium to bovine fibrinogen.

The determination of the number of calcium binding sites of fibrinogen was carried out by means of equilibrium experiments. At pH 7.5 fibrinogen has 3 binding sites of high affinity and several binding sites of low affinity. In the presence of MgCl2 (10(-2)M) the sites of low affinity are eliminated, suggesting that they are not specific and due to weak interactions. Study of the effect of the pH have demonstrated that the 3 sites of high affinity are not identical. At pH values below 7.5 one site is eliminated. This could be due either to an abnormal protonation or to a conformational change. No cooperativity between the sites was found. Partial identification of the binding site by means of direct titration of the calcium induced proton release have shown that the calcium is tightly bound through a chelate system. From the apparent dissociation constant obtained a possible involvement of histidine residues in this chelate system is suggested.

The proteolytic action of bacterial proteases from Clostridium histolyticum on bovine fibrinogen.

Bacterial proteases from Clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (I) of the solution is between 0.1 and 0.2. At I=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations. The striation pattern is different from that obtained with fibrin which shows a major period of 23 nm with three minor striations. The degree of degradation of fibrinogen monitored by disc gel electrophoresis shows that the Aalpha polypeptide chain (mol. wt.=68 000) is degraded into Aalpha1 (mol. wt.=32 000) and Aalpha2 (mol. wt.=29 000), whilst the mobility of Bbeta increases and gamma remains unchanged; the molecular weight is estimated between 272 000 and 269 000. These results emphasize that the charge distribution differences which are compatible with the loss of different portions of the molecule, lead to variations in fibre structures.

Electron microscopic studies of plasmic degradation products of fibrinogen. Implications for the disulfide structure of fibrinogen.

Fibrinogen, coagulable plasmic derivatives (Fragments X) and Fragments Y, D and E were studied by negative staining electron microscopy. Fragment X obtained from Stage 1 digests and fibrinogen were both globular, while Fragment X of Stage 2 digests appeared as a nodular filament. The Stage 1 and Stage 2 Fragment X preparations had approximately the same molecular weight, but could be differentiated by several subtle differences in polypeptide chain structure. Fragments Y and D were also filamentous, although shorter than Fragment X (Stage 2), and Fragment E appeared as a small, compact or folded filament. These results agree with the concept that fibrinogen consists of a strand of nodules connected by thin strands, folded into a compact, spherical shape. The molecule opens up when stabilizing bonds are disrupted or liberated by plasmin. The data are compatible with a fibrinogen molecule in which the two halves are linked by a single locus of disulfide bonds at the amino terminus and with the asymmetric hypothesis of plasmic degradation to Fragments X, Y, D and E.

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.

Soluble fibrin complexes: separation as a function of pH and characterization.

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH. 2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aalpha, Bbeta and gamma chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aalpha, alpha, Bbeta and gamma chains were found. In the peak containing the high polymers, only the presence of alpha, Bbeta and gamma chains was demonstrated. 3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

Reversible inhibition of the in vitro coagulation of human plasma by lectins.

Wheat germ agglutinin (WGA) and concanavalin A (Con-A) (also red kidney bean agglutinin, PHA) have been found to be inhibitors of plasma clotting in vitro. At 40 micrograms/ml and 250 micrograms/ml (4.4 MicroM and 10 microM in carbohydrate binding sites, final concentrations) respectively, WGA and Con-A are able to double the activated partial thromboplastin time of normal human control plasma. Their inhibitory effect is due to their capacity to interact with the carbohydrate portion of blood clotting factors. It is totally abolished in the presence of specific saccharides for WGA or Con-A and is attenuated in the presence of 4% (v/v, final concentration) of human erythrocytes. The action of WGA is mediated by its ability to interact with N-acetylneuraminic acid. When purified phospholipid vesicles plus kaolin are used as an activator instead of cephalidin, this effect persists to the same extent. These two lectins also prolong the plasma clotting time using Russell's viper venom plus purified phospholipid vesicles as an activator. Quick's time was also prolonged by WGA and Con-A but to a lesser extent in this case. WGA can interact directly with some purified blood clotting factors (IX, X and II) in a classical lectin-glycoprotein precipitin reaction. When assessed at individual factors level in whole plasma using clogging assays, direct inhibitions by WGA are only apparent.

The fibrinolytic action of flufenamate and its influence on plasminogen binding to fibrin.

The effects of the synthetic fibrinolytic agent flufenamate were investigated in a purified system made up of bovine fibrin and human plasminogen. The lysis of the fibrin clots was observed after a 12-hour incubation for flufenamate concentrations ranging from 0.25 to 3 mM. Below 0.25 mM and above 3 mM, no lysis occurred, even after a longer incubation. An increase in plasminogen adsorption on the fibrin clot was also observed in the presence of flufenamate. The amount of plasminogen bound was estimated using 125I labelled Glu-plasminogen or Lys-plasminogen, in the presence of a protease inhibitor to avoid the fibrinolysis induced at lytic concentrations of flufenamate. Maximum binding was observed with both types of plasminogen at a flufenamate concentration of 2.5 mM. The binding, relatively fast at the start of incubation, slowed down progressively, but a real plateau was not reached, even after a 6-day incubation. Plasminogen binding was not saturable, suggesting a non-specific binding type. Although Lys-plasminogen binding was always greater than that of Glu-plasminogen, the effect of flufenamate was more pronounced on Glu-plasminogen binding.