Release of B beta peptides from fibrinogen or fibrin in the presence of alpha 2 antiplasmin

When Glu-plasminogen (plg) was activated by urokinase (UK) in the presence of fibrinogen or fibrin, B beta peptides (B beta 1-42) were released faster from fibrinogen than from fibrin (B beta 15-42). These results were contrary to faster release of B beta 15-42 from fibrin in the UK-activated clotted plasma in comparison to the release of B beta 1-42 from UK-activated plasma. The addition of plasma or lysine-Sepharose pass through fraction to the above system resulted in faster release of B beta peptides from fibrin than fibrinogen. The addition of alpha 2 antiplasmin (alpha 2AP) to the mixture of Glu-pig, UK and fibrinogen or fibrin resulted in faster release of B beta peptides from fibrin than from fibrinogen. These results indicate that fibrin protected plasmin from inactivation by alpha 2AP, leading to cleavage of Arg(42)-Ala(43) bond in beta-chain of fibrin which seems to be less susceptible to plasmin than the same bond in fibrinogen.     

Kinetic analyses of the activation of Glu-plasminogen by urokinase in the presence of fibrin, fibrinogen or its degradation products

The kinetics of the activation of Glu-plasminogen (Glu-plg) and Lys-plasminogen (Lys-plg) by urokinase (UK) were studied in purified systems. The activation of plasminogen by UK in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant (Km) of 1.45 microM and a catalytic rate constant (kcat) of 0.93/sec for Glu-plg as compared to 0.25 microM (Km) and 0.82/sec (kcat) for Lys-plg. In the presence of fibrin and fibrinogen or its plasmin degradation products (fragment D and fragment E), Km for Glu-plg hardly changed, whereas kcat for Glu-plg increased. Effect on increase in kcat was in the order of fibrin greater than fibrinogen greater than D greater than E. Fibrin, fibrinogen, D and E did not influence the activation of Lys-plg by UK. These results indicate that Glu-plg bound to fibrin, fibrinogen, D or E becomes easily activatable by UK. The activation of Lys-plg, however, is not influenced in the presence of fibrin, fibrinogen, D or E.     

Augmentation of streptokinase activator activity by fibrinogen or fibrin

The presence of either human or bovine fibrinogen or soluble fibrin monomer, enhanced the plasminogen activator activity of streptokinase. The effect was due to an increased rate of plasmin formation as shown by SDS-gel electrophoresis. Plasminogen contamination of the purified fibrinogen was ruled out as a contributing factor. The activator activities of urokinase or other human tissue activators were not affected by fibrinogen, neither were the proteolytic activities of plasmin or trypsin. Since the degree of augmentation was not affected by ε-amino-n-caproic acid or L-lysine, and since no obvious physical binding of SK-plasminogen activator to fibrinogen could be demonstrated, this type of interaction does not appear to be responsible for the augmentation. The presence of a highly active but labile activator which could be visualized and stabilized only in the presence of fibrinogen appears to be the cause for the augmentation.     

The role of the lysine binding sites of human plasminogen in the fibrinogen stimulated rate of active site formation in the streptokinase-plasminogen equimolar complex

The effect of various concentrations of -amino caproic acid (EACH) on the rate of active site formation in the human plasminogen moiety of the streptokinase-plasminogen equimolar complex has been studied in the absence and presence of human fibrinogen fragment D₁(FD₁). In the absence of FD₁, the pseudo first order rate constant (k_obs) for active site development in this complex ranges from 8.4–17.9 × 10⁻³ sec⁻¹ with Glul-plasminogen (Glul-Pg), Lys₇₇-plasminogen (Lys₇₇-Pg), and Val₄₄₂-plasminogen (Val₄₄₂-Pg) at levels of EACA from 0–25 mM. In the presence of 2 μM FD₁, the k_obs for active site formation in the SK·Glu₁-Pg complex, of 60.1 × 10⁻³ sec⁻¹, was not altered significantly as the EACA level was increased to 25 mM. Similarly, in the SK· Lys₇₇-Pg complex, the k_obs for active site formation, of 62.1 × 10⁻³ sec⁻¹, was essentially unchanged as the EACA level was increased to 25 mM. Finally, the k_obs for active site formation in the SK·Val₄₄₂-Pg complex, of 113.6 × 10⁻³ sec⁻¹, was also unaffected at levels of EACA up to 5 mM, with a slight inhibition at 25 mM EACA.

These results show that the stimulation of active site formation in the equimolar SK·Pg complex by fibrinogen fragment D₁ is mediated by sites separate from the lysine binding sites of plasminogen.     

Affinity chromatography of soluble fibrin in human plasma on insolubilized fibrinogen and on fragment D from fibrinogen,non cross-linked or cross-linked fibrin

Normal human plasma containing approximately 2.5 mg of fibrinogen per ml, ¹³¹l-labelled fibrinogen (less than 0.1 mg per ml) and ¹²⁵l-labelled fibrin monomer (between 0.012 and 0.05 mg per ml) was subjected to affinity chromatography on insolubilized human fibrinogen (Fg-agarose), fragment D prepared from fibrinogen (FgD-agarose), fragment D from non cross-linked fibrin (FbD-agarose) or fragment D from cross-linked fibrin (D-D-agarose). The fibrin monomer was obtained by dissolving fibrin in 2 M NaBr. The fibrinogen and fragments D were highly purified materials which were coupled to CNBr-activated Sepharose in comparable molar amounts (8 mg per ml gel for the fragments D and 20 mg per ml gel for fibrinogen).The FgD- and D-D-columns had negligible affinity for fibrinogen in plasma, the Fg-column bound approximately 3 percent of the Fibrinogen and the FbD-column 1.5 percent.All columns showed very similar binding properties for soluble fibrin monomer both with respect to the amounts of fibrin adsorbed and the amounts eluted with 2 M NaBr. Thus the reported higher affinity of insolubilized fibrin monomer to fragment D prepared from fibrin compared to Fragment D from fibrinogen was not associated with a higher affinity of insolubilized fragments D from fibrin for soluble fibrin monomer.     

Effects of SK-potentiator and fibrinogen on SK-plasminogen complex in the presence of tranexamic acid

The addition of tranexamic acid to the mixture of plasminogen and streptokinase (called SK-activator activity) resulted in decreased extent of hydrolysis of TAMe (tosyl arginine methyl ester). The addition of SK-potentiator to the mixture of SK (streptokinase), plasminogen and tranexamic acid prevented the decrease in SK-activator activity caused by tranexamic acid, thus SK-potentiator counteracting with the effects of tranexamic acid. Fibrinogen potentiated SK-activator activity, but did not prevent the decrease of the activity caused by tranexamic acid. Fibrinogen added to SK and plasminogen prior to tranexamic acid prevented the decrease in SK-activator activity. From these data it is suggested that SK-potentiator and fibrinogen bind with lysine binding sites of plasminogen part of SK-activator, and SK-potentiator binds with SK-plasminogen (or -plasmin) complex faster than fibrinogen.     

Glu-plasminogen I and II: their activation by urokinase and streptokinase in the presence of fibrin and fibrinogen

Two isozymes of a native form of human plasminogen (plg), Glu-plg I and II, were isolated. Glu-plg I or II was activated by urokinase (UK) or streptokinase (SK) in the presence of fibrinogen or fibrin. The activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin. Fibrin caused better activation of both Glu-plg I and II than fibrinogen. When fibrinolysis or fibrinogenolysis was measured, fibrin was degraded faster than fibrinogen after the activation of Glu-plg I and II by UK. These results suggest that the activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin or to less extent fibrinogen.     

Crosslinking of soluble fibrin and fibrinogen

Monomeric 125I-desAA-fibrin and 125I-desAABB-fibrin were prepared by treating 125I-fibrinogen with thrombin. Preactivated factor XIII was added to 125I-fibrin/131I-fibrinogen mixtures, and after incubation for 2 and 6 hours, samples were investigated by SDS polyacrylamide gel electrophoresis under reducing conditions. The electrophoresis pattern showed a gamma-gamma band containing both 125I-fibrin and 131I-fibrinogen. These experiments indicate that a fibrin molecule polymerizes with a fibrinogen molecule in a similar way as a fibrin molecule polymerizes to a second fibrin molecule. This type of polymerization results in conformational changes of the molecules involved thus enabling FXIIIa for specific crosslinking reaction. The polymerization of fibrin and fibrinogen molecules as well as the crosslinking of fibrin to fibrinogen molecules seem to represent a mechanism for interrupting the process of further fibrin polymerization.     

Biological effects of the administration of an equimolar streptokinase-plasminogen complex in man

Two different lots of a 1:1 stoichiometric streptokinase-plasminogen (SK-Plg) complex were prepared. Some in vitro experiments suggested that the SK-Plg complex did not react with antisera against human plasminogen.Infusion of the streptokinase-human plasminogen complex in man (5 patients receiving lot SKPC-VIIIK and 4 patients receiving lot SKPC-XXIIK) was associated with signs of immediate antigenicity in 3 patients and of immunogenicity in all patients.     

Binding properties on Sepharose insolubilized fibrinogen and fibrin, of various species of fibrinogen and fibrin solubilized in plasma

Radiolabelled tracers of fibrinogen, fibrin des-AA and fibrin des-AABB were solubilized in recalcified, prothrombin depleted plasma, adding either 125I-fibrin des-AA or 125I-fibrin des-AABB together with 131I-fibrinogen, and subsequently subjected to affinity chromatography, utilizing short columns of Sepharose insolubilized preparations of fibrinogen, fibrin des-AA and fibrin des-AABB, respectively. Two naturally occurring fibrinogen species, of high molecular weight (HMW; m.w. 340.000) and of low molecular weight (LMW; m.w. 305.000) exhibited similar binding characteristics, as judged by adsorption and desorption experiments. In subsequent studies all tracer preparations were derived from HMW-fibrinogen. Sepharose insolubilized fibrinogen favoured the adsorption of soluble fibrins as compared to fibrinogen in solution; the adsorption of soluble des-AA fibrin was similar to that of soluble des-AABB fibrin. To insolubilized fibrin, adsorption of soluble tracers of fibrinogen and fibrins increased considerably, and soluble fibrins were no longer preferentially adsorbed. The latter observation was supported by similar desorption characteristics of these tracers. These findings may indicate that the E-domains of soluble fibrin become largely inaccessible to the D-domains of Sepharose insolubilized fibrinogen, probably due to complexing fibrinogen in plasma. Furthermore, adsorption was largely related to the a-epitope of insolubilized fibrin.     

Incorporation of fibrinogen into soluble fibrin complexes

Fibrinogen was incubated with traces of thrombin, and the reaction stopped with hirudin prior to visible gelation. When I₁₂₅-labelled fibrinogen was added to the mixture before chromatography on Sepharose 4B, labelled fibrinogen eluted not only in the main fibrinogen peak, but also in the earlier fractions containing high molecular weight complexes. This implies that fibrinogen not acted upon by thrombin, may form complexes with soluble fibrin. The complexes detected by gel chromatography could not be demonstrated by SDS-urea polyacrylamide gel electrophoresis, indicating that non-covalent bonds are involved.     

The effect of heparin and dipyridamole on the deposition of fibrin-like material in rabbits infused with soluble fibrin monomer or fibrinogen

¹²⁵I-labelled fibrinogen or fibrin monomer was infused into rabbits treated with saline, heparin or dipyridamole. Serial measurements of radioactivity in blood were performed and the quantity of radioactivity excreted in the urine in 5 $\text{1}\text{2}$ hours was determined. The radioactive material deposited in various organs was measured and characterized. The results indicate that some fibrin-like deposition occurs in fibrinogen infused animals, the amount being unaffected by either heparin or dipyridamole. After FM-infusion, fibrin deposition especially in the spleen was substantially greater, the extent being inhibited by dipyridamole but not by heparin. It is concluded that the deposition of fibrin-like material in organs of animals infused with FM or fibrinogen may occur in the absence of thrombin elaboration. Alternative mechanisms of FM and fibrinogen polymerization are discussed.     

Fibrinogen to fibrin transformation in umbilical cord blood and purified neonatal fibrinogen

Thrombin and reptilase times in umbilical cord plasma were prolonged as compared to adult plasma. Thrombin time of fibrinogen, purified from pools of cord or adult plasma respectively, showed no difference. Fibrin polymerization time were somewhat longer for cord fibrinogen than for adult, indicating a polymerization defect. There were no difference between the two types of fibrinogen concerning polyacrylamide gel electrophoresis and mapping of tryptic peptides of reduced and alkylated polypeptide chains.     

Comparison of fibrin networks in plasma and fibrinogen solution

Mass-Length ratio of fibrin fibres (microT and microP) derived independently from turbidity and permeability respectively, in networks made in fibrinogen solution and plasma, have been compared under similar conditions of clotting. Amount of fibrinogen conversion to fibrin was similar in both systems when high thrombin concentrations were used. But networks in plasma had significantly thicker fibres (higher microT and microP) than those in fibrinogen solution. This difference arises from differing kinetics of fibrin assembly in the two systems. When lower thrombin concentration is used, fibrin fibre thickness (as indicated by microP and microT) is increased in fibrinogen solution but the fibrin content of the network remains unaltered. In plasma, on the other hand, the fibrin content of the network is decreased, microT remains relatively unchanged while microP increases. The thrombin concentration dependence of the fibrin content of the network in plasma and the bimodal distribution of fibrin fibre thickness explain the breakdown in correlation between microP and microT.     

Adsorption of fibrinogen and fragment D of fibrinogen onto the insolubilized alpha-chain of fibrin.

After thrombin treatment insolubilized fibrinmonomer, which is obtained from insolubilized fibrinogen covalently bound to agarose, adsorbs soluble fibrin and its derivatives from solutions. The immobilized proteins are attached to the agarose by the 'A' alpha-chain. After reduction of the disulfide bridges the beta- and gamma-chains can be removed from the agarose. After thrombin treatment the immobilized alpha-chain adsorbs fibrinogen and fragment D. To some extent the beta- and gamma-chain do not seem necessary for the adsorption. The amount adsorbed increases, when thrombin treatment of the insolubilized protein follows the reduction process. This may indicate that the fibrinopeptides 'A' of the insolubilized alpha-chain are better accessible after the removal of the beta- and gamma-chains.     

The enhanced activation of Glu-plasminogen by urokinase in the presence of fibrin or des A fibrin as measured by the release of B beta peptide and FDP

Fresh plasma was incubated either with urokinase (UK) alone or the mixture of UK and human thrombin or Reptilase. In a purified system Glu-plasminogen (Glu-plg) was incubated with fibrinogen or fibrinogen plus thrombin in the presence of UK. At intervals, aprotinin was added to stop the reactions and the amounts of B beta peptide and fibrin(ogen) degradation products (FDP, FgDP) were measured by radioimmunoassay and enzyme immunoassay, respectively. Results obtained by using plasma showed that fibrin was degraded faster than fibrinogen upon the addition of UK to the plasma or plasma clotted with thrombin. B beta peptide was released faster from the clot than plasma. The clot formation caused by Reptilase (release of fibrinopeptide A) was accompanied by increase in the release of B beta 1-42 from des A fibrin (fibrin without fibrinopeptide A). In a purified system, fibrin was degraded faster than fibrinogen upon the activation of Glu-plg by UK. These results may correlate well with the observation that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen.