Characterisation of a soluble D dimer-E complex in crosslinked fibrin digests.

Further information is presented on the heterogeneity of the fragments released by plasmin digestion of crosslinked human fibrin. Six components (labelled initially 1 to 6) were detected by polyacrylamide gel electrophoresis and the two major components (3 and 4) which make up the D dimer complex were examined in relation to fragments D and E. The more cathodic component of the D dimer complex was shown to consist of E fragments non covalently bound to D dimer fragments, while the other fragment was shown to be D dimer. The lability of the soluble D dimer - E complex to freezing and thawing was observed as was the conversion of D dimer to lower molecular weight fragments. The plasmin induced release of D dimer - E, D dimer, fragments D and E and some very large molecular weight components from crosslinked fibrin should prove useful in understanding fibrin polymerisation and lysis.     

Like fibrin, (DD)E, the major degradation product of crosslinked fibrin, protects plasmin from inhibition by alpha2-antiplasmin

Plasmin generation is localized to the fibrin surface because tissue-type plasminogen activator (t-PA) and plasminogen bind to fibrin, an interaction that stimulates plasminogen activation over a hundred-fold. To ensure efficient fibrinolysis, plasmin bound to fibrin is protected from inhibition by alpha2-antiplasmin. (DD)E, a major soluble degradation product of cross-linked fibrin that is a potent stimulator of t-PA, compromises the fibrin-specificity of t-PA by promoting systemic activation of plasminogen. In this study we investigated whether (DD)E also protects plasmin from inhibition by alpha2-antiplasmin, facilitating degradation of this soluble t-PA effector. (DD)E and fibrin reduce the rate of plasmin inhibition by alpha2-antiplasmin by 5- and 10-fold, respectively. Kringle-dependent binding of plasmin to (DD)E and fibrin, with Kd values of 52 and 410 nM, respectively, contributes to the protective effect. When (DD)E is extensively degraded by plasmin, yielding uncomplexed fragment E and (DD), protection of plasmin from inhibition by alpha2-antiplasmin is attenuated. These studies indicate that (DD)E-bound plasmin, whose generation reflects the ability of (DD)E to stimulate plasminogen activation by t-PA, has the capacity to degrade (DD)E by virtue of its resistance to inhibition. This provides a mechanism to limit the concentration of (DD)E and maintain the fibrin-specificity of t-PA.     

Isolation of vasoactive peptides from human fibrin and fibrinogen degraded by plasmin

Human fibrin(ogen) was incubated for 60 min at pH 8.0 and at 37 °C in a clot-lysis system containing plasmin. The low molecular weight degradation products (lmw-fdp or LMW-FDP) thus formed were separated from the bulk of the material by membrane filtration. After concentration and subsequent chromatography on Bio-Gel P-6, the degradation products were separated into at least 12 distinct fractions of which 3 had permeability-increasing activity. The major active fraction was further separated into 8 subfractions by column zone electrophoresis; 2 had permeability-increasing and 2 had vasoconstrictor activity. These active fractions were further purified to a homogeneous state by electrophoresis at pH 1.9. The recovery of each active peptide was about 60 %. Both permeability-increasing factors, a pentapeptide (Ala₂, Pro, Lys, Arg) and an undecapeptide (Ser, Glx₃, Pro₂, Val, Leu, Trp, Lys₂) contain 2 basic amino acids per molecule while the vasoconstrictors contain only 1 each. This difference in composition might explain the differences in their physiological activity. Finally, the location of the active peptides in the fibrinogen molecule has been tentatively assigned on the basis of their amino acid composition and the known sequence of fibrinogen.     

The isolation and characterization of a ternary human plasmin B-chain-streptokinase-plasminogen complex

A ternary equimolar human plasmin B-chain-streptokinase-plasminogen complex was isolated from a mixture of the plasmin B-chain-streptokinase complex and human plasminogen at 0 degrees C and 37 degrees C. A ternary complex which was shown to be species specific, was identified and characterized by ultracentrifugal, acrylamide gel electrophoretic, and agarose double diffusion analyses. When mixed at a 1:1 molar ratio at 0 degrees C, 39.9% of the preparation existed as a plasmin B-chain-streptokinase-plasminogen complex; when mixed at 37 degrees C, 86.4% existed as a complex, which was identified by electrophoretic analyses to be a plasmin B-chain-streptokinase-plasmin complex. Sedimentation velocity analyses gave s degrees 20,w values of 3.79 for the plasmin B-chain-streptokinase complex, 4.10 for Lys-plasmin, and 6.23 for the plasmin B-chain-streptokinase-plasmin complex. Sedimentation equilibrium analyses gave molecular weights of 73,900 for the plasmin B-chain-streptokinase complex, 82,900 for Lys-plasmin, and 153,100 for the plasmin B-chain-streptokinase-plasmin complex. The diisopropylphosphorofluoridate (DFP)-inhibited and the p-nitrophenyl-p-guanidino-benzoate (NPGB)-inhibited plasmin B-chain-streptokinase complexes both retained their ability to form a ternary complex with human plasminogen, but this complex did not convert to a plasmin B-chain-streptokinase-plasmin complex. Thus, the active site serine residue is essential for the activator activity of the plasmin B-chain-streptokinase complex, but it is not necessary for the binding of the plasmin B-chain-streptokinase complex to plasminogen to form a ternary complex.     

The dissolution of human cross-linked plasma fibrin clots by the equimolar human plasmin-derived light (B) chain-streptokinase complex. The acceleration of clot lysis by pretreatment of fibrin clots with the light (B) chain.

The equimolar human light (B) chain·streptokinase activator complex was shown to be an effective fibrinolytic agent in the dissolution of human cross-linked plasma fibrin clots, in vitro; its activator activity was similar to that of the equimolar human plasmin·streptokinase complex. ¹²⁵I-radiolabelled preparations of the human plasmin-derived light(B) and heavy(A) chains, and Lys-plasminogen were shown to be adsorbed to cross-linked plasma fibrin clots invitro to the same extent. Clot lysis by the light(B) chain·streptokinase complex, the plasmin·streptokinase complex, streptokinase and urokinase was enhanced by a factor of at least 10-fold when the fibrin clots were pretreated with either the light(B) chain or Lys-plasminogen, but was not enhanced when the fibrin clots were pretreated with the heavy(A) chain. The activator activities of the light(B) chain·streptokinase complex, the plasmin·streptokinase complex and streptokinase were similar on these pretreated cross-linked fibrin clots. Limited proteolysis of fibrinogen by the light(B) chain and by plasmin showed the same specificity giving Fragments X, Y, D and E, but the rate of hydrolysis of fibrinogen by the light(B) chain was much slower.     

Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D

Prolonged degradation of fibrinogen by plasmin at calcium chloride concentrations of about 2 mM yields one major fragment D, designated as D(cate)¹. This fragment has a MW of 93,000 as derived from the MW of its subunits of 12,000, 38,000 and 43,000. Calcium ions protect D(cate) against further attack by plasmin. After sequestration of calcium ions with EGTA, fragment D(cate) is further degraded to a final fragment D, designated as D(EGTA), with a MW of 80,000 by breakdown of the γ-chain subunit⁷ 38,000 MW to 25,000 MW. The varying degrees of degradation of D(cate) in a medium containing no calcium or only traces may account for most of the $\text{in vitro}$ heterogeneity of fibrinogen fragment D described in the literature. Similarly, degradation of non-crosslinked fibrin by plasmin depends on the calcium concentration. Prolonged incubation of crosslinked fibrin with plasmin at 2 mM calcium chloride concentration resulted in the formation of a fragment D-dimer, with subunits of MW of 12,000, 43,000 and 75,000, indicating that it is comprised of two molecules of D(cate) which are crosslinked by the γ-subunit of 38,000 MW. The D-dimer also appeared to be plasmin-resistant by virtue of the presence of calcium ions. Both D(cate) and the D-dimer formed at calcium concentrations normally occurring in blood can be expected to represent physiologically important fragments D.     

Nephelometric determination of plasminogen and plasmin inhibitors in human plasma using fibrin suspension as a substrate

A nephelometric method is described for determination of plasminogen and two types of plasmin inhibitors in human plasma having different affinity toward plasmin. This method is based on the kinetic analysis of effects of whole plasma and plasmin inhibitor fraction obtained from plasma on the activity of exogenously added plasminogen which was determined by measuring the decrease of light scattering of fibrin suspension. With this method we have determined the activity of plasminogen and two types of inhibitors in the plasma of normal subjects and patients with high fibrinogen degradation product values. They include patients with various malignant tumors with DIC, chronic renal failure, sepsis, vascular diseases, and liver cirrhosis with hepatoma.     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

Spectrophotometrical determination of plasminogen and plasmin inhibitors in human plasma using fibrin suspension as a substrate

The method presented in this article is based upon the kinetic observation that plasmin molecule evolved from plasminogen by urokinase binds exclusively to α₂-plasmin inhibitor(α₂-PI)-like inhibitors in the presence of other inhibitors and substrate molecules. Using fixed amounts of urokinase and diluted plasma(or plasmin inhibitor fraction obtained from plasma) and changing the amount of plasminogen added, the activity of plasminogen was determined by measuring the absorbance decrease of fixed amount of aqueous suspension of non-cross linked fibrin. Two parallel straight lines are obtained, one from plasma and the other from plasmin inhibitor fraction, when the apparent activity of plasminogen is plotted against the amount of the enzyme added. The intersection between the abscissa and the straight line obtained with the plasmin inhibitor fraction would give the amount of inhibitors having very high affinity toward plasmin, like α₂-PI. The slope of the line would show the amount of inhibitors with relatively moderate affinity toward the enzyme. The distance between the two intersections of the two straight lines with the abscissa corresponds to the amount of plasminogen present in plasma.     

Degradation of non cross-linked and cross-linked fibrin clots by plasmin, trypsin, chymotrypsin and Brinase

Fibrin clots with either no or complete γ-dimerization and α-polymerization were digested by plasmin, trypsin, chymotrypsin and Brinase®. The degradation products in the supernatants were examined in crossed agarose immunoelectrophoresis (CAIE) with anti-serum against fibrinogen, fibrinogen-D fragments, fibrinogen-E fragments and a mixture of the two last anti-sera. The approximate mol wt of the non-reduced fragments were estimated according to their electrophoretic mobility in sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAE).D-antigenic fragments with some anodic migration in agarose, mol wt c. 170,000, appear during degradation of cross-linked fibrin with the four enzymes, and D-antigenic fragments with less migration in agarose, mol wt c. 80,000, in digests from non cross-linked fibrin. The D-antigenic fragments and the corresponding bands in SDS-PAE disappear on extended degradation by trypsin, chymotrypsin and Brinase.Plasmin sensitive, only E-antigenic fragments with some anodic migration in agarose are especially demonstrable in plasmin digests from cross-linked fibrin. E-antigenic plasmin resistant fragments with distinct anodic migration, mol wt c. 50,000 appear in digests from both kinds of fibrin after degradation by the four enzymes. The core fragments E after extended degradation by trypsin, chymotrypsin and Brinase show electrophoretic heterogeneity in CAIE and spur formation in double immunodiffusion against core fragments E in plasmin digests.     

The simpliRED D dimer test: a novel assay for the detection of crosslinked fibrin degradation products in whole blood

A new system for the detection of fibrin degradation products in whole blood has been developed. The test provides a clearly visible agglutination of the patient's red blood cells in the presence of elevated levels of the crosslinked fibrin derivative, D dimer. The test, which uses a bispecific reagent prepared from Fab' fragments of monoclonal antibodies, gives a positive result in 1-2 minutes. One monoclonal antibody (RAT-1C3/86) was raised against human red blood cells, and the second (DD-3B6/22) was specific to the crosslinked fibrin derivative, D dimer. Addition of the bispecific reagent to a drop of patient's whole blood resulted in red blood cell agglutination when elevated levels of D dimer were present in the sample. Clinical trials showed sensitivity equivalent to that of current commercial tests. Samples from patients with thrombotic disease states as well as normals were examined. The test was compared with commercial latex agglutination and enzyme immunoassay systems and showed good correlation with the presence of elevated levels of crosslinked fibrin degradation products. This technology represents an advance which allows rapid "on the spot" whole blood analysis, for the diagnosis of thrombotic disorders.     

Evidence for aggregation of fibrin fragments D and E in soluble fibrin complex formation

The capability of fibrin fragments D and E (Fb:D and Fb:E) to participate in soluble fibrin complex (SFC) formation was investigated by gel exclusion chromatography. Non-crosslinked fibrin extensively digested by plasmin and purified Fb:D and Fb:E did not form higher molecular weight aggregates upon incubation with thrombin and upon incubation with fibrinogen in the absence of thrombin. On the other hand, Fb:D-E mixtures, as well as purified Fb:D and purified Fb:E, upon incubation with fibrinogen and thrombin displayed moderate peak shifts toward a higher molecular weight range by agarose gel chromatography, suggesting the formation of complexes in the dimeric-size range. Fb:D and Fb:E differ in this respect from fibrinogen fragments D and E (Fg:D and Fg:E), which fail to form higher molecular weight aggregates under similar experimental circumstances (Smith, G. F. and Bang, N. U., Biochemistry, $\text{11}$:2958–2966, 1972).     

Release of N-terminal peptides from glu-plasminogen by plasmin in the presence of fibrin

The rate of plasmin-catalyzed Glu-plasminogen to Lys-plasminogen conversion was faster in the presence of fibrin than in its absence when assayed by SDS-polyacrylamide gel electrophoresis followed by quantification of stained bands by densitometer. Experiments with the isozymes Glu-plasminogen I and Glu-plasminogen II showed that plasmin catalyzed formation of Lys-plasminogen II was especially facilitated in the presence of fibrin.     

Stabilization of the plasmin digestion products of fibrinogen and fibrin by calcium ions.

The role of calcium in the stabilization of fibrinogen and fibrin plasmin digestion products, D-monomer and D-dimer has been studied. The largest D fragment, D1¹, and D-dimer are both stable to further plasmin degradation in the presence of calcium ions. Chelation of calcium leads to further plasmin digestion due to sequential cleavage of peptides from the C-terminal end of the γ-chain. This stabilization is due to calcium ions and not to occupation of the cross-link site. All the digestion products of D-dimer were also found to be stabilized by the re-addition of calcium ions.     

Degradation of human fibrinogen by plasmin: a kinetic study.

A mathematical study of the degradation of human fibrinogen by the action of plasmin is proposed by means of the variation of number of moles of X,Y,D and E fragments with incubation time, starting from the qualitative and quantitative study of each component in the digestion mixture by electrophoresis in 5% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate. From this study the existence of two moles of D fragment and one mole of E fragment for each mole of fibrinogen is determined, confirming the scheme suggested for the degradation of fibrinogen, in accordance with a symmetrical structure for the molecule.     

The effect of streptokinase on the fibrin binding site of plasmin(ogen)

When streptokinase binds to the light chain region of plasmin(ogen) it induces a change in the fibrin binding site of the heavy chain region such that the affinity of the complex for fibrin is increased.