Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

Hereditary dysfibrinogenemia.

Inherited qualitative abnormalities of fibrinogen have been documented in more than 100 families. These dysfibrinogenemias usually are clinically silent, but in some cases are associated with bleeding, thrombosis, or defective wound healing. Abnormalities of the fibrinogen molecule may impair any of the major steps involved in the conversion of fibrinogen into stabilized fibrin; i.e., cleavage of the fibrinopeptides by thrombin, polymerization, and cross-linking of fibrin. Biochemical studies of several abnormal fibrinogens have demonstrated that the functional defects are the result of single amino acid substitutions. The hereditary dysfibrinogenemias are the first coagulation disorder in which the pathophysiology has been elucidated on a molecular level. Studies of these "experiments of nature" have important implications in such diverse processes as wound healing and thrombosis.

Stabilized Fibrin Urea-Dispersion In Normal Plasma And Serum

Clots from human normal native platelet-poor-plasma are dispersed by the addition of urea, final concentration 2.5 M. Cross-linked fibrin, preincubated in undiluted plasma and serum, is also dispersed by urea. Clot-dispersion is not inhibited by EACA, added to plasma prior to clotting or preincubation, at the final concentration of 5×10-2 M which inhibits streptokinase-plasminogen activation in the same plasma sample.Characterization of this activity has been carried out by the following procedures: 1. Ammonium-suiphate-fractionation. The activity is recovered in the material precipitated at 13-17% concentration. 2. Sephadex chromatography (G 200, 150, 100). The activity is recovered in the protein material with 150.000-300.000 daltons, eluated at pH 7.2. 3. Dialysis of the ammonium-sulphate-precipitate against a solution buffered with TRIS or phosphate 0.1 M, pH range b-8.5 at 4°C. The activity is recovered in the gelatinous material selectively precipitated at pH 7.2. 4. Standard preparative electrophocusing on Sephadex plate (Ultrodex LKB). The activity is recovered only in some of the thirty protein-fractions.EACA does not inhibit the fibrin dispersing activity of these different materials.These results indicate the presence in normal plasma and serum of a fibrin-dispersing activity different from plasminogen.

Study of the Normal Serum Activity Sensitizing Stabilized Fibrin to Urea Dispersion by Sephadex G. 200 Chromatography

Clots from human normal native platelet-poor plasma are dispersed by the addition of urea at a final concentration of 2.5 M. The same clots, washed in buffered saline (pH 7.4), are not dispersed by urea, but are rendered susceptible to its dispersing action by prior incubation in normal undiluted plasma and serum or 33% ammonium sulphate fractions. The serum fraction was dissolved in and dialyzed against pH. 7.4 phosphate-buffered saline and analyzed by Sephadex G. 200 chromatography. Two peaks were obtained; the relative materials were dialyzed against pH. 7.4 phosphate-buffered distilled water, lyophilized and dissolved in saline. The activity sensitizing the stabilized fibrin to urea dispersion was recovered in the second peak material.

Fibrin and Fibrinogen Proteolysis Products: Comparison between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis

SummaryThe proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the “double D” cross-linked form.

Fibrin and Fibrinogen Proteolysis Products: Comparison Between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS Polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS Polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400, 000 to 800, 000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and non-stabilized fibrin in that fragment D was present in the “double D” cross-linked form.