[Functional and diagnostic significance of A-, Bbeta 1-42 and Bbeta 15-42 peptide fragments of fibrin(ogen)]. / O funktsional'nom i diagnosticheskom znechenii A-, Bbeta 1-42 i Bbeta 15-42 peptidnykh fragmentov fibrin(ogen)a.

A scheme of in vitro formation and hydrolysis of the fibrin clot is suggested. This scheme considers the data concerning a cofactor role of fibrin in activation of polymerization and fibrinolysis. Such parameters of a number of components as concentration of A-, B beta 1-42-and B beta 15-42 peptides and of other fragments of fibrinogen which allow characterizing a state of hyperfibrinolysis, hyperclotting or dynamic equilibrium of these systems are selected in the scheme.

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.

Role of fibrinopeptide B in early atherosclerotic lesion formation.

The development of atherosclerotic lesions involves many cell types, including macrophages. Fibrinopeptide B (FPB) has been shown to be a potent chemotactic agent for macrophages, which are abundant as intimal foam cells in atherosclerotic lesions, especially in cholesterol-fed rabbits. We hypothesize that intimal low-density lipoproteins also cause fibrinogen in the intima to release FPB and that FPB attracts macrophages in response to the high lipid levels associated with lesion development. To test our hypothesis, we used an atherosclerotic model. Silk sutures containing either FPB, fibrinopeptide A (FPA), lipopolysaccharide (LPS), or saline control were prepared. One suture of each type was placed in the adventitia of the femoral artery of a rabbit. Animals were killed at 1 or 2 weeks. Only vessels exposed to either FPB or LPS showed significant intimal thickening in the region adjacent to the suture site. Semi-thin electron microscopic sections indicated that the intimal wall was highly cellular and that many cells contained lipid vacuoles after 2 weeks. These sections also showed that the endothelium remained intact and that no injury to the media of the artery had occurred. Electron microscopy of the tissue samples showed the proliferation of smooth muscle cells and deposition of extracellular matrix in the 2-week animals, whereas foam cells were present in the 1-week animals. We conclude that FPB does indeed attract macrophages to the intima and that these macrophages may become foam cells. The model we have developed can be used to study possible mechanisms for the entry of macrophages into the intima during early lesion development and to further understand the complex interactions of FPB, fibrinogen, and lipids in atherosclerotic lesion development.

Growth factors for human fibroblasts in the solute remaining after clot formation.

Fibroblasts adhere to, and readily grow into, fibrin clots that form as a result of the cleavage of fibrinogen by thrombin. Subsequent fibroblast replication is believed to be stimulated by mitogens released by entrapped platelets, such as platelet-derived growth factor. We suggest that the supernatant remaining after the fibrinogen-thrombin reaction could stimulate fibroblast replication, even in the absence of other blood components. To examine this hypothesis we expressed liquid from a fibrin clot and measured its mitogenic activity on human lung fibroblasts, in serum-free conditions, using a colorimetric assay based on uptake and subsequent release of Methylene Blue. The clot supernatant caused a mitogenic response of 51 +/- 6% above control and was equivalent to about half that elicited by medium containing 10% newborn calf serum. On their own, both thrombin and fibrinopeptides A and B (small molecular weight cleavage products released from fibrinogen) showed some mitogenic activity, but there was also activity in higher molecular weight cleavage products, suggesting the presence of uncharacterized mitogens. It is proposed that these agents may play important roles in wound healing and diseases associated with vascular leakage and fibrosis, by stimulating fibroblast replication.

[Thrombin-fibrinogen interaction: release of fibrinopeptides and the effects of ATP]. / Interazione trombina-fibrinogeno: rilascio dei fibrinopeptidi ed effetto dell'ATP.

This study presents experimental data on the time dependence of the release of fibrinopeptides A and B following the interaction between thrombin and fibrinogen. It is found that the release of fibrinopeptide A is fast as compared to that of fibrinopeptide B. This process is modulated by ATP in a complex fashion. In fact, the release of fibrinopeptide A is first enhanced at low (less than 1 mM) ATP concentrations and then progressively inhibited at ATP concentrations greater than 1 mM. These results draw attention to a possible model for the in vivo modulation of thrombin activity by ATP.

Fibrin induces release of von Willebrand factor from endothelial cells.

Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.

The release of small amounts of fibrinopeptide-B (FPB) is of critical importance for the thrombin clotting time.

When fibrinogen is clotted with thrombin, mainly fibrinopeptide-A (FPA) is released at gel point, the FPB-release being delayed. The present study present evidence that the small amounts of des-AABB fibrin found at visible gelation improves polymerization, thereby determining the thrombin clotting time. 1. Purified fibrinogen was clotted with thrombin or Reptilase, peptide release monitored radioimmunologically. With thrombin, significantly less fibrin (released FPA) was necessary for gel formation than when Reptilase was used. Furthermore, approximately 10% of the available FPB had been released by thrombin at gel point. 2. Polymerization of monomers prepared by incubation of fibrinogen with Reptilase (des-AA) and thrombin (des-AABB) in urea, were examined by light scattering. Des-AA fibrin monomers polymerised after a lag-phase of 4 minutes. When 20% of these monomers were substituted with des-AABB monomers, the lag was reduced to 1 1/2 minutes. 3. Finally, polymerization of preformed des-AA monomers was studied with and without the addition of small amounts of thrombin, and the FPB-release monitored. The presence of 0.04 NIH U/ml of thrombin reduced the lag from 10 minutes to 3 minutes. At this time approximately 10% of FPB had been released. It is concluded that even small amounts of des-AABB fibrin substantially improves polymerization of des-AA fibrin.

Kinetic characterization of a saturable pathway for rapid clearance of circulating fibrin monomer.

The mechanism of clearance of circulating fibrin monomer was investigated in rabbits through (1) study of decay in plasma concentrations of 125I-labeled monomers with variant fibrinopeptide content and (2) concurrent analysis of decay of the monomers relative to coinjected 131I-fibrinogen. Under the conditions employed, essentially all of the fibrin became distributed in a soluble form in plasma and decayed independently of the coinjected fibrinogen. Among the species of fibrin studied, monomer lacking fibrinopeptide A alone (alpha-fibrin) underwent very rapid clearance by a saturable mechanism that was not evident in relatively sluggish clearance of monomer lacking either fibrinopeptide B alone (beta-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin). Decay of alpha-fibrin conformed with a kinetic mechanism involving first-order permeation of the fibrin into extravascular space at a rate equivalent to that of permeation of fibrinogen; unlike fibrinogen, however, the alpha-fibrin underwent immediate absorption in parallel with permeation (t1/2 = 2.6 hours) at doses below an apparent saturating level of 3 mg/kg. At doses near the absorptive limit, the uptake accompanying permeation diminished as in a second-order kinetic mechanism, and at very high doses the plasma decay of the alpha-fibrin approached that of fibrinogen. The beta- and alpha beta-fibrins also permeated extravascular space in parallel with fibrinogen, but absorption proceeded sluggishly (t1/2 = 11 and 16 hours, respectively) at low doses and did not change with increasing dose. The uniquely rapid and saturable clearance of alpha-fibrin is suggested to involve uptake through the fibrin aggregation site that is blocked by fibrinopeptide A in fibrinogen and beta-fibrin and by tight binding to fibrinogen in soluble complexes formed by alpha beta-fibrin. A corollary of this hypothesis is that rapid uptake depends on dissociability of fibrin complexes for access to the aggregation site, a mechanism that is just the converse of uptake through aggregation.

Fibrinogen Baltimore II: congenital hypodysfibrinogenemia with delayed release of fibrinopeptide B and decreased rate of fibrinogen synthesis.

A congenital hypodysfibrinogenemia, fibrinogen Baltimore II, was found in a young asymptomatic Caucasian female. Prothrombin, partial thromboplastin, and euglobulin lysis times were normal, as were platelet function and coagulation factor assays. Subnormal plasma fibrinogen levels were found using chronometric, rate-independent, and immunologic assay methods. Kinetic analysis of fibrinopeptide release revealed a delay in the thrombin-catalyzed release of fibrinopeptide B from the abnormal protein. Proteolysis of fibrinopeptide A by thrombin or Arvin, fibrin monomer polymerization, and fibrin polymer ligation occurred at normal rates. Catabolism of radiolabeled autologous and homologous fibrinogen was also normal, but the fibrinogen synthetic rate was less than half the normal value. Comparison of the coagulation characteristics of fibrinogen Baltimore II with those of other abnormal fibrinogens indicates that it represents a unique example of hypodysfibrinogenemia.

Molecular basis for measurement of circulating fibrinogen derivatives.

Fibrinogen plays a pivotal role in both the humoral and cellular mechanisms involved in hemostasis. In performing its hemostatic function, fibrinogen in turn is acted on by several independent enzyme systems that either modify its structure or cleave specific fragments of the molecule into the surrounding milieu. Measurements of enzymatically modified fibrinogen or its proteolysis products represent a means whereby the action of these specific enzymes can be quantitated both in vitro and in vivo. Advances in such techniques as protein purification, affinity chromatography, peptide synthesis, and radioimmunoassay technology permit the translation of recently acquired primary structural data on this important protein into sensitive and specific assays for its circulating derivatives. These assay systems are important tools for probing mechanisms of hemostasis and thrombosis.

Chemotaxis for human monocytes by fibrinogen-derived peptides.

Chemotactic activity for human peripheral blood monocytes was generated by the action of thrombin on human fibrinogen, a reaction known to release low molecular fibrinopeptides. Supernatants prepared by incubating Contortrix venom with fibrinogen, which cleaves the B peptide, were also chemotactic, whereas no activity was present in supernatants from Arvin venom, which cleaves peptides A, AP and AY. Thus fibrinopeptide B was chemotactic for the monocyte in addition to the neutrophil, as previously reported. Monocyte chemotactic activity was also generated by the action of plasmin on human fibrinogen and shown to be associated with the D and E fragments but not with a mixture of fragments X and Y. When plasmin digestion was stopped at time intervals up to 24 h, monocyte chemotactic activity corresponded with the appearance of the D and E fragments. The monocyte chemotactic activity, contained in a 24 h digest, eluted from Sephadex G-75 at VO, corresponding to the expected position of the D and E fragments whereas neutrophil chemotactic activity eluted with molecules of molecular size of approximately 30 000 daltons. Thus fragments D and E derived from plasmin digestion of fibrinogen attract the monocyte whereas only the small uncharacterized peptides were chemotactic for the neutrophil. These different profiles of chemotactic activity for the neutrophil and the monocyte in terms of plasmin digestion products of fibrinogen may be of significance in the events leading to the accumulation of these cells in vivo during fibrin deposition.