Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Synthetic peptide derivatives that bind to fibrinogen and prevent the polymerization of fibrin monomers

A series of small peptides corresponding to the amino termini of the fibrin alpha- and beta-chains has been synthesized. The peptides glycyl-L-prolyl-L-arginyl-L-proline and glycyl-L-prolyl-L-arginylsarcosine are potent inhibitors of fibrin polymerization. Moreover, these peptides have a natural stability stemming from their inherent resistance to proteolysis because of the involvement of amino acids in each of their peptide bonds. The peptide glycyl-L-prolyl-L-arginyl-L-proline binds to fibrinogen and to fragment D, in both cases with an association constant of approximately 5 x 10(4); it does not bind to fragment E. The number of binding sites is two for fibrinogen and one for fragment D. The tripeptide glycyl-L-prolyl-L-arginine binds less tightly and is less than half as effective in preventing polymerization. The peptide glycyl-L-histidyl-L-arginyl-L-proline, which corresponds exactly to the amino terminus of the fibrin beta-chain, does not inhibit the aggregation of fibrin monomers under the conditions used. It does bind weakly to fibrinogen, however, suggesting the involvement of sites other than those binding the alpha-chain analogues. Various other peptides were found not to inhibit polymerization; these included glycine-L-proline, L-prolyl-L-arginine and glycyl-L-prolyl-L-seryl-L-proline. The last-named corresponds to the serine/arginine amino acid replacement previously reported for a defective human fibrinogen.     

Analysis of fibrin formation and proteolysis during intravenous administration of ancrod

Ancrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of acute ischemic stroke. Treatment with ancrod leads to fibrinogen depletion. The present study investigated the mechanisms leading to the reduction of plasma fibrinogen concentration. Twelve healthy volunteers received an intravenous infusion of 0.17 U/kg body weight of ancrod for 6 hours. Blood samples were drawn and analyzed before and at various time points until 72 hours after start of infusion. Ancrod releases fibrinopeptide A from fibrinogen, leading to the formation of desAA-fibrin monomer. In addition, a considerable proportion of desA-profibrin is formed. Production of desA-profibrin is highest at low concentrations of ancrod, whereas desA-profibrin is rapidly converted to desAA-fibrin at higher concentrations of ancrod. Both desA-profibrin and desAA-fibrin monomers form fibrin complexes. A certain proportion of complexes carries exposed fibrin polymerization sites E(A), indicating that the terminal component of the protofibril is a desAA-fibrin monomer unit. Soluble fibrin complexes potentiate tissue-type plasminogen activator-induced plasminogen activation. Significant amounts of plasmin are formed when soluble fibrin in plasma reaches a threshold concentration, leading to the proteolytic degradation of fibrinogen and fibrin. In the present setting, high concentrations of soluble fibrin are detected after 1 hour of ancrod infusion, whereas a rise in fibrinogen and fibrin degradation products, and plasmin-alpha(2)-plasmin inhibitor complex levels is first detected after 2 hours of ancrod infusion. Ancrod treatment also results in the appearance of cross-inked fibrin degradation product D-dimer in plasma. (Blood. 2000;96:2793-2802)     

The fibrinolytic response to ancrod therapy: characterization of fibrinogen and fibrin degradation products

Ancrod is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, α₂ antiplasmin (α₂AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4–3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8–3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100–155)% to 79 (40–118)% at 49 h and α₂AP fell from 91 (75–107)% to 24 (10–35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.     

The comparative ultrastructure of fibrin induced by thrombin and by staphylocoagulase.

Fibrin induced by the action of thrombin and by staphylocoagulase was studied by transmission electron microscopy. Periodic striations were consistently observed in the negatively stained preparations of both fibrins. When 4200 major periods in the thrombin fibrin system were measured the mean length was 228 A. For 3666 major periods in the coagulase fibrin system the mean length was 223 A. While the T test analysis of these values gave a value of 10, it is noteworthy that the differences are well within the scatter of periodicity reported in the literature for thrombin-induced fibrin. Gross inspection of the preparations indicated that the coagulase-induced fibrin had a knottier appearance and was accompanied by a greater amount of background debris than the thrombin-induced fibrin.     

Polymerisation and crosslinking of fibrin monomers in diabetes mellitus

Summary Polymerisation and crosslinking of fibrin monomers was studied in 35 healthy volunteers and in 42 poorly controlled diabetic patients. Polymerisation did not show any difference between control subjects (n = 10) and diabetic patients (n = 11) (p>0.1), although fibrinogen was 35% more glycated in the diabetic patients (p<0.001). Alpha chain crosslinking in the diabetic patients, however, was impaired as is shown from an increase in intermediate alpha polymers with a concomitant decrease in alpha monomer disappearance. A significant positive correlation was found between the degree of glycation of fibrinogen and the defective alpha chain polymerisation (r = 0.86, p<0.005). These results were consistent with the results of thrombin and reptilase experiments. The reaction rate with reptilase did not show any difference between the two groups (p>0.1), whereas the reaction rate with thrombin was significantly slower in the diabetic group compared to the control subjects (p<0.001). Purified fibrin clots obtained from the diabetic patients were more susceptible to plasmin than clots obtained from control subjects. It is concluded that in poorly controlled diabetic patients polymerisation of fibrin monomers is normal, but crosslinking of the alpha chains is impaired, leading to a higher susceptibility of the clots to plasmin degradation.     

The procoagulant effect of thrombin on fibrin(ogen)-bound platelets.

In a final stage of activation, platelets become procoagulant because of the appearance of phosphatidylserine (PS) at the membrane outer surface. This PS exposure requires a rise in cytosolic [Ca(2+)](i), is accompanied by formation of membrane blebs, and stimulates the formation of thrombin from its precursor prothrombin. Here, we investigated whether thrombin, as a potent platelet agonist, can induce this procoagulant response in plasma-free platelets interacting with fibrin or fibrinogen through their integrin alpha(IIb)beta(3) receptors. First, in platelets that were stimulated to spread over fibrin or fibrinogen surfaces with adrenaline, addition of thrombin and CaCl(2) caused a potent Ca(2+) signal that in about 30% of the cells was accompanied by exposure of PS. At low doses, integrin alpha(IIb)beta(3) receptor antagonist (RGD peptide) inhibited platelet spreading as well as thrombin-evoked PS exposure. Second, in platelet-fibrinogen microaggregates that were preformed in the presence of adrenaline, thrombin/CaCl(2) induced PS exposure and bleb formation of about 35% of the cells. Third, a potent, thrombin-dependent stimulation of prothrombinase activity was measured in platelet suspensions that were incubated with a fibrin clot. These results indicate that, in the absence of coagulating plasma, thrombin is a moderate inducer of the procoagulant response of platelets, once integrin alpha(IIb)beta(3)-mediated interactions are stimulated (by adrenaline) and CaCl(2) is present.     

The acceleratory effect of thrombin on fibrin clot assembly

Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme:substrate ratio, 1:400 and 1:800) prior to the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin/fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system. Lowering the ionic strength from 0.14 to 0.09 amplified these differences. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions as a cofactor in the fibrin assembly process.     

Effects of divalent cations on the conversion of fibrinogen to fibrin and fibrin polymerization

Effects of divalent cations on fibrinogen and its reaction with thrombin were re-examined and correlated to improve definition of mechanisms underlying the acceleration of clot formation by the ions. The rate of release of fibrinopeptides from fibrinogen was not affected by any of the specific ions studied, but increased rates of fibrin monomer polymerization were obtained with all but magnesium ions. Maximal acceleration of monomer polymerization was observed with the divalent ions at concentration of 2.5–5 mM, and no added specific ion effects were observed with much higher levels. The acceleratory ions were found to have a corresponding effect on the solubility of fibrinogen, as judged from acceleration of its precipitation at low temperature. Although magnesium ions had no effect on fibrin monomer polymerization, they did have a low ranking effect on cryoprecipitation. These data confirm that the principal effect of divalent cations in the fibrinogen-fibrin transformation is to accelerate fibrin monomer polymerization, and suggest that the acceleration is associated with effects on the solubility of fibrinogen.     

Idiopathic autoantibody that inhibits fibrin monomer polymerization

S ummary . A 73-year-old female was found to have prolonged thrombin and reptilase times in the immediate post-operative period. These abnormalities were not corrected by the addition of normal plasma. They were subsequently shown to be due to an IgG immunoglobulin which inhibited fibrin monomer polymerization. The IgG immunoglobulin activity could be neutralized completely by prior incubation with either patient or normal fibrinogen, uncrosslinked fibrin monomers or IgG antisera. No inhibitory effect on thrombin activity, fibrinopeptide A release or on the fibrin cross-linking reaction of factor XIIIa could be detected. Purified patient fibrinogen was functionally normal as demonstrated by normal fibrinogen–fibrin polymerization and fibrinopeptide A release. No underlying cause for this phenomenon was found. The presence of the inhibitor was associated with excessive blood loss during the post-operative period.     

Localizaton of fibrin converted by thrombin from released platelet fibrinogen

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane "ghosts" for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII- detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.     

Qualitative changes in fibrinogen following exposure to agents used for preparation of fibrin monomers.

The solubility, clottability, thrombin clotting time and agarose gel chromatography pattern of human fibrinogen were studied after exposure to solvents commonly used to prepare fibrin monomers (0.0167 M acetic acid with 2.5 mM EDTA; 1 M NaBr, pH 5.2; 3.3 M urea, pH 7.4; 5 M urea, pH 7.4). When exposed to acetic acid at room temperature, fibrinogen precipitated almost immediately and quantitatively. Subsequent dialysis for 72 h against 0.3 M NaCl, pH 7.4, caused resolution of fibrinogen to a varying degree, the amount depending on the time of exposure. The redissolved fibrinogen showed reduced clottability, markedly shortened thrombin clotting time and a chromatographic profile that indicated large amounts of aggregates. Fibrinogen exposed to 1 M NaBr, pH 5.2, at room temperature for 1 h showed a slightly shortened thrombin clotting time and a broadened chromatographic profile. Exposure for 24 h to the same agent resulted in reduced solubility and clottability, a prolonged thrombin clotting time and progressive broadening of the chromatographic profile. Similar findings were obtained with fibrinogen exposed to 5 M urea, pH 7.4. Exposure to 3.3 M urea at pH 7.4 for 24 h, room temperature, led only to a moderate increase in solubility.     

Evidence for four different polymerization sites involved in human fibrin formation.

The mechanism of association and the organization of human fibrin were studied by using affinity chromatography. Insolubilized fibrinogen, fibrin monomer, and crosslinked fibrin were used to localize the binding sites on fibrinogen and fibrin derivatives. Four different polymerization sites have been distinguished. A binding site ("a"), available without thrombin action, is present on the fibrinogen fragment D domain. The complementary ("A") is inoperative in fibrinogen and requires thrombin for activation; it is located on the fibrinogen NH2-terminal domain. A third polymerization site ("b") appears to be formed by the alignment of the fragment D domains on two fibrin monomer molecules upon polymerization; this site functions without thrombin mediation and the alignment is stabilized by the Factor XIIIa-catalyzed crosslink bonds. The "b" site is complementary to another thrombin-activated site ("B") on the fibrinogen NH2-terminal domain. The two thrombin activable sites, "A" and "B", are distinguishable, although they are located in the same fibrinogen domain.