Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.     

New strategies in the determination of fibrin and fibrin(ogen) derivatives by monoclonal antibodies

Until recently only tests with a limited specificity were available for the assessment of the products of activated coagulation and/or fibrinolysis. Those assays were based on polyclonal antibodies, which crossreact with fibrinogen, and as a consequence they were performed on serum samples i.e. after removal of fibrinogen by clotting. Serum preparation, however, is a notorious source of artefactually high or low levels of fibrin(ogen) degradation products, and is not suitable for the determination of coagulation products. Recently, highly specific monoclonal antibodies (MoAb's) have been developed, the majority of which do not crossreact with fibrinogen. This has enabled new strategies to be developed, i.e. assays using these MoAb's on plasma samples. Furthermore, the new assays can discriminate between (individual) fibrin and fibrinogen degradation products, and coagulation products can be assessed in the same plasma samples.     

Kinetics of in vitro fibrinolysis by plasmin and a plasmin-streptokinase equimolar complex]

Kinetics of fibrinolysis by plasmin and plasmin streptokinase complex have been studied using fibrin gels formed from purified fibrin and human blood plasma. The gels were placed into buffer or blood plasma. The contributions of plasminogen and alpha 2-antiplasmin present or absent in both phases to the kinetics of fibrinolysis were quantitatively estimated. In the complex catalyzed fibrinolysis, plasminogen activation reaction dominated whereas in plasmin-catalyzed fibrinolysis, the inhibitor involved reaction, suppressing the process, prevailed.     

Babesia bovis (argentina): analysis of fibrinogen-like proteins during infection

Fibrinogen and fibrinogen-like proteins (FLP) were isolated from plasma and serum of cattle acutely infected with Babesia bovis. The sizes and chain structures of these proteins were examined and clotting assays performed. The results indicated that the blood was in a hypercoagulable state due mainly to enhanced production of hydrogen bonded fibrin and offset partly by slight inhibition of chain cross-linking. The latter appeared due to a Factor XIII inhibitor. Reduction of A alpha chains of plasma FLP was not evident, nor could lower molecular weight remnants be regularly detected strongly suggesting that fibrin(ogen) lysis rarely occurred. Similarly the size and chain structure of the majority of noncoagulable FLP of serum was consistent with their being the product of coagulation and not fibrinolysis. Only in heavily infected splenectomized cattle were products from lysed cross-linked fibrin detected and these constituted only about 3% of total serum FLP.     

Measuring the mechanical properties of blood clots formed via the tissue factor pathway of coagulation

Thrombelastography (TEG) is a method that is used to conduct global assays that monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (three times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI, and fibrinolysis was induced by adding tissue plasminogen activator (tPA). Changes in viscoelasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93 ± 0.23 min and in plasma in 5.12 ± 0.23 min (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intraassay variability (CVa<10%) for R time, angle, and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Effects of clot contraction on clot degradation: A mathematical and experimental approach

Thrombosis, resulting in occlusive blood clots, blocks blood flow to downstream organs and causes life-threatening conditions such as heart attacks and strokes. The administration of tissue plasminogen activator (t-PA), which drives the enzymatic degradation (fibrinolysis) of these blood clots, is a treatment for thrombotic conditions, but the use of these therapeutics is often limited due to the time-dependent nature of treatment and their limited success. We have shown that clot contraction, which is altered in prothrombotic conditions, influences the efficacy of fibrinolysis. Clot contraction results in the volume shrinkage of blood clots, with the redistribution and densification of fibrin and platelets on the exterior of the clot and red blood cells in the interior. Understanding how these key structural changes influence fibrinolysis can lead to improved diagnostics and patient care. We used a combination of mathematical modeling and experimental methodologies to characterize the process of exogenous delivery of t-PA (external fibrinolysis). A three-dimensional (3D) stochastic, multiscale model of external fibrinolysis was used to determine how the structural changes that occur during the process of clot contraction influence the mechanism(s) of fibrinolysis. Experiments were performed based on modeling predictions using pooled human plasma and the external delivery of t-PA to initiate lysis. Analysis of fibrinolysis simulations and experiments indicate that fibrin densification makes the most significant contribution to the rate of fibrinolysis compared with the distribution of components and degree of compaction (p < 0.0001). This result suggests the possibility of a certain fibrin density threshold above which t-PA effective diffusion is limited. From a clinical perspective, this information can be used to improve on current therapeutics by optimizing timing and delivery of lysis agents.     

Alpha 2-plasmin inhibitor inactivation by human granulocyte elastase

Plasminogen-binding human alpha 2-plasmin inhibitor is converted by human granulocyte elastase into its non-plasminogen-binding and finally into the inactive form of the inhibitor. This degradation of the plasmin inhibitor, described earlier as "spontaneously" occurring conversion, is shown in dodecyl sulfate polyacrylamide gel electrophoresis, in two-dimensional immunoelectrophoresis and by measuring the kinetics of plasmin inhibition. Experiments in the presence of normal human plasma required unphysiologically high concentrations of elastase to inactivate alpha 2-plasmin inhibitor, suggesting a role of elastase in this type of indirect fibrinolysis in a microenvironment only and not in systemic events.     

Pharmacologic influence on secondary generalized fibrinolysis

Secondary generalized hyperfibrinolysis was induced by thrombin infusion or batroxobin injection in rats. To follow intravascular fibrinolysis quantitatively, an electroimmuno-assay was used for determination of the fibrin degradation products formed. Anticoagulants (heparin, hirudin), antifibrinolytics (EACA, PAMBA, AMCA), and synthetic (APPA) and naturally occurring (aprotinin) protease inhibitors were studied with regard to their influence on secondary fibrinolysis. The potency and duration of action of the antifibrinolytics tested correspond to their antifibrinolytic activity measured in vitro and to their pharmacokinetics. Formation of degradation products is initiated after the appearance of fibrin monomer or fibrin, respectively. Due to their antithrombin action heparin, hirudin, and APPA prevent the thrombin-induced fibrin formation and thus the induction of secondary fibrinolysis. In contrast, formation of fibrin monomers caused by batroxobin is not influenced by thrombin inhibitors so that in this case formation of degradation products is not prevented.     

Usefulness of human coagulation and fibrinolysis assays in domestic pigs

Pigs are often used as animal models in research on blood coagulation and fibrinolysis. The usefulness of the assays applied within this field, and the knowledge of reference intervals are therefore essential and of utmost importance. In the study reported here, we investigated the applicability of commercial human coagulation and fibrinolysis assays for use with porcine plasma. In total, 22 functional and immunologic assays were applied to plasma obtained from domestic pigs, and the following blood coagulation and fibrinolysis variables were measured: prothrombin time, activated partial thromboplastin time, tissue factor, tissue factor pathway inhibitor, factor VII, protein C, protein S, prothrombin fragment 1+2, antithrombin, thrombin-antithrombin complexes, fibrinogen, soluble fibrin, urokinase-type plasminogen activator, plasmin inhibitor, plasminogen activator inhibitor 1, and D-dimer. We found that 11 of 12 functional assays, but only 3 of 10 immunoassays, were applicable to porcine plasma, and we determined the normal range of these variables. We conclude that human functional assays are useful in porcine plasma, whereas only a few immunologic assays can be used. However, precautions must be taken in interpretation of the results and in extrapolation toward human results because possible differences between porcine and human values can be due to species variations and/or methodologic errors.     

Reversible cross-linking of alpha 2-plasmin inhibitor to fibrinogen by fibrin-stabilizing factor

alpha 2-Plasmin inhibitor, a primary inhibitor of fibrinolysis, is cross-linked to fibrin by plasma transglutaminase (glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13, activated fibrin-stabilizing factor) when blood coagulation takes place. alpha 2-Plasmin inhibitor was found also to be cross-linked to fibrinogen by plasma transglutaminase. The inhibitor was corss-linked exclusively to the A alpha-chain of fibrinogen, and the cross-linking reaction proceeded very rapidly. The reaction was almost completed before the formation of the gamma-chain dimers of fibrinogen which precedes cross-linking polymerization of the A alpha-chain of fibrinogen. The maximum level of inhibitor cross-linking achieved was approx. 30% of the inhibitor present at the start of the reaction. The level of cross-linking of the inhibitor was not changed when the cross-linking reaction was preceded by dimerization of fibrinogen. The cross-linking reaction was found to be a reversible one, since the cross-linked complex of the inhibitor and fibrinogen was partly dissociated to each of its components when the complex was incubated with plasma transglutaminase. These results suggest that the self-limiting nature of the cross-linking reaction between alpha 2-plasmin inhibitor and fibrin(ogen) is due to the reaction equilibrium favoring dissociation of the complex, and not due to the development of structural hindrance in polymerizing fibrin(ogen).     

Menstrual Blood Loss and Fibrin Degradation Products

As part of a large-scale study of menstrual blood loss in the community serum fibrin degradation products were measured soon after menstruation in 331 women. No significant correlation was found between the amount of blood lost and the serum level of fibrin degradation products. These findings conflict with reports suggesting that excessive intrauterine fibrinolysis, which may play a part in menorrhagia, is associated with raised serum F.D.P. concentrations.     

Coagulation and Fibrinolytic Systems in Pre-eclampsia and Eclampsia

The coagulation and fibrinolytic mechanisms were investigated in a group of patients with severe pre-eclampsia and eclampsia and the findings were compared with those of healthy women in late pregnancy. In patients with pre-eclampsia the following significant differences were found: (1) greater depression of plasma fibrinolytic activity (euglobulin lysis time) than in normal pregnancy, (2) a higher level of inhibitor to urokinaseinduced lysis, (3) increased levels of serum fibrin degradation products, and (4) reduced platelet counts.In patients with eclampsia a progressive increase of the level of serum fibrin degradation products was found over the three days following eclamptic seizures. No such increase occurred after grand mal seizures in late pregnancy. The findings in this study support the view that intravascular clotting is taking place in pre-eclampsia and that this disturbance of the balance between coagulation and fibrinolysis may be localized to certain areas of the vascular compartment, particularly the placental and renal circulations. Fibrin deposition in the maternal vessels supplying the placenta would impair the placental blood flow, which may explain the placental insufficiency which occurs in pre-eclampsia. Likewise fibrin deposition in the renal vasculature will result in glomerular damage and proteinuria. Hypertension may be related to the renal ischaemic changes or a compensatory response to the presence of fibrin deposition in the vascular compartment. This evidence of intravascular fibrin deposition raises the question of the possible therapeutic value of antithrombotic agents to inhibit the clotting process. On a theoretical basis such treatment might be expected to improve blood flow to the placenta and thereby fetal growth.     

Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis

Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products under conditions closely resembling those in vivo. Plasma samples from 15 patients with arterial hypertension have been investigated. Coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by the clot turbidity assay. The coagulation/fibrinolysis cycle was accompanied by a significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated with the degree of increase of these amino acids: r _S = −0.733 and −0.761 for arginine and lysine, respectively (p < 0.05). Arginine generation had two maximums: one in the beginning of clot lysis and another one at the end of the lysis, whereas the lysine release occurred mainly in the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential amino acids originated from fibrin clot degradation products.     

Matrix metalloproteinases collagenase-2, macrophage elastase, collagenase-3, and membrane type 1-matrix metalloproteinase impair clotting by degradation of fibrinogen and factor XII

The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity.     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

Fibrinolysis in Cholestatic Jaundice

The fibrinolytic system was studied in primary biliary cirrhosis (16 patients) and large bile duct obstruction (10 patients, nine of whom had carcinoma). Plasma fibrinolysis (plasminogen activator activity) was decreased and fibrinogen increased in both groups of patients, particularly in those with large duct obstruction. These changes were related to the degree of cholestasis. Plasminogen activator activity was inversely related to serum triglyceride levels in patients with primary biliary cirrhosis. Urokinase inhibitors were decreased in both groups and antiplasmins increased in patients with large duct obstruction; fibrin/fibrinogen degradation products were normal in primary biliary cirrhosis and moderately increased in large duct obstruction. None of these fibrinolytic indices was related to the degree of cholestasis. Fibrinolytic activity and fibrinogen returned almost to normal levels after palliative surgery in the three patients with large duct obstruction who were studied. The decreased plasma fibrinolysis and increased fibrinogen may be due to altered lipid metabolism in cholestatic jaundice. In patients undergoing surgery for large duct obstruction there may be an increased risk of thrombosis.     

Hemostatic and fibrinolytic parameters in patients with acute myeloid leukemia: activation of blood coagulation, fibrinolysis and unspecific proteolysis

Blood coagulation, fibrinolytic and unspecific proteolytic parameters were investigated in 34 patients with acute myeloid leukemia. An increased activity of the coagulation system, documented by elevated thrombin-antithrombin III-complex (TAT) plasma levels, was found in 91% of the patients; 50% had increased elastase plasma levels. Hyperfibrinolysis, as shown by elevated fibrin split-product D-Dimer plasma levels, was detected in 91% of AML patients. Activation of these enzyme systems was not associated with relevant defects in blood coagulation or fibrinolysis in the majority of the patients investigated. In selected cases of promyelocytic M3 and monoblastic M5 leukemia, however, hypofibrinogenemia and alpha 2-plasmininhibitor deficiency was found, most likely due to depletion of these proteins in the course of disseminated intravascular coagulation and secondary hyperfibrinolysis. Significant correlations were calculated between TAT and fibrinogen (r = -0.57, P less than 0.005), TAT and D-Dimer (r = 0.89, P less than 0.0005), and D-Dimer and alpha 2-plasmininhibitor (r = -0.77, P less than 0.0005) levels. Indications of a pathogenetic importance of primary hyperfibrinolysis or unspecific proteolysis for hypofibrinogenemia and alpha 2-PI deficiency were not found.