A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.     

Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.     

Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.     

Human renal microvascular endothelial cells as a potential target in the development of the hemolytic uremic syndrome as related to fibrinolysis factor expression, in vitro

Renal glomerular microvascular endothelial cell damage is characteristic of Shiga toxin-associated hemolytic uremic syndrome (HUS). An impaired renal fibrinolysis may be responsible for renal microvascular fibrin accumulation during the course of HUS disease. This study examined the effect of Shiga toxin, bacterial lipopolysaccharide (LPS, endotoxin), and tumor necrosis factor (TNF) on the expression of fibrinolysis factors by human renal glomerular microvascular endothelial cells (HRMEC) in vitro. The results were compared to a previously better-characterized endothelial cell type, human umbilical vein endothelial cells (HUVEC). In HUVEC, the ratio of fibrinolysis antigens was antifibrinolytic, consisting of 55-fold more plasminogen activator inhibitor type 1 (PAI-1) than tissue-type plasminogen activator (tPA). Treatment of HUVEC with LPS or TNF accentuated this ratio by decreasing tPA and increasing PAI-1 expression. In contrast, HRMEC produced urokinase-type plasminogen activator (uPA) in a 24-fold excess to PAI-1 and were thereby profibrinolytic with regard to fibrinolysis antigen expression. LPS and TNF further decreased PAI-1 antigen expression by HRMEC. These results argue against a role for LPS or TNF in decreasing renal fibrinolysis at the level of fibrinolysis factor expression by renal endothelial cells. Nevertheless, HUVEC and HRMEC were responsive to the same LPS analogs in the same order of potency. Shiga toxin decreased fibrinolysis factor expression to a greater extent in HRMEC than in HUVEC. Since HRMEC fibrinolysis antigen expression was profibrinolytic, the Shiga toxin-mediated decrease in renal endothelial uPA synthesis may predispose renal microvasculature to thrombosis and may have implications for the development of HUS.     

Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator-induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.     

Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis

The thrombin thrombomodulin dependent activation of the plasma protein TAFI (Thrombin Activatable Fibrinolysis Inhibitor) and Subsequent Inhibition of Fibrinolysis by the TAFIa is described. Work to date indicates that TAFIa is a carboxypeptidase B enzyme that suppress fibrinolysis most likely by down regulating the cofactor functions of partially degraded fibrin. The existence of TAFI provides the explanation for the apparent profibrinolytic effect of activated protein C. and implies the existence of an explicit molecular connection between the blood coagulation of fibrinolytic cascades that is expressed through the thrombin thrombomodulin dependent activation of TAFI. Thus, thrombin generation can, in principle, result in the suppression of fibrinolysis.     

Involvement of protease inhibitors in staphylokinase-induced fibrin-specific fibrinolysis

We compared the fibrinolytic properties of recombinant staphylokinase (SAK), a fibrin-specific plasminogen activator, with those of streptokinase and tissue-type plasminogen activator (t-PA) by means of the amidolytic method. We also investigated the involvement of alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin in SAK-induced fibrin-specific fibrinolysis. Both SAK and t-PA activated plasminogen efficiently in the presence of fibrin in human plasma. Although t-PA activated plasminogen dependently on fibrin in the reconstituted plasma system, SAK activated plasminogen independently of fibrin without alpha 2-plasmin inhibitor (alpha 2-antiplasmin, alpha 2-PI). These findings suggest that fibrin and alpha 2-PI play important roles in plasminogen activation by SAK but not by t-PA. Furthermore, protease inhibitors such as alpha 2-PI, alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin inhibited plasminogen activation by SAK and the inhibitory actions of these protease inhibitors disappeared in the presence of fibrin. This shows that alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin, other than alpha 2-PI, contribute to the fibrin-specificity of SAK.     

Intraventricular and brachial artery thrombosis in nephrotic syndrome

PURPOSE: To investigate the influence of hyperthermia up to 45 degrees C on fibrinolysis with recombinant tissue-type plasminogen activator (rt-PA). METHODS: Standardized fibrin clots were incubated in a water bath for 5 hr with either rt-PA (test group) or 0.9% sodium chloride (control group) and blood plasma at temperatures of 30-45 degrees C. Concentrations of D-dimer and time to complete clot lysis were measured. RESULTS: The activity of fibrinolysis with rt-PA rose with increasing temperature: time to lysis approximately halved from 30 degrees C to 40 degrees C and the concentration of D-dimer tripled. In the control group clot size did not change. CONCLUSIONS: Activity of rt-PA-induced fibrinolysis rises distinctly with higher temperatures. Since even healthy subjects show a physiologic decline in body temperature in the extremities, in patients with occlusive arterial disease decreased activity of fibrinolysis with rt-PA can be expected. Controlled hyperthermia may improve fibrinolysis with rt-PA and should be investigated in vivo.     

Enhancement of fibrinolysis with 40-kHz ultrasound

BACKGROUND: Ultrasound at frequencies of 0.5 to 1 MHz and intensities of > or =0.5 W/cm2 accelerates enzymatic fibrinolysis in vitro and in some animal models, but unacceptable tissue heating can occur, and limited penetration would restrict application to superficial vessels. Tissue heating is less and penetration better at lower frequencies, but little information is available regarding the effect of lower-frequency ultrasound on enzymatic fibrinolysis. We therefore examined the effect of 40-kHz ultrasound on fibrinolysis, tissue penetration, and heating. METHODS AND RESULTS: 125I-fibrin-radiolabeled plasma clots in thin-walled tubes were overlaid with plasma containing tissue plasminogen activator (tPA) and exposed to ultrasound. Enzymatic fibrinolysis was measured as solubilization of radiolabel. Tissue attenuation and heating were examined in samples of porcine rib cage. Fibrinolysis was increased significantly in the presence of 40-kHz ultrasound at 0.25 W/cm2, reaching 39+/-7% and 93+/-11% at 60 minutes and 120 minutes, compared with 13+/-8% and 37+/-4% in the absence of ultrasound (P<0.0001). The acceleration of fibrinolysis increased at higher intensities. Attenuation of the ultrasound field was only 1.7+/-0.5 dB/cm through the intercostal space and 3.4+/-0.9 dB/cm through rib. Temperature increments in rib were <1 C/(W/cm2). CONCLUSIONS: These findings indicate that 40-kHz ultrasound significantly accelerates enzymatic fibrinolysis at intensities of > or =0.25 W/cm2 with excellent tissue penetration and minimal heating. Externally applied 40-kHz ultrasound at low intensities is a potentially useful therapeutic adjunct to enzymatic fibrinolysis with sufficient tissue penetration for both peripheral vascular and coronary applications.     

Annexin II tetramer inhibits plasmin-dependent fibrinolysis

In this paper, we have characterized the regulation of plasmin activity by annexin II tetramer (AIIt). Plasmin activity was measured by a fibrin lysis assay in which a fibrin polymer was produced from purified components and the extent of polymer lysis was determined by following changes in turbidity. Extrinsic lysis of the fibrin polymer, initiated by addition of tissue plasminogen activator (t-PA), was totally blocked if AIIt was present during fibrin polymer formation. Furthermore, fibrin polymer formed in the presence of AIIt was resistant to extrinsic lysis initiated by addition of plasmin. AIIt bound to fibrin polymer under conditions in which polymer lysis was inhibited. Plasmin-dependent extrinsic lysis of the fibrin polymer was also blocked if AIIt was present in the incubation medium, and under these conditions the amidolytic activity of plasmin, measured with an artificial substrate, was inhibited about 5-fold. In contrast, in the absence of fibrin, and at an AIIt/plasmin molar ratio of 526, the amidolytic activity of plasmin was inhibited by only 22.3% +/- 7.4% (mean +/- SD, n = 5) by AIIt. Plasmin-dependent fibrinolysis was only slightly inhibited if fibrin polymer was formed in the presence of annexins I, II, V, or VI. These results identify AIIt as an in vitro regulator of plasmin activity.     

The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.     

Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties

Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with kcat = 0.55 s-1, K(m) = 0.54 microM, and Kd = 6.0 nM for rTAFI and kcat = 0.61 s-1, K(m) = 0.55 microM, and Kd = 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37 degrees C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to approximately 62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (delta Ho++ approximately equal to 45 kcal/mol and delta So++ approximately equal to 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and epsilon-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.     

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.     

Endothelium, thrombosis and fibrinolysis

Vascular endothelium is involved in the control of thrombosis by influencing among other things, platelet functions, coagulation and fibrinolysis. Fibrinolysis is due to plasminogen conversion into plasmin by activators, which are efficiently and rapidly controlled by a specific inhibitor. Endothelium plays a pivotal role in these associations. It is a constitutive and inducible source of activators and inhibitor. It provides a favorable microenvironment for plasmin generation at the vessel surface. Furthermore, the fibrinolytic system participating in angiogenesis is implicated in plaque survival. Subjected to a pathological environment, endothelial activation leads to change in endothelial properties especially by increasing the production of inhibitor of plasminogen activators. These modifications could combine to create locally or distantly, a state predisposing to thrombosis.     

Gamma interferon administration to patients with atopic dermatitis inhibits fibrinolysis and elevates C1 inhibitor

Recombinant human gamma interferon was used to treat 10 atopic dermatitis patients. Recombinant gamma interferon was administered weekly for three consecutive days at 50 microg/M2 SQ for four weeks. All patients' dermatitis improved with recombinant gamma interferon therapy and plasma tumor necrosis factor-alpha levels rose with treatment. Recombinant gamma interferon treatment positively correlated with reduced total plasma fibrinolysis as measured by the fibrin lysis plate, plasmin-alpha2antiplasmin complexes, and tissue type plasminogen activator levels. Accordingly, plasminogen activator inhibitor levels increased. Treatment also was associated with a transient increase in thrombin-antithrombin III complexes. Recombinant gamma interferon resulted in a significant increase in C1 inhibitor antigen but not activity. Plasma prekallikrein, high molecular weight kininogen, and factor XII levels were not decreased. However, 5 of the 10 atopic dermatitis patients before therapy had circulating cleaved plasma high molecular weight kininogen detected on immunoblot, indicating prior kallikrein formation. The cleaved, circulating plasma high molecular weight kininogen disappeared in four out of the five original patients who were reexamined at one year after treatment. These combined data indicated that recombinant gamma interferon treatment reduced total plasma fibrinolysis. In untreated atopic dermatitis, circulating cleaved high molecular weight kininogen also may be a presenting manifestation.     

Evidence against an effect of endothelin-1 on blood coagulation, fibrinolysis, and endothelial cell integrity in healthy men

On the basis of an array of preclinical experimental results, it has been widely assumed that endothelin-1 (ET-1) may affect blood coagulation, fibrinolysis, and endothelial cell function, thereby playing a pathophysiological role in various cardiovascular diseases in humans. However, confirmation of this assumption is still lacking. ET-1 or placebo was administered intravenously to 12 healthy volunteers in a prospective, randomized, double-blind, crossover trial. Pathophysiologically relevant concentrations of ET-1 (an approximate threefold increase of normal blood levels) causing hemodynamic effects were reached by continuous intravenous infusion for 6 hours. Components of the coagulation (thrombin-antithrombin complexes, prothrombin fragment F1 + 2, activated factor VII, and factor VII antigen) and fibrinolytic (fibrin split product D-dimer, plasmin-plasmin inhibitor complex, tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1) systems and markers of endothelial cell perturbation/dysfunction (von Willebrand factor and thrombomodulin) were measured before the start of infusion and after 2, 6, 12, and 24 hours. Comparing changes in the plasma concentrations of these parameters during and after infusion of ET-1 and placebo, we found no specific effects of ET-1. In contrast to previous reports from preclinical experiments, ET-1 does not appear to affect coagulation or fibrinolysis, nor does this peptide induce relevant endothelial cell perturbations in humans.     

Retinoids and fibrinolysis

Vitamin A and its analogues have been reported to increase the release of tissue plasminogen activator in vitro. The aim of the present study was to reevaluate these findings and to investigate whether retinoids in doses used in dermatological therapy could enhance the release of endothelial fibrinolytic factors. Our results showed that endothelial cells incubated in vitro with retinoic acid increased the release of tissue plasminogen activator to the supernatant without concomitant secretion of plasminogen activator inhibitor-1. In patients treated with isotretinoin or etretinate these findings were confirmed, showing enhanced baseline tissue plasminogen activator concentrations in plasma in association with unchanged levels of plasminogen activator inhibitor-1 and von Willebrand factor. These findings are consistent with chronically augmented tissue plasminogen activator secretion without evidence of endothelial cell damage and may be of importance for the interpretation of the safety of lon-term therapy with regard to retinoid-induced hyperlipemia and the development of cardiovascular disease.     

Decreased fibrinolytic activity in porcine-to-primate cardiac xenotransplantation

BACKGROUND: One major barrier to successful xenotransplantation is acute vascular rejection, a process pathologically characterized by microvascular thrombosis and diffuse fibrin deposition in transplant blood vessels. This pathologic picture may result from a disturbance in the coagulant or fibrinolytic pathways that regulate normal vascular patency. This study evaluated the regulation of fibrinolytic activity defined by tissue plasminogen activator and plasminogen activator inhibitor-1 as it may exist in the setting of acute vascular rejection. MATERIALS AND METHODS, RESULTS: Serial biopsies from cardiac xenotransplants evaluated by immunofluorescence microscopy demonstrated progressive decreases in tissue plasminogen activator and increases in plasminogen activator inhibitor-1. In vitro studies measuring fibrinolytic activity of cell culture medium from porcine aortic endothelial cells stimulated with human serum or autologous porcine serum revealed that human serum triggered as much as 93% increase in antifibrinolytic activity. CONCLUSIONS: These findings demonstrate that porcine vascular endothelial cells change toward an antifibrinolytic state following stimulation with human xenoreactive antibodies and complement. The shift is at least partly explained by an increased ratio of plasminogen activator inhibitor-1 to tissue plasminogen activator, and is at least in part mediated by the activation of complement. This increased antifibrinolytic activity may contribute to the thrombotic diathesis seen in acute vascular rejection in pig-to-primate xenografts.     

Acceleration of plasminogen activation by tissue plasminogen activator on surface-bound histidine-proline-rich glycoprotein

Histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, is a major plasminogen-binding protein. In this work we characterized extensively the circumstances under which HPRG accelerates plasminogen activation and the specificity of this effect. Soluble HPRG did not significantly influence plasminogen activation. In contrast, native HPRG bound to hydrazide or nickel chelate surfaces strongly stimulated the activation of plasminogen by tissue plasminogen activator, but not by urokinase or streptokinase. The efficiency of activation on surface-bound HPRG was increased for Glu-plasminogen (41-fold), Lys-plasminogen (17-fold), and cross-linked Glu-plasminogen (11-fold) but not for mini-plasminogen, and was mainly due to a decrease in the apparent Km. A reduced susceptibility to inhibition by chloride ions contributed to the higher activation rate of Glu-plasminogen on an HPRG surface. The immobilized N- and C-terminal domains, but not the histidine-proline-rich domain of HPRG, also bound plasminogen and stimulated its activation. HPRG-enhanced plasminogen activation was proportional to the quantity of HPRG immobilized and was abolished by anti-HPRG antiserum, by low concentrations of epsilon-aminocaproic acid, by methylation of lysine residues in HPRG, and by treatment of HPRG with carboxypeptidase B. Soluble HPRG and a plasminogen fragment, kringle 1-2-3, acted as competitive inhibitors by binding to plasminogen and immobilized HPRG, respectively. The interaction of the conserved C-terminal lysine of HPRG with the high affinity lysine binding site of plasminogen is necessary and sufficient to accelerate plasminogen activation. Unlike other stimulators of plasminogen activation, the effect of HPRG on fibrinolysis is modulated by factors that influence the equilibrium between solution and surface-bound HPRG.     

Alpha 2-antiplasmin causes thrombi to resist fibrinolysis induced by tissue plasminogen activator in experimental pulmonary embolism

BACKGROUND: In patients with pulmonary embolism, thrombi resist fibrinolysis induced by plasminogen activators. Because the molecular basis of this thrombus resistance is poorly understood, we used a potent inhibitor to examine the potential role of alpha 2-antiplasmin (alpha 2AP) in experimental pulmonary embolism. METHODS AND RESULTS: Lysis of experimental pulmonary emboli was measured 4 hours after embolization in anesthetized ferrets. All animals received heparin (100 U/kg). Five experimental groups were studied: (1) no recombinant tissue plasminogen activator (rTPA); (2) rTPA at 1 mg/kg; (3) rTPA at 2 mg/kg; (4) rTPA at 1 mg/kg plus a control monoclonal antibody (MAb); and (5) rTPA at 1 mg/kg plus an alpha 2AP inhibitor (MAb 77A3). In comparison with ferrets receiving no rTPA (15.6 +/- 10.5% lysis, mean +/- SD), rTPA-treated groups showed significantly greater lysis (P < .01). Animals treated with rTPA and alpha 2AP inhibitor (56.2 +/- 4.7% lysis) showed significantly greater lysis than all other treatment groups, including ferrets treated with the same dose of rTPA alone (38.5 +/- 6.3%, P < .01), with twice the rTPA dose alone (45.0 +/- 6.5%, P < .05), or with a control MAb (35.2 +/- 4.6%, P < .01). The combination of rTPA treatment and alpha 2AP inhibition caused no consumption of fibrinogen. CONCLUSIONS: Inhibition of alpha 2AP significantly amplified the lysis of experimental pulmonary emboli by rTPA without increasing fibrinogen consumption. These results suggest that alpha 2AP may play an important role in thrombus resistance in patients with venous thromboembolism.