Role of annexin II tetramer in plasminogen activation

The enzymatic cascade triggered by activation of plasminogen has been implicated in a variety of normal and pathologic events, such as fibrinolysis, wound healing, tissue remodeling, embryogenesis, and the invasion and spread of transformed tumor cells. Recent data established that the Ca(2+)- and phospholipid-binding protein, annexin II heterotetramer (AIIt) binds tissue-type plasminogen activator (tPA), plasminogen, and plasmin, and dramatically stimulates the tPA-dependent conversion of plasminogen to plasmin in vitro. Interestingly, the binding of plasmin to AIIt can inhibit the activity of the enzyme, suggesting that plasmin bound to the cell surface is regulated by AIIt. The existing experimental evidence suggests that AIIt is the key physiological receptor for plasminogen on the extracellular surface of endothelial cells.     

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ(42) specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ(42)-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ(42), and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ(42). Our results indicate the existence of 2 mechanisms of Aβ(42) involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)-fibrin binding.     

Modulation of fibrinolysis by the combined action of phospholipids and immunoglobulins.

Because both immunoglobulin G (IgG) and phospholipids interfere with fibrinolysis, their combined modulating effects were investigated in experimental models of three consecutive steps of the fibrinolytic process [diffusion of tissue-type plasminogen activator (tPA) into the clot, plasminogen activation on fibrin surface and fibrin dissolution by plasmin] using IgGs isolated from healthy subjects and from patients with antiphospholipid syndrome in combination with mixtures of synthetic dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine. In fibrin clots containing phospholipids the normal IgG enhanced the barrier function of the phospholipids with respect to the diffusion of tPA and plasminogen activation, but did not modify the lysis by plasmin. One of the examined antiphospholipid syndrome-IgGs also restricted the diffusion of tPA, but it accelerated the plasminogen activation on the fibrin surface and slowed down the lysis of fibrin by plasmin. Another antiphospholipid syndrome IgG, which did not affect significantly the tPA penetration into the fibrin gel, did not modify the plasminogen activation on its own, but it partially opposed the inhibiting effect of phospholipids on plasmin formation and accelerated the end-stage lysis of fibrin containing phospholipids. The IgGs from the two examined antiphospholipid syndrome patients did not show consistent deviation from the pattern of normal IgG effects on fibrinolysis in phospholipid environment. Thus, a high degree of heterogeneity with respect to the profibrinolytic or antifibrinolytic effects of the pathological IgGs can be expected in the antiphospholipid syndrome patient population, which may contribute to the variable thrombotic symptoms in this clinical syndrome.     

The role of the annexin A2 heterotetramer in vascular fibrinolysis

The vascular endothelial cells line the inner surface of blood vessels and function to maintain blood fluidity by producing the protease plasmin that removes blood clots from the vasculature, a process called fibrinolysis. Plasminogen receptors play a central role in the regulation of plasmin activity. The protein complex annexin A2 heterotetramer (AIIt) is an important plasminogen receptor at the surface of the endothelial cell. AIIt is composed of 2 molecules of annexin A2 (ANXA2) bound together by a dimer of the protein S100A10. Recent work performed by our laboratory allowed us to clarify the specific roles played by ANXA2 and S100A10 subunits within the AIIt complex, which has been the subject of debate for many years. The ANXA2 subunit of AIIt functions to stabilize and anchor S100A10 to the plasma membrane, whereas the S100A10 subunit initiates the fibrinolytic cascade by colocalizing with the urokinase type plasminogen activator and receptor complex and also providing a common binding site for both tissue-type plasminogen activator and plasminogen via its C-terminal lysine residue. The AIIt mediated colocalization of the plasminogen activators with plasminogen results in the rapid and localized generation of plasmin to the endothelial cell surface, thereby regulating fibrinolysis.     

Measurements of plasmin-alpha 2 plasmin inhibitor complex and FDP.D dimer levels in the fibrinolytic therapy of acute pulmonary thromboembolism]

The key enzyme for fibrinolysis is plasmin, which is converted from plasminogen by plasminogen activator. Activated plasmin lyses fibrinogen and fibrin to make fibrin degradation products(FDPs) and plasmin is inactivated immediately by alpha 2 plasmin inhibitor. As FDP.D dimer is derived solely from insoluble fibrin, FDP.D dimer is thought of as an index for clot lysis. We measured plasmin-alpha 2 plasmin inhibitor complex(PIC) and FDP.D dimer plasma levels in 3 patients with acute pulmonary thromboembolism treated with recombinant tissue plasminogen activator(tPA). Fifteen million units of tPA(TD-2061) were infused in one hour on the first, second and third hospital days. PIC and FDP.D dimer before tPA infusion showed slightly elevated values as compared to normal ranges. They increased markedly after tPA infusion. These findings suggest that the fibrinolytic system is slightly activated in the acute phase of pulmonary thromboembolism and also strongly activated by tPA infusion. Increased FDP D dimer suggests that fibrin clots are dissolved by activated plasmin. Improvement of arterial oxygen tension was observed after tPA infusion. As sustained higher FDP.D dimer means the existence of fibrin clots, heparin treatment should be continued for prevention of clot formation as long as FDP.D dimer shows higher value. In conclusion, PIC and FDP.D dimer are useful indices not only to detect the activated state of the fibrinolytic system but also to know clot lysis in tPA treatment.     

Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis.

In this study, we investigated tissue-type plasminogen activator (tPA)-induced lysis of glutamic acid (glu)-plasminogen-containing or lysine (lys)-plasminogen-containing thrombin-induced fibrin clots. We measured clot development and plasmin-mediated clot disintegration by thromboelastography, and used scanning electron microscopy (SEM) to document the structural changes taking place during clot formation and lysis. These events occurred in three overlapping stages, which were initiated by the addition of thrombin, resulting first in fibrin polymerization and clot network organization (Stage I). Autolytic plasmin cleavage of glu-plasminogen at lys-77 generates lys-plasminogen, exposing lysine binding sites in its kringle domains. The presence of lys-plasminogen within the thrombin-induced fibrin clot enhanced network reorganization to form thicker fibers as well as globular complexes containing fibrin and lys-plasminogen having a greater level of turbidity and a higher elastic modulus (G) than occurred with thrombin alone. Lys-plasminogen or glu-plasminogen that had been incorporated into the fibrin clot was activated to plasmin by tPA admixed with the thrombin, and led directly to clot disintegration (Stage II) concomitant with fibrin network reorganization. The onset of Stage III (clot dissolution) was signaled by a sustained secondary rise in turbidity that was due to the combined effects of lys-plasminogen presence or its conversion from glu-plasminogen, plus clot network reorganization. SEM images documented dynamic structural changes in the lysing fibrin network and showed that the secondary turbidity rise was due to extensive reorganization of severed fibrils and fibers to form wide, occasionally branched fibers. These degraded structures contributed little, if anything, to the structural integrity of the residual clot, and eventually collapsed completely during the course of progressive clot dissolution. These results provide new perspectives on the major structural events that occur in the fibrin clot matrix during fibrinolysis.     

Inhibition of vascular endothelial cell prostacyclin synthesis by plasmin

Vascular endothelial cells (EC) play an active role in the synthesis and assembly of components of the fibrinolytic system and the generation of the major fibrinolytic protease plasmin. However, the reciprocal effects of plasmin on EC function have not been previously examined. We have studied the actions of plasmin on the production of prostacyclin (PGI2) by cultured human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells. Plasmin causes little or no direct stimulation of PGI2 formation by EC. Preincubation of EC with plasmin, however, produces a time- and concentration-dependent inhibition of ionophore A23187-, thrombin-, and histamine-induced PGI2 synthesis; a smaller inhibitory effect on arachidonate- and PGH2-induced PGI2 synthesis is found. Incubation of HUVEC or BAEC with a physiologic concentration of plasminogen (180 micrograms/mL) and recombinant tissue plasminogen activator (tPA) generates tPA dose-dependent plasmin activity that exceeds that generated in the absence of EC. In the presence of plasminogen, tPA also causes a tPA dose-dependent inhibition of thrombin- and ionophore A23187-stimulated PGI2 production. PGI2 inhibitory plasmin activity is generated within the concentration range of tPA achieved in plasma during pharmacologic therapy with tPA. These findings suggest that vascular endothelial cells not only regulate activation of the fibrinolytic system but may also be targets of plasmin action on PGI2 synthesis in the modulation of hemostasis and thrombosis.     

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.     

ENDOTHELIAL CELL SURFACE ACTIN SERVES AS A BINDING SITE FOR PLASMINOGEN, TISSUE PLASMINOGEN ACTIVATOR AND LIPOPROTEIN(a)

One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45 kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45 kD plasminogen receptor (pI = 6.3) whereas the other component (pI = 5.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitro studies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle-dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70–80% of binding inhibited by lysine. Lipoprotein(a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low-density lipoproteins inhibited Lp(a) binding to actin in a dose-dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti-actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.     

Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis enhance the activation of the fibrinolytic system of the host

Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat.     

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.     

Cellular receptors for plasminogen activators recent advances

The generation of the broad-specificity protease plasmin by the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodeling, and cell migration. A mechanism for the regulation of plasmin generation is through binding of the plasminogen activators to specific cellular receptors: uPA to the glycolipid-anchored membrane protein urokinase-type plasminogen activator receptor (uPAR) and tPA to a number of putative binding sites. The uPA-uPAR complex can interact with a variety of ligands, including plasminogen, vitronectin, and integrins, indicating a multifunctional role for uPAR, regulating not only efficient and spatially restricted plasmin generation but also having the potential to modulate cell adhesion and signal transduction. The cellular binding of tPA, although less well characterized, also has the capacity to regulate plasmin generation and to play a significant role in vessel-wall biology. (Trends Cardiovasc Med 1997;7:227-234). ? 1997, Elsevier Science Inc.     

Activation of the fibrinolytic system

The fibrinolytic system is activated either directly or indirectly by proteins that convert plasminogen to plasmin in the circulation, within the interstitices, and on the surface of fibrin clots, or both. Agents that activate circulating and clot-bound plasminogen comparably induce a systemic lytic state accompanying clot lysis. Agents that activate plasminogen in the domain of fibrin preferentially exhibit clot selectivity. Fibrinolytic activators are assayed by detection of protein, generally immunologically, or of functional activity, generally by visualization of lysis of fibrin or by spectrophotometric determination of amidolytic activity.     

Stimulation of cell surface plasminogen activation by heparin and related polyionic substances.

The functional operation of the cell surface pro-u-PA and plasminogen activating system has previously been shown to depend on the assembly of u-PA receptors, plasminogen binding sites, and their respective ligands at the focal adhesions of cell extensions. We now show that additional factors operate that affect the persistence of functional activity and that evidently involve charge interactions mediated by polyanions, such as those found in the cell surface proteoglycans. Heparin-like compounds and protamine were identified as fast-acting stimulators of cell surface plasminogen activation. Heparin stabilized surface u-PA activity during plasminogen activation, and we propose that a heparin binding site exists in the kringle structure of u-PA. Heparin at 40 micrograms/ml could reduce u-PA loss to only 20% compared with 60% on control cells activating plasminogen. Protamine (25 micrograms/ml) exerted a strong stimulatory effect on the level of generated bound plasmin and notably prolonged the persistence of this activity, so that 100 minutes after addition of plasminogen the level of plasmin on protamine-treated cells was five times higher than on control-treated cells. The effect of protamine on plasmin clearance suggests that an unknown plasmin inhibitor may be produced by rhabdomyosarcoma cells, whose action is accelerated by endogenous polyanions, in an analogous manner to thrombin inactivation by antithrombin III and protease nexin on endothelial cells and fibroblasts, respectively. The stimulatory effects of heparin and protamine do not affect the inactivation of cell surface u-PA by recombinant PAI-2.     

An Analysis of Mechanisms Underlying the Antifibrinolytic Properties of Radiographic Contrast Agents

Background: Radiographic contrast agents inhibit fibrinolysis, although by poorly defined pathways. The purpose of this study was to define specific mechanisms by which contrast agents inhibit clot lysis. Methods and Results: Diatrizoate (high osmolar ionic agent), ioxaglate (low osmolar ionic), and ioversol (nonionic) were studied in vitro. Diatrizoate inhibited clot lysis by 81.3±0.6% vs. control (p<0.001). Ioxaglate inhibited clot lysis by 41.7±11.9%, which was of borderline significance (p=0.07). Ioversol did not significantly inhibit clot lysis (14.9±11.5% decrease vs. control; p>0.3). Inhibition of fibrinolysis was not explained by the high osmolarities of contrast agents, by their iodine content, or by their effects on the amidolytic activities of t-PA, urokinase, or plasmin. However, plasminogen activation by t-PA, urokinase, or streptokinase was significantly inhibited by contrast agents. Diatrizoate, ioxaglate, and ioversol inhibited plasminogen binding to plasma clots by 51±4% (p<0.001), 30.1±4% (p<0.01), and 19.4±7% (p=0.07), respectively. Plasma clots formed in the presence of contrast agents were resistant to lysis by plasmin. Diatrizoate produced the most potent effect, inhibiting clot lysis by 40±5.7% (p<0.03). Contrast agents did not inhibit plasminogen binding to fibrin or plasmin-mediated fibrinolysis if they were added after clot formation. Contrast agents altered clot turbidity, an index of fibrin structure, if present during clot formation, but not if added to preformed clots. Contrast agents did not affect plasminogen activator inhibitor-1 or ₂-antiplasmin function. Conclusions: Contrast agents inhibit clot lysis by inhibiting plasminogen activation and by disrupting interactions of plasminogen and plasmin with fibrin by altering fibrin structure. Significant variation in antifibrinolytic properties exists between different contrast agents. Abbreviated Abstract. The purpose of this study was to define specific mechanisms by which contrast agents inhibit clot lysis. In both a purified clot lysis system and a plasma clot lysis system, diatrizoate, an ionic agent, produced the most potent inhibition of fibrinolysis. Contrast agents did not inhibit the active sites of plasminogen activators or plasmin, but did inhibit plasminogen activation. Binding of plasminogen to fibrin and lysis of fibrin by plasmin were inhibited by contrast agents if they were present during clot formation, but not if they were added after clot formation was complete. Contrast agents altered clot turbidity, an index of fibrin structure, if present during clot formation, but not if added to preformed clots. Contrast agents did not affect plasminogen activator inhibitor-1 or ₂-antiplasmin function. The effects of contrast agents on fibrinolytic parameters were not explained by their high osmolarities. These results suggest that contrast agents inhibit clot lysis by inhibiting plasminogen activation and by disrupting interactions of plasminogen and plasmin with fibrin by altering fibrin structure.     

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.     

Dependence of human vascular cell surface proteolysis on expression of the urokinase receptor

To delineate the role of binding of urokinase type plasminogen activator (uPA) to its receptor (uPAR) in the local generation of plasmin by endothelium, we transfected spontaneously transformed immortalized human vascular endothelial cells that express high levels of uPA but low levels of uPAR with human uPAR complementary DNA. Compared with nontransfected cell, the stably transformed clonal cell line exhibited (a) a > 10-fold increase in steady-state uPAR mRNA levels documented with Northern blot analysis (n = 3), (b) a 2.8-fold increase in cell surface expression of uPAR protein quantified by enzyme linked immunosorbent assay (n = 3), (c) a 2.9-fold increase in specific binding of radiolabeled single chain uPA (n = 4), and (d) markedly increased matrix adhesion. The participation of uPAR in cell surface proteolysis was apparent based on a 3.0-fold increase in cell associated plasmin activity (n = 3) and a 2.3-fold increase in lysis of noncrosslinked fibrin clots (n = 5). Thus, local generation of plasmin and consequent degradation of fibrin are likely to be promoted by cell surface localization of uPA by uPAR in cellular constituents of the vessel wall. Furthermore, genetic engineering of endothelium to enhance expression of uPAR may confer resistance to thrombosis or restenosis associated with endovascular stents.     

A faster-acting and more potent form of tissue plasminogen activator.

Current treatment with tissue plasminogen activator (tPA) requires an intravenous infusion (1.5-3 h) because the clearance of tPA from the circulation is rapid (t 1/2 approximately 6 min). We have developed a tPA variant, T103N,N117Q, KHRR(296-299)AAAA (TNK-tPA) that has substantially slower in vivo clearance (1.9 vs. 16.1 ml per min per kg for tPA in rabbits) and near-normal fibrin binding and plasma clot lysis activity (87% and 82% compared with wild-type tPA). TNK-tPA exhibits 80-fold higher resistance to plasminogen activator inhibitor 1 than tPA and 14-fold enhanced relative fibrin specificity. In vitro, TNK-tPA is 10-fold more effective at conserving fibrinogen in plasma compared to tPA. Arterial venous shunt models of fibrinolysis in rabbits indicate that TNK-tPA (by bolus) induces 50% lysis in one-third the time required by tPA (by infusion). TNK-tPA is 8- and 13-fold more potent in rabbits than tPA toward whole blood clots and platelet-enriched clots, respectively. TNK-tPA conserves fibrinogen and, because of its slower clearance and normal clot lysis activity, is effective as a thrombolytic agent when given as a bolus at a relatively low dose.     

Plasminogen assay by means of the lysis time method.

The lysis time method for the determination of plasminogen has been investigated using plasminogen-free thrombin and fibrinogen preparations. The experiments have shown that the lysis of a fibrin clot is the result of two consecutive reactions: the formation of fibrin which proceeds as a first order reaction and the degradation of fibrin which proceeds as a zero order reaction. Plasminogen is activated in a separate reaction. If the rate of the fibrin formation is much greater than the rate of degradation, the lysis of the fibrin clot is approximately of zero order in fibrin. The lysis time will then be inversely proportional to the plasmin concentration and proportional to the fibrinogen concentration. In a double logaritmic system the correlation between lysis time and plasmin activity is a straight line with a slope of 135 degrees. Plasminogen is rapidly activated with streptokinase. Maximal activation is obtained only with a certain streptokinase concentration. Higher concentrations inactivate plasmin and with lower concentrations, the maximal activity is never reached. A spontaneous inactivation is seen after about 30 minutes. With urokinase, a higher maximal plasminogen activity is obtained than with streptokinase. Urokinase in higher concentrations does not inactivate plasmin. A standard assay for determination of plasminogen by the lysis time method has been worked out and is based on these results.     

Targeting of tissue plasminogen activator to the regulated pathway of secretion

Tissue plasminogen activator is a serine protease that plays the dominant role in removal of fibrin from the vascular tree by activating plasminogen to the primary fibrinolytic enzyme, plasmin. Tissue plasminogen activator has a widespread neuroendocrine distribution in addition to its expression by endothelial cells. Within neuroendocrine cells, secretory proteins are sorted into one of two pathways: regulated or constitutive. Proteins entering the regulated pathway are concentrated and stored in vesicles, and subsequently released upon stimulation by a secretagogue. In contrast, in the constitutive pathway, newly synthesized protein is not stored but is transported directly to the cell surface and secreted even in the absence of an extracellular signal. The focus of this article is to review recent studies demonstrating that tissue plasminogen activator is targeted to the regulated secretory pathway in neuroendocrine cells and to discuss the physiological implications of the trafficking of tissue plasminogen activator to regulated secretory vesicles.