Plg Activation Research Articles

Selective behaviors altered in plasminogen-deficient mice are reconstituted with intracerebroventricular injection of plasminogen

In vitro studies demonstrate a role for the plasminogen (Plg) system in neurological function and recently in vivo studies show a role of the Plg system in neurodegeneration after the injection of an excitotoxic agent. Differences in the development of neurological function, however, have not been demonstrated in the Plg-deficient (Plg−/−) mice compared to wild-type (WT) mice. The role of Plg system in neurological function may relate to remodeling which occurs in response to various environmental challenges. In this study, behaviors (open field, grooming, hind-leg gait, water maze, and acoustic startle reflex) were tested in the Plg-deficient and WT mice at 6–8 weeks of age. Grooming, a response to the stress of an open field or fur moistening, was increased in the Plg−/−-deficient mice compared to WT mice, and the acoustic startle reflex (ASR) was markedly decreased in the Plg−/− mice. The reduced ASR in Plg−/− mice occurred in mice with a mixed C57BL:129 background or in mice with a C57BL background. Plg was required for the ASR, since a deficiency of the Plg activators, urokinase (uPA) or tissue Plg activator (tPA), did not cause a reduction in the ASR compared to their WT control. Infusion of Plg directly into the brain was effective in restoring the ASR in the Plg−/− mice, but had no effect on the ASR of WT mice. Peripheral bolus injections of Plg or infusion into the jugular vein were ineffective in restoring the ASR in the Plg−/− mice. These results indicate that Plg is required for the appropriate response to the environmental challenge of a sudden loud sound, and that the response can be restored in Plg−/− mice by directly infusing Plg into the brain.

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation.

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.

Prevalence of Circulating Antibodies against a Streptokinase C-Terminal Peptide in Normal Blood Donors

Streptokinase (SK) is a streptococcal protein widely used as a thrombolytic agent. Anti-SK antibodies (Abs) are found in most individuals due to common streptococcal infections. The presence of these Abs increases the possibility of allergic reactions and may reduce the thrombolytic efficacy of SK upon a first therapy. Previous studies report on the immunodominance of the SK C-terminus and the role of this region in plasminogen (Plg) activation. The aim of this study was to assess the prevalence of circulating Abs to the SK C-terminus in normal blood donors. Sera from 1008 subjects aged 30 to 60 years were tested by Ultra-Micro-ELISA using a synthetic peptide resembling the SKC-2 C-terminus. An overall prevalence of 30.4% was found. Prevalence was significantly higher among male than among female donors (RR = 1.70, 1.13 < CI < 2.55). No age effect was observed. This is the first extensive study about Abs directed against a particular region of SK in normal subjects.

Analysis of the interactions between streptokinase domains and human plasminogen

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.

Function of the plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes.

The matrix metalloproteinase (MMP) system, which may be activated via the plasminogen (Plg)/plasmin system, is claimed to play a role in matrix degradation and smooth muscle cell migration. To test the role of both systems, expression of fibrinolytic and gelatinolytic activity was quantified after vascular injury in mice with targeted inactivation of tissue-type Plg activator (tPA-/-), urokinase-type Plg activator (uPA-/-), or Plg (Plg-/-). Neointima formation 1 week after vascular injury was impaired in uPA-/- and Plg-/- mice compared with wild-type (WT) mice or tPA-/- mice (reduction of neointimal area to 30% and 10% of WT, respectively). Cell accumulation at the borders of the injury was significantly (P<0.01) impaired compared with that in WT mice. One week after injury of the femoral artery, tPA-mediated fibrinolytic activity in arterial sections or extracts of WT, uPA-/-, or Plg-/- mice was not altered, whereas uPA activity levels in tPA-/- and Plg-/- mice were 2- to 3-fold higher than in uninjured controls. Total levels (latent plus active) of MMP-2 (gelatinase A) were increased by 2- to 4-fold, whereas the contribution of active MMP-2 represented 38% to 63% of the total in the different genotypes. MMP-9 (gelatinase B) was not detectable in the majority of control arteries, whereas total MMP-9 levels after injury were dramatically increased (up to 50-fold above the detection limit). Active MMP-9 represented 20% to 46% of total MMP-9 in WT, tPA-/-, and uPA-/- mice but was not consistently detectable in Plg-/- mice. Similar results were obtained in carotid arteries. Thus, the unaltered ratios of active and latent MMP-2 suggest that proMMP-2 activation may occur in the absence of tPA, uPA, or Plg, whereas no active MMP-9 was detected in the absence of Plg. The data of this study confirm a role for uPA and Plg but not for tPA in smooth muscle cell migration and neointima formation after vascular injury and indicate that impairment of these phenomena may occur despite the observed increases in MMP-2 or MMP-9 levels after vascular injury.

114 The effect of argatroban on staphylokinase activity in the presence of endothelial cell injured by thrombin

When Glu-plasminogen (Glu-plg) was incubated with urokinase (UK) in the presence of various concentrations of tranexamic acid and S-2251, the hydrolysis of S-2251 increased in the presence of 1 mM of tranexamic acid, but decreased in the presence of more than 10 mM of tranexamic acid. Use of SDS-polyacrylamide gel electrophoresis indicated: 1) Glu-plg was converted to plasmin better in the presence of 1 mM of tranexamic acid, but in the presence of more than 10 mM of tranexamic acid it was less converted. 2) Plg I (of higher molecular weight) was more easily converted than plg II by UK, but binding of tranexamic acid with lysine binding sites of plasminogen caused almost equal rate of activation of plg I and II.

112 Effect of heparin and hirudin on endotoxin-induced PAI-1 mRNA expression by human umbilical vein endothelial cells

When Glu-plasminogen (Glu-plg) was incubated with urokinase (UK) in the presence of various concentrations of tranexamic acid and S-2251, the hydrolysis of S-2251 increased in the presence of 1 mM of tranexamic acid, but decreased in the presence of more than 10 mM of tranexamic acid. Use of SDS-polyacrylamide gel electrophoresis indicated: 1) Glu-plg was converted to plasmin better in the presence of 1 mM of tranexamic acid, but in the presence of more than 10 mM of tranexamic acid it was less converted. 2) Plg I (of higher molecular weight) was more easily converted than plg II by UK, but binding of tranexamic acid with lysine binding sites of plasminogen caused almost equal rate of activation of plg I and II.

Homozygous type I plasminogen deficiency.

Homozygous type I plasminogen (Plg) deficiency has not been described in human subjects so far. Ligneous conjunctivitis is a rare and unusual form of chronic pseudomembranous conjunctivitis of unknown etiology. Here we report for the first time on homozygous type I Plg deficiency in three unrelated female patients who suffered from ligneous conjunctivitis and additional pseudomembranous lesions of other mucous membranes. The disease is caused by massive fibrin depositions within the "extravascular space" of mucous membranes because of absent clearance by plasmin. Infusions of albumin, fresh frozen plasma, or Lys-plasminogen (Lys-Plg) into two of the three patients revealed normal Plg activation capacity in these patients. The absence of fibrinolytic activity could therefore be shown to be due to Plg deficiency. Similar studies in the third patient have not been completed. In the two patients studied so far, infusions of Lys-Plg resulted in prompt and adequate Plg recovery with a short half-life and high amounts of plasmin-antiplasmin complexes and D-dimer. One patient additionally revealed an inherited partial factor XII deficiency. Functionally, this factor XII deficiency did not interfere with Plg activation. However, there may be a pathway of Plg activation in this patient via the prekallikrein C1-INH system.

IMPAIRED CLOT LYSIS BY rt-PA CATALYZED MINI-PLASMINOGEN ACTIVATION

The fibrinolytic system contains a proenzyme plasminogen (Plg) which is converted to plasmin (Plm) by the action of Plg activators. Physiological Plg activators are: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plg was shown to be further cleaved by leukocyte elastase producing several fragments, one of wich is called mini-plasminogen (mini-Plg) or neo-plasminogen Val 442. In this paper we studied whether mini-Plg is able to produce clot lysis when it is activated by rt-PA in purified systems and in Plg depleted normal plasma. We found that mini-Plg clot lysis time was longer than that of Plg. Clot lysis times were 2.3 minutes ± 0.06 for Plg and 9.8 minutes ± 0.1 for mini-Plg. Mini-Plg is less efficient than Plg in producing clot lysis at all studied concentrations (0.1–1.2μM). In Plg depleted normal human plasma mini-Plg is unable to produce complete clot lysis in presence of rt-PA. Although mini-Plg can be activated to mini-Plm by rt-PA, these results show that the activation process is insufficient to produce an efficient clot lysis. © 1997 Elsevier Science Ltd

Characterization of plasminogen system on rat yolk sac carcinoma (L2) cells

The plasminogen (Plg) system on rat yolk sac carcinoma (L2) cells was characterized by zymography, Western and immunoprecipitation analysis, reverse-transcriptase polymerase chain reaction, binding, and activity assays. The L2 cells produced tissue Plg activator but not urokinase Plg activator and contained RNA for Plg activator inhibitor 1, but Plg activator inhibitor 1 was not detectable by zymography or Western analysis and contained the receptor for urokinase Plg activator. Plg bound to the cells in a saturable manner when plasmin inhibitors were present with a dissociation constant of 1.34 +/- 0.18 x 10(-6) M and 1.54 +/- 0.25 x 10(7) sites/cell. Immunoprecipitation analysis showed that Plg was binding to gp330, a known Plg receptor. Once bound to the L2 cells, Plg was activated by tissue Plg activator to plasmin in a time- and concentration-dependent manner. Under saturating Plg conditions, most of the plasmin produced was released into the medium. Inhibition of plasmin activation occurred when Plg activator inhibitor 1, anticatalytic tissue Plg activator antibody, or Heymann nephritis autoantibody was present.

Cell-surface plasminogen activation causes a retraction of in vitro cultured human umbilical vein endothelial cell monolayer

Vascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.

Cell-Surface Plasminogen Activation Causesa Retraction of In Vitro Cultured Human Umbilical Vein Endothelial Cell Monolay

AbstractVascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell and cell-matrix contacts. We conclude that the cell-surface activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.

Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.

An anti-plasminogen antibody preparation that inhibits the activation of human plasminogen but enhances the activator activity of the B-chain-streptjkinase complex

An anti-Glu-plasminogen (GLU-PLG) polyclonal antibody antiserum was prepared in the goat, and specific IgG anti-GLU-PLG antibodies were purified by affinity chromatography using a sepharose-GLU-PLG column. In chromogenic assay studies, the anti-GLU-PLG antibody preparation was found to be an effective inhibitor of the activation of PLG, and it produced different inhibition curves with four different PLG activators. 80% inhibition of streptokinase (SK) activation of GLU-PLG occurred with an anti-GLU-PLG antibody/GLU-PLG molar ratio of 1:1, wnereas at tnis ratio only 28% and 36% inhibition of the plasmin-streptokinase (PLN-SK) and the B-chain-streptokinase (B-SK) complexes occurred. At a 1:1 molar ratio of antibody to PLN, no inhibition of PLN activity occurred. When the anti-GLU-PLG antibody preparation was incubated with each PLG activator, an enhancement in the activator activity of the B-SK complex, but not the other activators was observed with mini-plasminogen (MINI-PLG). Enhancement occurred at a molar ratio of 1:1 and reached a peak of 97% enhancement at a molar ratio of 10:1. Enhanced activator activity of the B-SK complex of 49% occurred at a molar ratio of 1:1 when GLU-PLG was the substrate. At higher molar ratios innibition of activator activity on GLU-PLG was observed, but not on MINI-PLG.These results indicate that there are multiple activator binding sites on PLG, that these four activators all bind differently to GLU-PLG and MINI-PLG, that some antibody populations that are specific for the PLN B-chain can stimulate activator activity, while other antibody populations that are specific for either the PLN A-chain or the B-chain are inhibitory.

Activation of Val 442-plasminogen (mini-plasminogen) by urokinase, streptokinase and tissue plasminogen activator

Glu-plasminogen (Glu-plg), Lys-plg and Val 442-plg (mini-plg) were activated by urokinase (UK), streptokinase (SK) or tissue plasminogen activator (t-PA). Their activation rates were kinetically analyzed. UK activated Lys-plg with smaller Km and nearly identical Vmax as Glu-plg. Mini-plg was activated by UK with higher Vmax but with the same Km as Glu-plg. On the other hand, t-PA activated Gluplg, Lys-plg and mini-plg with the same Vmax, but Km was in the order of Glu-plg>mini-plg-Lys-plg. Fibrin hardly enhanced the UK activation of mini-plg, and fibrinogen,fragment D or E did not enhance the activation of mini-plg by UK. Only D domain, but not E domain, complexed with K5 of mini-plg and SK hydrolysed S-2251 faster than a dimolecular complex of mini-plg and SK, thus a trimolecular mixture of D, SK and mini-plg having a better activator activity than SK-mini-plg complex. In the t-PA activation of mini plg, fibrin and fibrinogen enhanced the activation, but fragment D or E was ineffective, suggesting that a molecule containing more than one domain was required for the enhanced activation of mini-plg by t-PA.

Thrombolytic function and extrinsic fibrinolysis in the domestic chicken.

Chicken thrombolytic function was observed in vivo, and the activation of chicken plasminogen by tissue plasminogen activator (t-PA) extracted roughly from chicken heart was studied. The chicken blood clot embedded subcutaneously or the thrombus clotted by thrombin injection in the brachial vein were removed promptly. The t-PA could be extracted when the fresh heart was homogenized with 2M KSCN, O.3M CH3COOK, 5M (NH2)2CO, 3.9-62.5mM and 0.5-2 M MgSO4 solution. The reactivity of t-PA extracted with 62.5mM MgSO4 solution was remarkably enhanced by the addition of CaCl2 in chicken plasminogen (Plg) and t-PA mixture after incubation at 37°C for 4hours. Human urokinase (UK) activation under the same conditions reached to the maximum in one hour. Changing the additional order and the addition of CaCl2 beyond the range of 31.3-125mM were weakened the reaction. Epsilonaminocaproic acid (EACA) inhibited these activation system, being especially effective in activation of Plg. These findings suggest that chicken has a thrombolytic mechanism and a t-PA activation system in fibrinolysis.

Monoclonal antibodies inhibitory to human plasmin. Definitive demonstration of a role for plasmin in activating the proenzyme of urokinase-type plasminogen activator.

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high-Mr urokinase to its low-Mr form. None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti-plg 1 inhibited (a), (b) and (c), while anti-plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti-plg 3 and anti-plg 4 inhibited only activity (c). Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase-type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.

Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin.

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.

Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation.

Ternary complex formation of tissue plasminogen activator (TPA) and plasminogen (Plg) with thrombospondin (TSP) or histidine-rich glycoprotein (HRGP) has been demonstrated using an enzyme-linked immunosorbent assay, an affinity bead assay, and a rocket immunoelectrophoresis assay. The formation of these complexes was specific, concentration dependent, saturable, lysine binding site-dependent, and inhibitable by fluid phase plasminogen. Apparent Kd values were approximately 12-36 nM for the interaction of TPA with TSP-Plg complexes and 15-31 nM with HRGP-Plg complexes. At saturation the relative molar stoichiometry of Plg:TPA was 3:1 within the TSP-containing complexes and 1:1 within HRGP-containing complexes. The activation of Plg to plasmin by TPA on TSP- and HRGP-coated surfaces was studied using a synthetic fluorometric plasmin substrate (D-Val-Leu-Lys-7-amino-4-trifluoromethyl coumarin). Kinetic analysis demonstrated a marked increase in the affinity of TPA for plasminogen in the presence of surface-associated TSP or HRGP. Compared to fluid phase activation or activation on fibronectin- or Factor VIII-related antigen-coated surfaces there was a 35-fold increase in efficiency of plasmin generation. A substantial amount (up to 71%) of the plasmin formed remained surface-associated and was found to be protected from inhibition by alpha 2-plasmin inhibitor. Greater than 200-fold increase in inhibitor concentration was required to effect 50% inhibition. Complex formation of locally released tissue plasminogen activator with Plg immobilized on TSP or HRGP surfaces may thus play an important role in effecting proteolytic events in nonfibrin-containing microenvironments.

Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator.

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of platelets, binds fibrinogen, fibronectin, heparin, and histidine-rich glycoprotein and thus may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a complex with purified human plasminogen (Plg). Complex formation was detected by rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins. Significant complex formation of fluid-phase Plg with adsorbed TSP was also demonstrated by enzyme-linked immunosorbent assay (ELISA). The complex formation was specific, saturable, and inhibited by excess fluid-phase TSP, with an apparent KD of approximately 35 nM. In both ELISA and rocket immunoelectrophoresis systems, complex formation was inhibited by 10 mM epsilon-amino-n-caproic acid, implying that there is a role for the lysine binding sites of Plg in mediating the interaction. TSP also formed a complex with plasmin as detected by ELISA but did not directly inhibit plasmin activity measured with a synthetic fluorometric substrate or with a 125I-fibrin plate assay. TSP, when incubated with Plg before addition to 125I-fibrin plates significantly inhibited the generation of plasmin activity by tissue plasminogen activator (TPA) in a manner that was calcium dependent. A kinetic study of Plg activation by TPA in the presence of TSP demonstrated that Michaelis-Menten kinetics were followed and that TSP acted as a noncompetitive inhibitor. These studies support the hypothesis that TSP, acting as a multifunctional regulator in focal areas of active hemostasis, could serve as a prothrombotic influence, leading to increased deposition of fibrin.