The primary inhibitor of plasmin in human plasma.

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.     

On the interaction of alpha2-plasmin inhibitor and proteases. Evidence for the formation of a covalent crosslinkage and non-covalent weak bondings between the inhibitor and proteases.

alpha2-plasmin inhibitor is a proteinase inhibitor in plasma which efficiently inhibits the lysis of fibrin clots induced by plasminogen activator. The nature of the binding of the inhibitor to trypsin or plasmin was studied by the chemical treatment of the enzyme-inhibitor complex with 7.5 M hydrazine at pH 10.0. With the hydrazine treatment, the complexes were degraded to proteins corresponding to the respective enzyme and inhibitor moieties. These results indicate that the covalent bond between the inhibitor and the enzymes is a carboxylic ester. The binding reaction of the inhibitor to active site-modified trypsin was also studied. The inhibitor formed complexes with anhydrotrypsin and carboxyamidomethylated trypsin. The complexes were dissociated in the presence of 1% sodium dodecyl sulfate, to the individual components: the respective enzyme and inhibitor moieties. The inhibitor, however, did not form a complex with diisopropylphosphorylated trypsin regardless of the presence or absence of the denaturing reagent. These results suggest the contribution of non-covalent interactions to the complex formation between the inhibitor and native enzymes.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.     

Thrombospondin is a slow tight-binding inhibitor of plasmin.

Thrombospondin is a multifunctional glycoprotein of platelet alpha-granules and a variety of growing cells. We demonstrate that thrombospondin is a slow tight-binding inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to cleave fibrinogen, and decreased lysis zones in fibrin plate assays. Stoichiometric titrations indicate that approximately 1 mol of plasmin interacts with 1 mol of thrombospondin, an unexpected result considering the trimeric nature of thrombospondin. Plasmin in a complex with streptokinase or bound to epsilon-aminocaproic acid is protected from inhibition by thrombospondin, thereby implicating the lysine-binding kringle domains of plasmin in the inhibition process. Thrombospondin also inhibits urokinase plasminogen activator, but more slowly than plasmin, stimulates the amidolytic activity of tissue plasminogen activator, and has no effect on the amidolytic activity of alpha-thrombin or factor Xa. These results, therefore, identify thrombospondin as a new type of serine proteinase inhibitor and potentially important regulator of fibrinolysis.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma.

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.     

The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin.

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).     

Partial purification and characterization of a new fast-acting plasmin inhibitor from human platelets. Evidence for non-identity with the known plasma proteinase inhibitors.

An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human blood platelets by (NH4)2SO4 fractionation and affinity chromatrography on Sepharose-linked purified plasminogen. The material contained none of the known plasma proteinase inhibitors when studied by crossed-immunoelectrophoresis and electroimmunoassay, but inhibited a clot-lysis-time assay and an esterolytic assay that used the synthetic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide). The inhibitory activity had the same mobility as the alpha 2-plasma proteins on preparative agarose-gel electrophoresis. Titration of the inhibitor preparation by active-site-titrated plasmin demonstrated a dissociation constant of approx. 0.1 nM. The inhibition was complete within 1 min. The inhibitor increased the mobility in agarose-gel electrophoresis of purified activator-free plasmin or 125I-labelled plasmin, as demonstrated by crossed-immunoelectrophoresis against specific immunoglobulins against plasminogen or by radioautography. The results strongly suggest the presence in platelets of a plasmin inhibitor different from the known plasma proteinase inhibitors.     

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.     

Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a K(D) value of 0.18 by gel filtration and post beta(1) mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a K(D) value of 0 and alpha(2) mobility appeared, which was probably plasmin in a complex with alpha(2) macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same K(D) value by gel filtration as plasminogen (0.35), but beta(1) and gamma mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with gamma mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin-alpha(2) macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin-alpha(2) macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, alpha(1) antitrypsin, inter-alpha-inhibitor, antithrombin III, and C(1)-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The beta(1) and post beta(1) components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.     

Reaction of thrombin and proteinases of the fibrinolytic system with a mechanism-based inhibitor, 3,4-dihydro-3-benzyl-6-chloromethylcoumarin

Thrombin, plasmin and tissue plasminogen activator (one- and two-chain forms) were examined with respect to their reaction with the suicide substrate, 3,4-dihydro-3-benzyl-6-chloromethylcoumarin, at 4 degrees C. The enzymes were irreversibly inhibited and the apparent second-order rate constants ki/Ki were 31,000, 316, 187 and 250 M-1.s-1, respectively. The extent of fibrin clot lysis induced by urokinase and two-chain tissue plasminogen activator was considerably decreased after treatment of these enzymes with the dihydrocoumarin derivative (molar excess of inhibitor over enzyme ranging from 6 to 21 for urokinase and 50 to 1500 for tissue plasminogen activator). This inhibitor has been tested as anticoagulant in human plasma and was effective at prolonging the prothrombin time from 12 to 40 s.     

Mechanism of action of inter-alpha-trypsin inhibitor

Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.     

Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

Kinetics of plasmin inhibition in the presence of a synthetic tripeptide substrate. The reaction with pancreatic trypsin inhibitor and two forms of alpha 2-plasmin inhibitor.

The progressive inhibition of plasmin by pancreatic trypsin inhibitor and by alpha 2-plasmin inhibitor in the presence of D-valyl-L-leucyl-L-lysine 4-nitroanilide was investigated. The kinetics with plasmin were compared with those with miniplasmin. The kinetic properties of two functionally different forms of alpha 2-plasmin inhibitor described by Clemmensen [(1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen. D., Wiman, B & Verstraete, M., eds.), pp 131-136, Elsevier, Amsterdam] were characterized. The two forms differ in their plasminogen-binding capability, and this difference can account for a difference in secondary site interaction suggested from the kinetics. The binding of inhibitor to miniplasmin is a simple pseudo-first-order reaction with both pancreatic trypsin inhibitor and the two alpha 2-plasmin inhibitor forms. Such simple kinetics are also observed for the reaction between plasmin and the non-plasminogen-binding form of alpha 2-plasmin inhibitor. More complicated kinetics are obtained for the reaction between plasmin and the alpha 2-plasmin inhibitor form that binds to plasminogen. With both forms of the alpha 2-plasmin inhibitor, a complex stable to acetic acid/urea and gel electrophoresis is present and fully developed 15 s after initiation of the reaction with plasmin.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

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

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.     

Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor

Endotoxin-stimulated macrophages hydrolyze fibrin by a plasmin-mediated process in the absence of detectable soluble plasminogen activator (PAs). The data show that macrophages also activate plasmin by a membrane-associated plasminogen activator (PAm). In the presence of endotoxin, PAm activity increases, and plasmin is formed only by PAm. In addition, endotoxin stimulates macrophages to secrete a proteinase inhibitor that blocks PAs activity but not PAm or plasmin activity. The increased PAm activity and the PA inhibitor secretion in response to endotoxin explains the ability of intact macrophages to hydrolyze fibrin in the absence of detectable PAs. Endotoxin, 100 ng/ml, induced an intracellular PA inhibitor in cultured macrophages, and this correlated with accumulation of inhibitor in medium over the cells. The intracellular PA inhibitor was found to be 50--60 kilodaltons by gel chromatography, to be of anionic charge at pH 7.4 and to inhibit urokinase esterolytic and proteolytic activity but not preformed plasmin. These results define two pathways of plasmin formation by intact macrophages and identify the macrophage cell surface as a site of PA activity relatively protected from soluble proteinase inhibitors.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

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.