Purification and reaction mechanisms of the primary inhibitor of plasmin from human plasma.
ABSTRACT: The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH(4))(2)SO(4) precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen-CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor-plasminogen complex was determined to be approx. 3mum at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme-inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26mum) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor-plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.
Project description:Plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) is regulated by the vascular components fibrin(ogen) fragments, plasminogen and lipoprotein (a). Kinetic analysis demonstrates that CNBr-derived fibrinogen fragments completely protect plasmin from alpha 2AP. Plasminogen and 6-aminohexanoic acid decrease the rate of inhibition by 5- and 10-fold respectively. These studies show that CNBr-derived fibrinogen fragments and 6-aminohexanoic acid bind plasmin kringle(s) with binding constants of 2 micrograms/ml and 120 microM respectively, and that plasminogen binds to alpha 2AP with an affinity of 0.5 nM. The unmodulated inhibition is not effected by the presence of lipoprotein (a), but in the presence of protective CNBr-derived fibrinogen fragments the rate of inhibition is increased by the presence of the lipoprotein. The kinetics demonstrate that lipoprotein (a) binds to CNBr-derived fibrinogen fragments with an affinity of 4 nM, displacing plasmin from the protective surface. In addition, tissue-type plasminogen activator and trypsin inhibition by alpha 2AP is not slowed by the presence of CNBr-derived fibrinogen fragments or plasminogen (Pg), respectively. These kinetics suggest that the initial reversible interaction between plasmin and alpha 2AP is mediated by binding of the inhibitor to the kringle 1 domain of plasmin, with a reversible inhibition constant (Ki) of 5.0 x 10(-10) M. Under conditions where this kringle-inhibitor interaction is blocked, the reversible inhibition still occurs between the plasmin and alpha 2AP, but the initial Ki is increased to 5.0 x 10(-9) M. These data suggest that, in the circulation, plasmin inhibition by alpha 2AP may be down-regulated by fibrin, fibrin(ogen) fragments and Pg, but up-regulated by lipoprotein (a) in the presence of fibrin or fibrin(ogen) fragments. The lipoprotein (a)-mediated promotion of plasmin inhibition may provide an additional mechanism by which the lipoprotein impairs fibrinolysis and promotes atherosclerosis.
Project description: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.
Project description:A new affinity column for renin was prepared by coupling the isosteric peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr, where R is a reduced isosteric bond, -CH2-NH-), to activated 6-aminohexanoic acid-Sepharose 4B. Chromatography of a crude extract of human kidney cortex on this material resulted in a 5500-fold purification of renin in 76% yield. The purified enzyme (specific activity 871 units/mg) was free of non-specific acid-proteinase activity and was stable at pH 6.8 and -20 degrees C over a period of several weeks.
Project description:An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.
Project description:A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.
Project description:Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1-790)]. This binding is decreased by alpha(2)-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, alpha(2)-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Lys(76), Glu(1)-Arg(67) or Glu(1)-Lys(77))display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, alpha(2)-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300-305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of alpha(2)-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Val(441)) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by alpha(2)-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than alpha(2)-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.
Project description:The ability of oleic acid to modulate fibrinolysis was measured by following the urokinase-mediated and plasminogen-dependent cleavage of 125I-labelled fibrin clots. Oleic acid levels within the physiological range exerted a concentration-dependent inhibition of urokinase-mediated fibrinolytic activity. SDS/PAGE revealed that oleic acid enhances urokinase activity but simultaneously increases the autolytic cleavage of the newly formed low-molecular-mass subunit of plasmin. Oleic acid-induced cleavage of this subunit containing the catalytic site of plasmin was suppressed by the plasmin substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251) and was prevented by alpha 2-antiplasmin. A concentration-dependent inhibition of the activity of purified plasmin on 125I-labelled fibrin clot was also observed; 93% and 50% inhibition was noted with 150 microM and 32 microM oleic acid respectively. Oleic acid at 200 microM also effectively displaced plasmin prebound to a polylysine-Sepharose column. Examination of the fatty acid specificity showed that a minimal chain length of 16 carbon atoms and the presence of at least one double bond, preferably in a cis configuration, were required for inhibition of the fibrinolytic activity of plasmin. Oleic acid at a concentration that produced only a minimal inhibition of plasmin activity induced a marked inhibition by palmitic acid, while palmitic acid alone is ineffective. The findings suggest that oleic acid stimulates plasminogen activation and modulates the fibrinolytic and autolytic activities of plasmin.
Project description:The amidolytic activity of plasmin with the chromogenic substrate H-D-valyl-L-leucyl-L-lysine p-nitroanilide (S-2251) is stimulated by oleic acid in a dose-dependent and saturable fashion. The activity of plasmin on S-2251 in the presence of oleic acid followed a sigmoidal kinetic pattern, with an almost 4-fold stimulation of activity at 60 microM-oleic acid. Half-maximal stimulation occurred at an oleic acid level of 19.5 microM. The amino acid analogue 6-aminohexanoic acid (AHA), which is known to bind to lysine-binding sites in plasmin, suppressed the stimulatory effect of oleic acid in a concentration-dependent manner; at 0.3 mM-AHA, about 70% of the oleic acid-dependent enhancement of plasmin activity was abolished. The l/v versus 1/[S] plot for plasmin changed in the presence of oleic acid from a linear to a non-linear curve, suggesting positive co-operativity. 14C-labelled oleic acid bound to plasmin, and the bound ligand was displaced by an excess of unlabelled oleic acid. Oleic acid also produced a marked (40-fold) stimulation of the plasminogen-dependent cleavage of S-2251 by urokinase. A half-maximal effect on plasminogen activation was obtained at 40 microM-oleic acid. The present findings suggest that the ability of oleic acid to stimulate plasmin activity and to enhance the conversion of plasminogen to plasmin depends on the interaction of oleic acid with specific lysine-binding sites in plasmin.
Project description:The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.
Project description:Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.