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Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for 'second-site' interactions.

ABSTRACT: The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.

SUBMITTER: Chmielewska J

PROVIDER: S-EPMC1149006 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

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Project description:Background and aimsPlasmin in human milk partially hydrolyzes milk proteins within the mammary gland and may enhance the hydrolysis of milk proteins within the infant's stomach. This study examined the effects of extremely preterm (EP)-, very preterm (VP)-, and term-delivery on plasmin activity and the concentrations of plasminogen activators [urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA)], plasminogen activator inhibitor type 1 (PAI-1) and the complexes of PAI-1/uPA and PAI-1/tPA in human milk.Materials and methodsHuman milk samples were collected from mothers who delivered extremely preterm infants [24-27 weeks gestational age (GA), n = 20], very preterm infants (28-32 weeks GA, n = 12), and term infants (38-39 weeks GA, n = 8) during 2-72 days postnatally. Plasmin activity was determined using fluorometric substrate assay, whereas concentrations of uPA, tPA, PAI-1, the PAI-1/uPA complex and the PAI-1/tPA complex were quantified by ELISA.ResultsPlasmin activity, uPA and tPA were detected in all human milk samples, PAI-1 and the PAI-1/uPA complex were present in 42.5 and 32.5% of milk samples, respectively, and the PAI-1/tPA complex was not detected. Plasmin activity was correlated negatively with postnatal age and postmenstrual age (PMA) in the VP group and positively with postnatal age in the term group. uPA and tPA concentrations decreased with increasing postnatal age in both EP and VP groups but did not correlate in the term group. uPA concentration was correlated positively with GA in the VP group and tended to be elevated with increasing GA in the combined three groups. In contrast, tPA concentrations were correlated negatively with GA and PMA in the combined three groups (P < 0.008) and with PMA in the EP and VP groups. PAI-1 concentration tended to be correlated positively with postnatal age in the combined three groups. No correlation was detected with the PAI-1/uPA complex.ConclusionPremature delivery impacted the plasmin activity and the concentrations of uPA, tPA, and PAI-1 in human milk. Whether these changes in milk plasminogen activators and inhibitors have a role in balancing the proteolytic digestion of premature infants remains to be investigated.

Project description:The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma.

Project description:Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

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Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for 'second-site' interactions.

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