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H R Lijnen

Publications and source records attributed to H R Lijnen.

At least 19 recordsLinked to original sources
PubMed1995

Structure-function relationships in staphylokinase as revealed by "clustered charge to alanine" mutagenesis.

Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine ("clustered charge-to-alanine scan") were characterized. Fifteen of these mutants had specific plasminogen-activating activities of > 20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had specific activities of 3%. SakSTAR13 had an intact affinity for plasminogen and a normal rate of active site exposure in equimolar mixtures with plasminogen. The plasmin-SakSTAR13 complex had a 14-fold reduced catalytic efficiency for plasminogen activation but was 5-fold more efficient for conversion of plasminogen-SakSTAR13 to plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward plasminogen. Thus, plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants. These findings support a mechanism for the activation of plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR to plasmin-SakSTAR.

Alanine↗
PubMed1995

Tissue-type plasminogen activator (tPA) interacts with urokinase-type plasminogen activator (uPA) via tPA's lysine binding site. An explanation of the poor fibrin affinity of recombinant tPA/uPA chimeric molecules.

Differential scanning calorimetry was used to study the domain structure and intramolecular interactions of tPA/uPA chimeras. A high temperature transition centered near 90 degrees C was observed upon melting of the tPA/uPA chimera (amino acids 1-274 of tPA and 138-411 of uPA) and its variant lacking the finger and epidermal growth factor-like modules (residues 1-3 and 87-274 of tPA and 138-411 of uPA). Since neither of the two parent plasminogen activators display such a stable structure, one may suggest that a new stabilizing intramolecular interaction occurs in the chimeras. We found that occupation of the lysine binding site of tPA by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties. All uPA species, single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by epsilon-aminocaproic acid indicating that the lysine binding site of tPA is involved. The binding of uPA with the fluorescein-labeled A-chain of tPA, registered by changes in fluorescence anisotropy, was estimated to have a Kd range of 1-7 microM. The interaction of tPA with uPA determined by solid-phase assays appeared to be tighter, with a Kd range of 50-300 nM. Two synthetic peptides, with and without carboxyl-terminal lysine, corresponding to urokinase residues 144-158 and 144-157, were approximately 100-fold more potent than epsilon-aminocaproic acid with respect to inhibition of the tPA-uPA interaction, indicating that the tPA binding site on urokinase is located within this sequence, close to the activation site Lys158-Ile159. The discovered intermolecular interaction may be related to the reported synergistic effect of simultaneous administration of these two plasminogen activators.

Amino Acid Sequence↗
PubMed1995

Structural domains of streptokinase involved in the interaction with plasminogen.

Two fragments of recombinant streptokinase, comprising amino acids Val143-Lys293 (17-kDa rSK) or Val143-Lys386 (26-kDa rSK), were cloned and expressed in Escherichia coli, purified to homogeneity and their interactions with plasmin(ogen) were evaluated. Both 17-kDa rSK and 26-kDa rSK bound to plasminogen with a 1:1 stoichiometry and with affinity constants of 3.0 x 10(8) M-1 and 12 x 10(8) M-1, respectively, as compared to 6.3 x 10(8) M-1 for the binding of intact recombinant streptokinase to plasminogen. Binding of 17-kDa rSK to plasminogen-Sepharose was displaced by addition of increasing concentrations of recombinant streptokinase, whereas bound recombinant streptokinase was not displayed by 17-kDa rSK. In equimolar mixtures of plasminogen and 26-kDa rSK, the appearance of amidolytic activity as monitored with a chromogenic substrate, was significantly delayed compared to the equimolar mixture with recombinant streptokinase (60% of the maximal activity after 30 min, compared to maximum activity within < or = 2 min). In contrast, no amidolytic activity was generated in equimolar mixtures of plasminogen and 17-kDa rSK. Plasminogen was rapidly activated by catalytic amounts (1:100 molar ratio) of recombinant streptokinase (60-70% within 10-15 min), whereas only 4% of the plasminogen was activated within 60 min with 26-kDa rSK, and no plasmin was generated with 17-kDa rSK. Complexes of plasmin with 17-kDa rSK or with 26-kDa rSK were very rapidly inhibited by alpha 2-antiplasmin (apparent second-order inhibition rate constant of approximately 2 x 10(7) M-1 s-1), whereas the complex with recombinant streptokinase was resistant to inhibition. With 26-kDa rSK, inhibition by alpha 2-antiplasmin resulted in dissociation of the complexes and recycling of functionally active 26-kDa rSK to other plasminogen molecules; 17-kDa rSK, in contrast, remained associated with the plasmin-alpha 2-antiplasmin complex. These findings suggest that different regions of the streptokinase molecule are involved in binding to plasminogen, in active-site exposure, and in impairment of the inhibition of plasmin by alpha 2-antiplasmin. Thus, the 17-kDa region spanning Val143-Lys293 in streptokinase mediates its binding to plasminogen but does not induce activation. Furthermore, this region does not interfere with the inhibition of the complex with plasmin by alpha 2-antiplasmin.

Amino Acid Sequence↗
PubMed1995

Mechanisms of physiological fibrinolysis.

The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, which degrades fibrin. Two immunologically distinct plasminogen activators (PA) have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA mediated plasminogen activation is mainly involved in the dissolution of fibrin in the circulation, whereas u-PA mediated plasminogen activation mainly plays a role in pericellular proteolysis. Plasminogen activation is regulated by specific molecular interactions between its main components, such as binding of plasminogen and t-PA to fibrin, or to specific cellular receptors resulting in enhanced plasminogen activation, inhibition of t-PA and u-PA by plasminogen activator inhibitors (PAI) and inhibition of plasmin by alpha 2-antiplasmin. Controlled synthesis and release of PAs and PAIs primarily from endothelial cells also contributes to the regulation of physiological fibrinolysis. The lysine binding sites situated in the kringle structures of plasminogen play a crucial role in the regulation of fibrinolysis by modulating its binding to fibrin and to cell surfaces, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin.

Fibrinolysin↗
PubMed1995

New thrombolytic agents and strategies.

Despite their widespread use in patients with acute myocardial infarction, all currently available thrombolytic agents suffer from a number of significant limitations, including resistance to reperfusion, the occurrence of acute coronary reocclusion, and bleeding complications. Several lines of research towards improvement of thrombolytic therapy are being explored, including strategies to enhance the fibrinolytic potency of plasminogen activators and to improve conjunctive antiplatelet or antithrombotic agents. Mutants and variants of plasminogen activators, chimeric plasminogen activators, and conjugates of plasminogen activators with monoclonal antibodies have been constructed, and plasminogen activators from animal or bacterial origin have been evaluated. Some of these new thrombolytic agents have shown promise in animal models of venous or arterial thrombosis and in pilot studies in patients with acute myocardial infarction. Such molecules include mutants of tissue-type plasminogen activator (t-PA) with prolonged half-life and/or resistance to protease inhibitors and staphylokinase. Antiplatelet strategies include the use of platelet glycoprotein IIb/IIIa receptor blocking agents, of thromboxane synthase inhibitors and endoperoxide receptor antagonists. Antithrombotic strategies include the use of selective inhibitors of thrombin, tissue factor or factor Xa. The efficiency and safety of these new agents in man will have to be carefully evaluated.

Amino Acid Sequence↗
PubMed1995

Thrombolytic agents in development.

The quest continues for thrombolytic agents with a higher thrombolytic potency, specific thrombolytic activity and/or a better fibrin selectivity. Several lines of research towards improvement of thrombolytic agents are being explored, including the construction of mutants and variants of plasminogen activators (PAs), chimaeric PAs, conjugates of PAs with monoclonal antibodies, and PAs from animal or bacterial origin. Some of these new thrombolytic agents have shown promise in animal models of venous or arterial thrombosis and in pilot clinical studies. Such molecules include numerous mutants of tissue-type PA (t-PA) with prolonged in vivo half-life and/or resistance to protease inhibitors, and chimaeric PAs consisting of different regions of t-PA and of urokinase-type PA (u-PA). Several molecular forms of the thrombolytic substance in the saliva of the vampire bat have been characterised and cloned. Vampire bat PA exhibits 85% homology to human t-PA but lacks kringle 2 and the plasmin-sensitive cleavage site. A thrombolytic enzyme of 203 amino acids is present in the venom of a southern copperhead snake. This polypeptide, termed fibrolase, is now produced by recombinant technology. Fibrolase does not activate plasminogen or protein C, but directly degrades the alpha and beta chains of fibrin and fibrinogen. Recombinant staphylokinase is not an enzyme, but it forms a 1:1 stoichiometric complex with plasminogen, which becomes active after conversion of plasminogen to plasmin. It is a potent and highly fibrin specific thrombolytic agent in animals and patients.

Amino Acid Sequence↗
PubMed1995

Differential inhibition with antifibrinolytic agents of staphylokinase and streptokinase induced clot lysis.

The inhibitory effects of antifibrinolytic amino acids on clot lysis induced with recombinant staphylokinase (SakSTAR) or with streptokinase (SK) were evaluated in a human plasma milieu in vitro and in a hamster pulmonary embolism model in vivo. Addition of tranexamic acid to a system composed of 60 microliters 125I-fibrin-labeled plasma clots submerged in 0.5 ml human plasma, caused dose-dependent inhibition of lysis; complete lysis in 120 min required 30 nM SakSTAR or 100 nM SK and was reduced to 50% with 0.015 mM or with 0.07 mM tranexamic acid, respectively. Aprotinin also produced dose-dependent inhibition; lysis with SakSTAR or with SK was reduced to 50% of the control value with 8 KIU/ml or with 10 KIU/ml aprotinin, respectively. Thus, in human plasma in vitro the antifibrinolytic potency of tranexamic acid was 5-fold higher towards SakSTAR than towards SK, whereas that of aprotinin was comparable towards both agents. In hamsters with pulmonary embolism given 0.063 mg/kg SakSTAR or 0.20 mg/kg SK over 30 min, the antifibrinolytic potency of tranexamic acid, administered as a single bolus injection or as a bolus injection followed by continuous infusion, was 8- to 10-fold higher towards SakSTAR than toward SK (50% reduction of clot lysis with SakSTAR at 12.5 mg/kg, as compared to 100-150 mg/kg with SK). In contrast, aprotinin was equipotent towards SakSTAR and SK (50% reduction of clot lysis with 2,000 to 2,700 KIU/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Staphylokinase: fibrinolytic properties and current experience in patients with occlusive arterial thrombosis.

Staphylokinase is a profibrinolytic agent that forms a 1:1 stoichiometric complex with plasminogen which, following conversion to plasmin, activates other plasminogen molecules to plasmin. The plasmin, staphylokinase complex, unlike the plasmin, streptokinase complex, is rapidly inhibited by alpha 2-antiplasmin. In a plasma milieu, staphylokinase is able to dissolve fibrin clots without associated fibrinogen degradation. This fibrin-specificity of staphylokinase is the result of reduced inhibition by alpha 2-antiplasmin of plasmin, staphylokinase complex bound to fibrin, recycling of staphylokinase from the plasmin, staphylokinase complex following inhibition by alpha 2-antiplasmin, and prevention of the conversion of plasminogen, staphylokinase to plasmin, staphylokinase by alpha 2-antiplasmin. In several experimental animal models, staphylokinase appears to be equipotent to streptokinase for the dissolution of whole blood or plasma clots, but significantly more potent for the dissolution of platelet-rich or retracted thrombi. The feasibility of fibrin-specific coronary thrombolysis with an intravenous infusion over 30 min of 10 mg recombinant staphylokinase was demonstrated in two small pilot studies in patients with acute myocardial infarction with angiographically confirmed total occlusion of the infarct-related coronary artery. However, neutralizing antibodies against staphylokinase were demonstrable from the third week on in all patients. Definition of the therapeutic benefit of recombinant staphylokinase will require more detailed dose-finding studies followed by randomized efficacy studies against other thrombolytic agents. An interim analysis after 50 patients of a randomized trial of recombinant tissue-type plasminogen activator versus staphylokinase in patients with acute myocardial infarction revealed similar rates of coronary patency at 90 minutes but a significantly higher fibrin specificity of the latter compound.

Animals↗
PubMed1995

Interactions of staphylokinase with human platelets.

The interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR. In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin-activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 +/- 2.7% or 101 +/- 1.7% of control in the presence of 0.1 to 20 microM SakSTAR, with corresponding values of 95 +/- 2.8% or 90 +/- 4.6% of control in the presence of 0.1 to 4 microM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 +/- 0.26 microM for SakSTAR (at concentrations of 0.1 to 20 microM) and 4.4 +/- 0.35 microM for SK (at concentrations of 0.1 to 4 microM), as compared to 3.4 +/- 0.70 microM in the absence of plasminogen activator. Fifty % lysis in 2 h (C50) of 60 microliters 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Characterization of the murine plasma fibrinolytic system.

The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, alpha 2-antiplasmin, tissue-type plasminogen activator and plasminogen activator inhibitor-1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts. Initial activation rates of murine and human plasminogen by autologous tissue-type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM-1 s-1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue-type plasminogen activator (k2/Km = 0.01 mM-1 s-1). Plasminogen activation by tissue-type plasminogen activator was stimulated 100- and 160-fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 microM, respectively, of CNBr-digested fibrinogen. Nearly quantitative binding (85-90%) of tissue-type plasminogen activator to fibrin was observed both in autologous and heterologous systems. Murine and human plasmin were very rapidly inhibited by autologous and heterologous alpha 2-antiplasmin (second-order inhibition rate constants, k1,app, of 2.1-2.3 x 10(7) M-1 s-1) and murine and human tissue-type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor-1 (k1,app of 1.8-4.9 x 10(7) M-1 s-1). Two-chain murine tissue-type plasminogen activator (added at a concentration of 1 microgram/ml) was inhibited in normal or plasminogen activator inhibitor-1-deficient murine plasma with half-lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue-type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor-1 which efficiently inhibit autologous tissue-type plasminogen activator. Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20-30% clot lysis in 2 h with 100 nM tissue-type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue-type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half-life of murine tissue-type plasminogen activator. Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are > 30-fold more resistant to lysis with autologous tissue-type plasminogen activator than human plasma clots.

Amino Acid Sequence↗
PubMed1994

Characterization of the binding of urokinase-type plasminogen activator (u-PA) to plasminogen, to plasminogen-activator inhibitor-1 and to the u-PA receptor.

Binding parameters [association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57 x 10(6) M-1 and 1.7 x 10(6) M-1, compared to 14-22 x 10(6) M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser356 converted to dehydroAla) were 81 x 10(6) M-1 and 670 x 10(6) M-1, respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHAla]rtcu-PA occurred with KA values of 3.7 x 10(6) M-1 and 0.33 x 10(6) M-1, compared to 670 x 10(6) M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHAla]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 x 10(6) M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA values of 26 x 10(6) M-1 and 28 x 10(6) M-1, compared to 0.77 x 10(6) M-1 and 3.2 x 10(6) M-1 for binding to the corresponding single-chain u-PA species, and 450 x 10(6) M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5 x 10(9) M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA < or = 0.2 x 10(6) M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Characterization of the interaction between plasminogen and staphylokinase.

Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka = ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu-plasminogen was 0.93 x 10(8) M-1 as compared to 2.0 x 10(8) M-1 and 1.6 x 10(8) M-1, respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with Ka values of 4.0 x 10(8) M-1 and 8.4 x 10(8) M-1, respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with Ka values of 1.0 x 10(8) M-1 and 0.49 x 10(8) M-1, respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1-3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2 x 10(5) M-1 and 2.9 x 10(5) M-1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10-20-fold lower than that of native staphylokinase. These results indicate that: (a) the affinity of staphylokinase for Glu-plasminogen and Lys-plasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1-4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.

Adsorption↗
PubMed1994

Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26.

Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76,000 +/- 10,000, 75,000 +/- 2400 and 78,000 +/- 9700 HU/mg, respectively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 microM) mixtures plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 microM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 microM-1 s-1, 0.16 microM-1 s-1 or 0.12 microM-1 s-1, respectively). Comparable dose-dependent lysis of 0.06 ml 125I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC50, of 17 +/- 1.6 nM, 19 +/- 1.4 nM and 14 +/- 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in position 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.

Amino Acid Sequence↗
PubMed1994

Novel thrombolytic agents.

The fibrinolytic system comprises an inactive pro-enzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, that degrades fibrin. Two immunologically distinct plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Plasminogen activation is regulated by specific molecular interactions between its main components, as well as by controlled synthesis and release of plasminogen activator inhibitors, primarily from endothelial cells. The observed association between abnormal fibrinolysis and a tendency toward bleeding or thrombosis demonstrates the (patho)physiological importance of the fibrinolytic system. Transgenic animals are a suitable experimental model to examine the in vivo impact of fibrinolytic components in thrombosis and thrombolysis. Inactivation, by homologous recombination, of the tissue-type plasminogen activator genes in mice impairs thrombolysis in a significant manner whereas inactivation of the plasminogen activator-1 gene enhances the rate of spontaneous lysis. Despite their widespread use all currently available thrombolytic agents suffer from a number of significant limitations, including resistance to reperfusion, the occurrence of acute coronary reocclusion and bleeding complications. Therefore, the quest for thrombolytic agents with a higher thrombolytic potency, specific thrombolytic activity and/or a better fibrin-selectivity continues. Several lines of research toward improvement of thrombolytic agents are being explored, including the construction of mutants and variants of plasminogen activators, chimeric plasminogen activators, conjugates of plasminogen activators with monoclonal antibodies, or plasminogen activators from animal or bacterial origin.

Amino Acid Sequence↗
PubMed1994

Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog.

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗