Deficiency of vascular endothelial growth factor-D does not affect murine adipose tissue development.

Vascular endothelial growth factor (VEGF)-D deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a standard fat (SFD) or a high fat diet (HFD) for 15 weeks. The composition of SC and GON adipose tissues of VEGF-D deficient mice in terms of size and density of adipocytes or blood vessels was also comparable to that of wild-type control mice. Staining of lymphatic vessels in adipose tissue sections did not reveal marked differences between both genotypes. The absence of an effect of VEGF-D deficiency could not be explained by compensatory increases of VEGF-C expression in adipose tissues of the deficient mice. Thus, our data do not support an important role of VEGF-D in (lymph) angiogenesis or in adipose tissue development.

Stromelysin-2 (MMP-10) deficiency does not affect adipose tissue formation in a mouse model of nutritionally induced obesity.

Adipose tissue development is associated with angiogenesis, adipogenesis and extracellular matrix degradation. The class of matrix metalloproteinases contributes to these processes, but little information is available on the role of individual proteinases. We report that stromelysin-2 (MMP-10) deficiency has no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a high fat diet for 15 weeks. The adipocyte size and density in SC and GON adipose tissues were also comparable in MMP-10 deficient and wild-type control mice. Similarly, blood vessel size and density in obese SC and GON adipose tissues was not affected by MMP-10 deficiency. Metabolic parameters and blood cell composition were similar for both genotypes. Stromelysin-1 (MMP-3) expression was significantly reduced in adipose tissues of the deficient mice as compared to the wild-type controls. These data indicate that MMP-10 does not significantly contribute to adipose tissue development and associated angiogenesis in a mouse model of nutritionally induced obesity.

Inhibition of vascular endothelial growth factor receptor tyrosine kinases impairs adipose tissue development in mouse models of obesity.

We have studied the effect of PTK787 (Vatalanib), an inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, on adipose tissue development. Oral administration of PTK787 for 4 weeks (2 mg/g high fat diet, HFD) to C57Bl/6 mice resulted in a significant reduction in total body weight and of subcutaneous (SC) and gonadal (GON) adipose tissue mass, as compared to control animals fed HFD only (all p<0.0005). In the GON adipose tissue adipocytes were hypertrophic after PTK787 treatment. Blood vessel size and density were not significantly affected by PTK787 treatment. Expression of Flk-1 (VEGFR-2) mRNA was significantly reduced in SC and GON adipose tissues of PTK787 treated mice. De novo fat pad formation following injection of preadipocytes in NUDE mice was significantly (p<0.005) impaired by PTK787 administration (2 mg/g HFD for 4 weeks), without associated effect on blood vessel size or density. Thus, in nutritionally induced murine obesity models, oral administration of the VEGFR tyrosine kinases inhibitor PTK787 resulted in reduced adipose tissue development.

Plasminogen activator inhibitor-1 contributes to the deleterious effect of obesity on the outcome of thrombotic ischemic stroke in mice.

BACKGROUND: It is widely accepted that obesity is a risk factor for ischemic heart disease, but the association with stroke is less clear. Adipose tissue is an important source of plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activation. OBJECTIVE: To test the hypothesis that elevated PAI-1 levels associated with obesity negatively affect the outcome of thrombotic ischemic stroke. METHODS: Middle cerebral artery (MCA) occlusion was induced photochemically in mice with nutritionally induced or genetically determined obesity and their lean counterparts. RESULTS: The MCA occlusion time (to obtain complete occlusion) was significantly shorter in obese (nutritionally induced) than in lean wild-type (WT) C57Bl/6 mice, whereas the infarct size was significantly larger and intracranial hemorrhage (ICH) was enhanced (all P < 0.05). Similar observations were made in genetically obese ob/ob mice, as compared to lean WT littermates. In both strains, obesity was associated with markedly elevated circulating PAI-1 levels, probably originating from the fat tissue. In contrast, PAI-1-deficient lean and obese mice did not display significant differences in MCA occlusion time, infarct volume or ICH. CONCLUSIONS: Plasminogen activator inhibitor-1 may play a functional role in the deleterious effect of obesity on the outcome of thrombotic ischemic stroke in mice.

Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator.

Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escherichia coli (recombinant scu-PA). The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics. Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs with a high affinity (Km below 0.8 microM) for both scu-PAs, while the catalytic rate constant (k2) is 0.017 s-1 for recombinant scu-PA but only 0.0009 s-1 for natural scu-PA. Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity (Km about 5 microM) for natural and recombinant scu-PA and with a k2 of 0.23 s-1 for natural and 1.2 s-1 for recombinant scu-PA. Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity (Km 30-50 microM) but with a high catalytic rate constant (k2 about 5 s-1) for both natural and recombinant tcu-PA. The differences in the kinetic parameters of the activation of plasminogen by natural or recombinant scu-PA are thus mainly due to differences in turnover rate in the first reaction. Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recombinant scu-PA than for natural scu-PA. Thus, surprisingly, the artificial, unglycosylated recombinant scu-PA molecule has a better catalytic efficiency than its natural glycosylated counterpart.

Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met(374)-Ser(375) (P3-P2), Glu(416)-Leu(417) and Ser(432)-Leu(433) peptide bonds in human alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37 degrees C, alpha(2)-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. alpha(2)-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro(19)-Leu(20) peptide bond in alpha(2)-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional alpha(2)-AP variant (Ala-alpha(2)-AP or alpha(2)-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37 degrees C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of alpha(2)-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.

On the molecular interactions between plasminogen-staphylokinase, alpha 2-antiplasmin and fibrin.

The molecular interactions between the plasminogen-staphylokinase complex, alpha 2-antiplasmin and fibrin were studied by measuring the effect of CNBr-digested fibrinogen on the inhibition rate of the plasminogen-staphylokinase complex by alpha 2-antiplasmin. The second-order rate constant for the inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin was 2.7 +/- 0.3.10(6) M-1 s-1 (mean +/- S.D.; n = 7). Addition of CNBr-digested fibrinogen, but not of fibrinogen, resulted in a concentration-dependent reduction of the apparent inhibition rate constant, with a 50 percent reduction at a concentration of 5 nM CNBr-digested fibrinogen. The second-order rate constant for the inhibition of the low-Mr plasminogen-staphylokinase complex (plasminogen lacking the kringle structures comprising the lysine-binding sites) by alpha 2-antiplasmin was about 30-fold lower (9.3 +/- 0.7.10(4) M-1 s-1, mean +/- S.D.; n = 4) than that of plasminogen-staphylokinase and was not affected by addition of CNBr-digested fibrinogen. Inhibition of the plasminogen-staphylokinase complex by the chloromethylketone D-Val-Phe-Lys-Ch2Cl is 9-fold less efficient than that of plasmin (k2/Ki of 700 M-1 s-1 versus 6300 M-1 s-1). Our results confirm and establish that rapid inhibition of plasminogen-staphylokinase by alpha 2-antiplasmin requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. Fibrin, but not fibrinogen, reduces the inhibition rate by alpha 2-antiplasmin by competition for interaction with the lysine-binding site. Protection of the plasminogen-staphylokinase complex bound to fibrin from rapid inhibition by alpha 2-antiplasmin thus appears to contribute to the fibrin-specificity of clot lysis with staphylokinase in a plasma milieu, by allowing preferential plasminogen activation at the fibrin surface, while the free complex is rapidly inhibited in plasma.

Effects of plasminogen activator inhibitor-1 on ischemic brain injury in permanent and thrombotic middle cerebral artery occlusion models in mice.

BACKGROUND AND OBJECTIVES: Tissue plasminogen activator (t-PA) improves the outcome of ischemic stroke by recanalization of occluded vessels, but has neurotoxic side effects in experimental stroke models. Here, the effect of plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of t-PA, on ischemic infarct volume was studied. METHODS: After either permanent ligation or thrombotic occlusion of the middle cerebral artery (MCA), infarct volume, spontaneous reperfusion of thrombosed MCA, t-PA/PAI-1 complex level, and blood-brain barrier (BBB) permeability in the ischemic region was studied in transgenic mice with overexpression of PAI-1 and wild-type littermate controls and in mice with intracerebroventricular injection of human PAI-1. RESULTS: Infarct volume was smaller in PAI-1 transgenic mice (2.9 +/- 3.7 mm3, mean +/- SD) than in controls (8.9 +/- 5.0 mm3, P < 0.05) after permanent MCA ligation (plasma PAI-1 level 39 +/- 23 ng mL(-1) in transgenic mice vs. 1.5 +/- 0.6 ng mL(-1) in controls), whereas after MCA thrombosis it was larger in transgenics (13.1 +/- 3.1 mm3) than in controls (8.0 +/- 3.2 mm3, P < 0.05). Spontaneous reperfusion of the thrombosed MCA was significantly delayed in transgenic vs. control mice. In the ligation model, t-PA/PAI-1 complex levels were higher and BBB disruption was more pronounced in the ischemic region. Human PAI-1 injection reduced infarct volume by about 50% in wild-type mice but not in t-PA gene deficient mice. CONCLUSIONS: High PAI-1 levels reduced infarct volume in the permanent MCA ligation model, but enhanced it in the MCA thrombosis model.

On the role of plasminogen activator inhibitor-1 in adipose tissue development and insulin resistance in mice.

OBJECTIVES: To investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue development and insulin metabolism. METHODS: Aged male wild-type (WT) or transgenic mice with adipose tissue overexpression of PAI-1 (45-55 weeks) in 50% C57Bl/6: 50% Friend Virus B-strain (FVB) genetic background, kept on normal chow, were used without or with administration of a synthetic low molecular weight PAI-1 inhibitor (PAI-039) to the food (1 mg g(-1)) for 4 weeks. RESULTS: The PAI-1 transgenic mice showed somewhat lower body weight and adipose tissue mass than WT mice, whereas fasting insulin levels were higher. Glucose and insulin tolerance tests did not reveal significant differences between both genotypes. Addition of PAI-039 to the food did not significantly affect total body fat, weight of the isolated s.c. and gonadal fat territories or their adipocyte size and blood vessel composition in either genotype. Fasting glucose levels and glucose tolerance tests were, for both genotypes, comparable with those without inhibitor treatment. Insulin levels and insulin tolerance tests in WT, but not in PAI-1 transgenic mice, suggested a higher insulin sensitivity after inhibitor treatment (insulin level 30 min after glucose injection of 2.0 +/- 0.17 ng mL(-1) vs. 3.2 +/- 0.48 ng mL(-1) without inhibitor treatment; P = 0.028). CONCLUSIONS: In this model, overexpression of PAI-1 moderately impaired adipose tissue formation without affecting glucose or insulin tolerance. Administration of a synthetic PAI-1 inhibitor for 4 weeks did not affect adipose tissue development in WT or PAI-1 transgenic mice, but induced a higher insulin sensitivity in WT mice.

Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface.

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner. This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasmin (microPli), the 28 kDa serine protease domain of plasmin. Biopanning indicated that residues Arg94 and Gly174 in microPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled mutations Glu94<-->Lys75 reversed and Gly174<-->Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1.8 to 2.3 kcal mol-1, respectively) and the plasminogen activation ability of the mutated complexes were partially restored. These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of microPli, that is in agreement with the structure of the Sak-microPli complex as recently derived by other methods.

Matrix metalloproteinase inhibition impairs adipose tissue development in mice.

The effect of galardin, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, was studied in mice kept on a high fat diet (HFD). Five-week-old male wild-type mice were fed the HFD (42% fat) for up to 12 weeks and were daily injected intraperitoneally with the inhibitor (100 mg/kg) or with vehicle. After 12 weeks of the HFD, the body weights of both groups were comparable, but the weight of the isolated subcutaneous (SC) or gonadal (GON) fat deposits was significantly lower in the inhibitor-treated group than in the control group (88 +/- 11 versus 251 +/- 66 mg, respectively, for SC fat [P<0.05]; 90 +/- 24 versus 217 +/- 30 mg, respectively, for GON fat [P<0.02]). The number of adipocytes was somewhat higher and the diameter was somewhat smaller (but not significantly) in adipose tissues of the inhibitor-treated group. Adipose tissue of the inhibitor-treated mice contained more collagen than did that of the vehicle-treated mice (Sirius red-stained area of 42 +/- 2.6% versus 22 +/- 4.4%, respectively, for SC fat [P<0.05]; 21 +/- 5.1% versus 4.7 +/- 0.92%, respectively, for GON fat [P<0.01]); a distinct collagen-rich cap was formed around the inhibitor-treated tissue. In situ zymography with casein- or gelatin-containing gels confirmed a reduced MMP activity in SC and GON adipose tissues of inhibitor-treated mice. Thus, in this model, growth and development of adipose tissue appears to be limited by the formation of a collagen-rich matrix cap around the inhibitor-treated tissue. These data suggest a functional role for MMPs in the development of adipose tissue.

Neointima formation and thrombosis after vascular injury in transgenic mice overexpressing plasminogen activator inhibitor-1 (PAI-1).

The controversial role of plasminogen activator inhibitor-1 (PAI-1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI-1 (PAI-1 Tg) and in wild-type (WT) controls. Despite the high circulating PAI-1 levels in the PAI-1 Tg mice (52 +/- 9.8 ng mL-1 vs. 0.76 +/- 0.17 ng mL-1 in WT mice), no significant fibrin deposition was observed in non-injured femoral arteries of 8- to 12-week-old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situ fibrinolytic activity in arterial sections of the PAI-1 Tg mice. The neointimal and medial areas were similar in WT and PAI-1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 +/- 0.25 and 1.04 +/- 0.17 at the center of the injury). Nuclear cell counts in cross-sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI-1 Tg mice (224 +/- 63, 233 +/- 20), and the distribution pattern of alpha-actin-positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI-1 does not affect neointima formation.

Recombinant human microplasmin: production and potential therapeutic properties.

The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen ( micro Plg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin ( micro Pli) and equilibrated with 5 mmol L(-1) citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg(-1) micro Pli reduced infarct size at 24 h from 27 (26-30) to 25 (21-28) mm3 (median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg(-1) rt-PA and 40 mg kg(-1) micro Plg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14-30) to 9.1 (3.1-25) mm3 with 5.0 mg kg(-1) micro Pli (n = 15 each, P < 0.002) and to 11 (5.2-27) mm3 with 4.0 mg kg(-1) rt-PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with micro Pli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg(-1) micro Pli over 2 h, induced 51 +/- 15% lysis (mean +/- SD, n= 11) vs. a control value of 23 +/- 5.5%. micro Pli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of micro Pli in both models was similar to that of intact plasmin. These findings indicate that recombinant micro Pli may be useful for treatment of ischemic stroke and arterial thrombosis.

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)

Interaction of heparin with histidine-rich glycoprotein and with antithrombin III.

The interaction between heparin, histidine-rich glycoprotein and antithrombin III was studied in purified systems. Histidine-rich glycoprotein binds heparin and thereby interferes with its interaction with antithrombin III, resulting in neutralization of the anticoagulant activity. This interaction occurs with clinical grade heparin as well as with high affinity (for antithrombin III) heparin and with a high affinity heparin fragment with Mr 4,300. Low affinity heparin competes with high affinity heparin for the binding to histidine-rich glycoprotein which results in an apparent increase of the anticoagulant activity of high affinity heparin. The interaction between heparin and histidine-rich glycoprotein is counteracted by Ca2+-binding anticoagulants, indicating that it is dependent on the presence of divalent metal ions. Ethylenediaminetetraacetate is a much more potent inhibitor of the interaction between heparin and histidine-rich glycoprotein than citrate.

Histidine-rich glycoprotein modulates the anticoagulant activity of heparin in human plasma.

Addition of 0.05 IU of heparin per ml normal plasma prolongs its thrombin time from 20 to 27s , but that of plasma specifically depleted in histidine-rich glycoprotein (by immunoadsorption) from 20 to 180s . Reconstitution of the depleted plasma with normal plasma or with purified histidine-rich glycoprotein normalizes its thrombin time. In 54 plasma samples from hospitalized patients a significant negative correlation was found between the anticoagulant activity of heparin measured by thrombin inhibition and the plasma level of histidine-rich glycoprotein (r = 0.69). It is concluded that the level of histidine-rich glycoprotein modulates the anticoagulant activity of heparin in human plasma to an extent which appears to be pharmacologically and possibly clinically relevant.

Functional properties of p-anisoylated plasmin-staphylokinase complex.

The kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p'-anisate-HCl was 52 M-1 s-1 and its deacylation rate constant 1.2 x 10(-4) s-1 (t1/2 of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min. The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

Biological and thrombolytic properties of proenzyme and active forms of human urokinase--I. Fibrinolytic and fibrinogenolytic properties in human plasma in vitro of urokinases obtained from human urine or by recombinant DNA technology.

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) obtained by expression of the human urokinase cDNA in E. coli, were compared with those of natural urokinase (Nat-UK) of urinary origin and of tissue-type plasminogen activator (t-PA) in a system, composed of a radioactive human plasma clot immersed in citrated human plasma. The specific fibrinolytic effects of Nat-UK, Rec-pro-UK and Rec-UK were very similar, causing significant clot lysis at concentrations of 100 IU/ml plasma or more. t-PA caused equivalent degrees of clot lysis at 10-fold lower concentrations. Activation of the fibrinolytic system in the plasma (fibrinogenolysis), was not observed with t-PA in concentrations which induced complete clot lysis within 5 hr (20-30 IU/ml plasma). With Nat-UK and Rec-UK, all concentrations which caused significant clot lysis (100-200 IU/ml plasma) also caused extensive activation of the plasma fibrinolytic system. With Rec-pro-UK an intermediate response was obtained. The highest amounts required for complete clot lysis in 5 hr (200 IU/ml plasma) also caused significant fibrinogenolysis. At intermediate concentrations (50-100 IU/ml), however, significant clot lysis (40-80%) was observed without systemic fibrinolytic activation.

Effect of fibrin-like stimulators on the activation of plasminogen by tissue-type plasminogen activator (t-PA)--studies with active site mutagenized plasminogen and plasmin resistant t-PA.

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenized to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA-Glu275, a recombinant single chain t-PA in which the Arg of the plasmin sensitive Arg275-Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s-1) per 1 nM enzyme. In the absence of fibrin stimulation, the v0 for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s-1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s-1). In the presence of 1 microM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and 33 pM s-1 respectively), whereas with 1 microM CNBr-digested fibrinogen, the v0 for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s-1 respectively). In contrast, the v0 for nPlg and rPlg-Ala740 by single chain rt-PA-Glu275, two-chain rt-PA-Glu275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition in purified systems and in human plasma of chimaeric plasminogen activators consisting of the NH2-terminal region of tissue-type plasminogen activator and the COOH-terminal region of urokinase-type plasminogen activator.

Recombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J. Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37 degrees C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37 degrees C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t1/2 of approximately 45 min.(ABSTRACT TRUNCATED AT 250 WORDS)