bluuebunny/crossref_metadata_2025 huggingface dataset:
ANIONIC INHIBITION OF PLASMINOGEN ACTIVATION
Authors: Castellino F J , Urano T , Chibber B , de Serrano Sator V
Published: August 2018

We have found that anions play a significant role in the activation rates of Gluj-plasminogen (GlujPg) by streptokinase (SK) and urokinase (UK), and that these properties of anions can be correlated with their effects on the structure of GlujPg. In the case of activation by SK, a species of activator complex consisting of equimolar SK and GlujPg (SK-Glu]Pg*), that is sensitive to the presence of Cl- and fibrinogen (Fg), exists which decays to another complex of SK and GlujPg (SK-GlujPg'), the activity of which is not influenced by these effector molecules. The overall activation of GlujPg by SK-GlujPg* is inhibited by Cl- and stimulated by Fg. Kinetic studies indicate that Cl- is a mixed type inhibitor of the process, and Fg functions as a mixed type activator.

The activation of GlujPg by both high- and low-molecular weight forms of UK is also inhibited by CL-, but is stimulated by e-amino caproic acid (EACA). The inhibition by Cl- does not occur in the presence of concentrations of EACA that saturate its weak binding sites on GlujPg, and the stimulation by EACA is maximally exhibited in the presence of CL-. Based upon structural studies of GlujPg in the presence of a variety of anions, such as Cl-, F-, I-, CNS-, OTs-, IO3
- and OAc-, we find that GlujPg can adopt a conformation (4.8S form) that is optimal to activation by UK, and one that is much less susceptible to such activation (5.8S form). The former occurs in certain salts and appears to correlate inversely with their ability to bind to the protein, or in the presence of any salt, plus saturating levels of EACA. The latter conformation is adopted in direct proportion with the ability of anions to interact with the protein, according to the Hofmeister series.

In conclusion, we find that anions serve as inhibitors of activation of GlujPg, effects that are reversed by Fg (for SK) and EACA (for UK). This inhibition, which occurs at physiological concentrations of Cl-, serves to inhibit significantly plasminogen activation in normal circulation. Activation of plasminogen is enhanced upon its binding to surfaces (e.g., fibrin), via its lysine binding sites, an effect which may well contain a contribution from sequestration of Cl- from the protein.
CHARACTERIZATION OF LIMITED PROTEOLYTIC DIGESTS OF TISSUE PLASMINOGEN ACTIVATOR
Authors: Teshima G , Harris R , Keck R , Meunier A , Burnier J , Keyt B
Published: August 2018

Tissue plasminogen activator (tPA) is a single chain glycoprotein of 527 amino acids consisting of structural domains homologous to other plasma proteins ("finger","epidermal growth factor", "kringles" and "protease"). Unlike zymogens of other serine proteases, tPA in the single chain form (1-527), has amidolytic and fibrinolytic activity. However, the amidolytic activity is enhanced when tPA is cleaved by plasmin at the Arg275-Ile276 bond to yield the disulfide bonded two chain form. We used trypsin to study the structure and function of tPA by limited digestion. Aliquots of tPA (1 mg/ml) were digested at pH 7 with varying amounts of trypsin (1:10,000, 1:1000, 1:100 and 1:10; enzyme to substrate ratio). The dilute solutions of trypsin (1:10,000) were effective at completely converting one chain tPA to the two chain form, but little additional proteolysis was observed on SDS-PAGE. The proteolytic fragments of tPA were isolated by reduction and carboxymethylation (RCM), SDS gel electrophoresis and reversed phase HPLC. The RCM polypeptides were identified by amino acid composition and sequence. Specific antisera were prepared against peptide antigens of tPA including (1-27), (1-275), (276-527) and (502525). Immunoblotting experiments with the tryptic digests of tPA indicated that the region (1-275) is more susceptible to proteolytic attack than the protease (275-527). Specific cleavage sites were identified at positions 7, 10, 27 and 40. Partially digested tPA preparations were tested for enzymatic activity as determined by hydrolysis of the peptide substrate S-2288 or by clot lysis. Limited proteolysis at the amino terminus was correlated with significant loss of fibrinolytic . activity but minimal effect on the amidolytic activity. Increased tryptic digestion resulted in complete loss of amidolytic activity and significant reduction in antigenic activity as determined by polyclonal anti-tPA ELISA. These results areconsistent with the amino terminal "finger" domain being in part responsible for the fibrin-binding specificity of tPA. Limited tryptic digest of tPA, cleaves first at Arg-275, then subsequently cleaves the "finger" with associated loss of fibrinolytic activity.
New high capacity lysine-agarose based on divinylsul-fone activated agarose
Authors: Lihme A , Nielsen C S , Christensen U
Published: August 2018

It is generally acknowledged that plasminogen is best purified on some kind of immobilized lysine. We here present results on plasminogen purification using a new type of lysine-agarose affinity chromatography.

Divinylsulfone activated agarose1 were used for alpha-N -immobilization of L-lysine ("Mini leak", KernEuTec, P.O. Box 613, DK-2200). The intrinsic features of this matrix are 2,3:

a) High density of active groups, high coupling capacities ( 40- 50mM ) of -NH2,-SH,-OH-groups.

b) Very high mechanical stability

c) Practically no leaking of immobilized ligands.

he matrix is useful for fast and effeicient protein-purification in automated systems. Using a FPLC-system and a variety of exp. conditions lysine-mini1eak, compared with lysine-Sepharose (Pharmacia) consistently yielded more than tvice the amount of plasminogen from plasma and serum.

1) L. Sundberg and J. Porath, (1975) Portides Biol. Fluids, Proc. Colloq., 23, 49-62.

2) A. Lihme et a 1.,( 1 986 ) J. Chromatogr., 376 , 299-305.

3) H.J. Allen and E.A.Z. Johnson,( 1 975 )Carbohydr . Res. 58, 253-265.
INTERACTION OF PLASMIN WITH ALPHA-2 MACROGLOBULIN (α2 M): EFFECT OF ANTIFIBRINOLYTIC AGENTS
Authors: Steiner J , Strickland D
Published: August 2018

Harpel (Harpel, P.C. (1981) J. Clin. Invest 68, 46-55) reported that levels of α2M-plasmin complexes are elevated in patients receiving urokinase. He found that the distribution of plasmin between the two inhibitors, α2M and α2-plasmin inhibitor (α2PI) is dependent upon whether plasmin is added directly to plasma, or whether plasminogen in plasma is activated to plasmin by urokinase. In order to investigate possible mechanisms regulating the distribution of plasmin between these two inhibitors, a study was initiated to examine the effects of antifibrinolytic agents on the reaction of plasmin with α2M. The kinetics of the reaction were measured by monitoring conformational changes in the inhibitor resulting from exomplex formation. In order to minimize nonspecific proteolysis of the inhibitor by plasmin, the reaction was performed under conditions where the concentration of α2M was greater than that of the enzyme. The reaction between Lys77-plasmin and α2M followed second order kinetics with a rate constant of 1.8 X 105M-1 s-1. This rate was not affected 1 mM EACA or by 10 uM histidine rich glycoprotein (HRG). Further, it was found that the rate of Val442-plasmin was essentially the same as that found for Lys77-plasmin. Therefore, the binding of these ligands to the lysine binding sites of plasmin do not affect the association rate between plasmin and α2M. This is in contrast to the reaction of plasmin with α2-PI, where the binding of ligands to the lysine binding sites of plasmin reduce the rate of the reaction (Petersen & Clerrmensen (1981) Biochem. J. 199, 121-127). The kinetic constants measured predict that under conditions when the lysine binding sites of plasmin are occupied, α2M will effectively compete with α2PI in inhibiting plasmin. Further, these studies inplicate HRG as a molecule capable of regulating the distribution of plasmin between these two inhibitors.
PLATELET DISAGGREGATION IN PLASMA--A NOVEL EFFECT OF TISSUE PLASMINOGEN ACTIVATOR
Authors: Vaughan D E , Loscalzo J
Published: August 2018

Platelet aggregates are thought to play a significant role in many clinically important ischemic vascular events. Recently it has been shown that the platelet surface binds plasminogen and, in so doing, enhances its conversion to plasmin by tissue plasminogen activator (tPA). Since fibrinogen, an alternative substrate for plasmin, serves as the cohesive link among platelets induced to aggregate by a variety of agonists, we hypothesized that the local production of plasmin at the platelet surface may be important in promoting the disaggregation of aggregated platelets. When added to a suspension of human platelets induced to aggregate in plasma with adenosine 5'-diphosphate, tPA promoted disaggregation over several minutes. The rate of disaggregation and its extent were dependent on the concentration of tPA as well as on its time of addition. Preincubation of platelet-rich plasma with excess alpha-2-antiplasmin inhibited disaggregation by tPA. While platelet surface fibrinogen receptors did not appear to be proteolyzed by plasmin in this plasma system, platelet-bound cohesive fibrinogen was selectively proteolyzed compared with proteolysis of ambient fibrinogen. The rate of disaggregation correlated best with the rate of loss of platelet-bound fibrinogen and not with the rate of ambient fi-brinogenolysis. These data demonstrate that tPA facilitates platelet disaggregation through the plasmin-mediated proteolysis of cohesive fibrinogen. This phenomenon may be important in the dispersal of circulating platelet aggregates and may be operative in the thrombolysis of platelet-rich clots.
TISSUE PLASMINOGEN ACTIVATOR (T-PA) PRODUCTION BY HUMAN EMBRYONIC FIBROBLASTS, IMR-90, STIMULATED BY PROTEOSE PEPTONE
Authors: Itagaki Y , Suzuki A , Higashio K
Published: August 2018

In order to study the mechanisms by which t-PA production by IMR-90 cells are induced, lactalbumin hydrolysates, yeast extracts, and peptones were tested for their ability to induce t-PA production by IMR-90 cells. IMR-90 cells were grown to confluency in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum at 37°C in 5% CO2 in air. And the cells were maintained in serum free medium containing 1% of each additive. The plasminogen activator activity was determined by fibrin plate method, using urokinase or t-PA from WHO as a standard. It was found that proteose peptone (Difco) and neopeptone (Difco) strongly induced the t-PA production by IMR-90 cells. The t-PA production in DMEM containing 1% proteose peptone reached approx. 200IU/ml after incubation at 37°C for 6 days and was from twenty to fifty times higher than that in DMEM only (control medium). The t-PA production by IMR-90 cells stimulated by proteose peptone was strongly inhibited by RNA synthesis inhibitor(actinomycin D) or prorein synthesis inhibitor (cycloheximide). Hence, t-PA production by IMR-90 cells stimulated by proteose peptone was mediated by de novo synthesis. Chelating reagent (EGTA), Ca2+ entry blocker (verapamil), inhibitor of phospholipase A2 (quinacrine) and inhibitor of lipoxygenase (NDGA) strongly inhibited the t-PA production by IMR-90 cells stimulated by proteose peptone. Inhibitor of cyclooxygenase (indomethacin) was inert. On the contrary, activators of phospholipase A2(Ca2+,melittin) and hydroxy-unsaturated fatty acid (5-HETE) derived from arachidonic acid by lipoxygenase strongly enhanced t-PA production by IMR-90 cells stimulated by proteose peptone. These results suggest that the t-PA production by IMR-90 cells stimulated by proteose peptone is mediated by arachidonate cascade involving the following pathway; (1) proteose peptone stimulates the membrane of IMR-90, (2) this stimulus causes Ca2+ influx, (3) Ca2+ ion activates phopholipase A2, (4) activated phospholipase A2 liberates arachidonic acid from phospholipids in ceil membrane and (5) lipoxygenase converts arachidonic acid into the hydroxy-unsaturated fatty acid.
HETEROGENOUS EXPRESSION OF PLASMINOGEN ACTIVATOR (PA) GENES IN THE HUMAN SARCOMA CELL LINE HT1080
Authors: Laug Walter E
Published: August 2018

Tumor cell derived PA activities are of crucial importance for tissue invasion and destruction by tumor cells. Therefore, we studied the expression of the PA genes in HT1080 cells using immunoenzymatic methods and specific PA gene probes.

Immunenzymatic methods allowed only for the detection of urokinase like PA (u-PA) activities in HT1080 cells which was suppressed by treatment of the cells with dexamethasone (10-7 m). Despite the lack of u-PA activities, the cells still degraded extracellular tissue glycoproteins. Northern blot analysis with specific PA gene probe showed that HT1080 cells express both tissue type PA (t-PA) and u-PA. The enzymatic activities of t-PA were most likely masked by the simultaneous production of inhibitors of PA (PAI). Treatment of HT1080 cells with dexamethasone resulted in increased transcription of t-PA and decreased expression of the u-PA gene, explaining the unchanged tissue destruction by dexamethasone treated HT1080 cells.

Cell clones secreting either large or small amounts of enzymatic PA activities were isolated from the parental HT1080 cell line using a fibrin agarose overlay technique.

The expression of the u-PA gene was enhanced in high secreting PA clones compared to low secreting PA clones when analyzed on Northern blots. This heterogenous expression of the u-PA gene within the HT1080 cell line was confirmed by in situ hybridization with a specific u-PA gene probe.

These findings demonstrate that PA gene expression can be missed with immunenzymatic methods due to simultaneous production of inhibitors of PA. In addition our results show that the expression of a given PA gene may be heterogenous on the cellular level within an established tumor cell line. These findings, therefore, suggest cellular variation of PA gene expression in tumor which may be of fundamental importance for tissue invasion and metastasis by cancer cells.
ANALYSIS OF THE SECRETION MECHANISM OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN A HUMAN MELANOMA CELL LINE (BOWES)
Authors: Sakai T , Okada K , Bando H , Ueshima S , Tanaka N , Matsuo O
Published: August 2018

The human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA) into the culture medium. As reported previously, the secretion of t-PA was depressed and t-PA was accumulated in the intracellular compartment at alternative sodium and potassium ion concentrations and also in the presence of monensin, an ionophore for monovalent cations. In the present study, the secretion and intracellular distribution of t-PA were investigated by radioisotope labeling and fractionation of the cell organelles under normal and monensin-treated conditions. Cell homogenate was fractioneted by discontinuous sucrose density gradient ultracentrifugation. The plasminogen activator (PA) activity or t-PA antigenicity in these fractions was not uniformly distributed, but rather localized in those fractions where cell organelles were rich. This implied that t-PA was enclosed by the intracellular membranous system. Cells were incubated with 35s-methionine and/or 3H-mannose to produce labeled intracellular glycoproteins including t-PA and its premature intermediates, which were separated by immunological adsorption to rabbit anti t-PA IgG-protein A Sepharose. Pulse labeling with 35s-methionine (3 min) demonstrated that intracellular t-PA was transported from the heavier fractions (rER), via intermediate ones (Golgi complex), to lighter ones. The radioactivity of the intracellular t-PA reached a maximum in 30 min, while that of secreted t-PA was observed in 30 min and increased linearly at least for the following 20 min. Enzymography revealed the major intracellular PA activity at 72 kDa and minor activity at 50 kDa. Monensin treatment (10 μM, 6 hr) caused accumulation of the 72 kDa component which was immunologically homologous to secreted t-PA. After long term (3 hr) simultaneous 35s-methionine and 3H-mannose labeling, the radioactivity ratio (3H/35S) the intracellular t-PA was increased in the presence of monensin. These results suggest that some sugar chain(s) in the t-PA molecule are of high mannose type, which is interfered with by monensin in the course of the intracellular processing and transport of t-PA.
DOSE-DEPENDENT PHARMACOKINETICS OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA) IN ANESTHETIZED DOGS FOLLOWING INTRAVENOUS INFUSION
Authors: Fong K L L , Crysler C S , Mico B A , Boyle K E , Kopia G A , Lynn R K
Published: August 2018

The pharmacokinetics of SK&F recombinant two-cha1n tissue-type plasminogen activator (tPA) following Intravenous (i.v.) infusion were characterized in anesthetized, open chested mongrel dogs in which artificial Intracoronary thrombi were formed. SK&F tPA was Infused at rates of 0.5, 1, 2, 4, and 8 μg/kg/min (n=3 to 5 per dose) for 90 min and arterial blood samples were withdrawn during and after Infusion for determination of functionally active tPA concentrations using a modified and validated S-2251 chromogenlc assay. At all doses studied, steady state active tPA plasma concentrations were achieved 10-20 min after the onset of Infusion. Upon cessation of Infusion, active tPA plasma concentrations declined rapidly with a t1/2 of 2-3 min. The active tPA plasma concentration at steady state (Css) and the area under the tPA plasma concentration-time curve (AUC) Increased linearly with dose in the range of 0.5-4 μg/kg/min. However, as the dose was Increased 2-fold from 4 to 8 μg/kg/min, the AUC and the Css Increased 2.5 fold. The systemic clearance ranged from 15-16 ml/min/kg at doses of 0.5-4 μg/kg/min but decreased to 11.7 ml/min/kg at the 8 μg/kg/min dose. With exceptions in 3 dogs, the volume of distribution at steady state approached or slightly exceeded the blood volume. Plasma tPA antigen concentrations were also determined in the dogs receiving the 2 μg/kg/min dose. At steady state, active tPA accounted for 40-60% of the total tPA antigen. The post-infusion t1/2 of the tPA antigen was considerably longer (13.46 ± 5.94 min) than that of active tPA. These results suggested that non-plasminogen activating metabolites (e.g., tPA-inhlbltor complex(es)) are present in the plasma of dogs receiving tPA. It 1s also concluded from the present study that (1) distribution of the i.v. administered tPA molecule was limited primarily to the Intravascular space, and (2) tPA may display dose-dependent pharmacokinetics within the optimum thrombolytic dose range.
PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA
Authors: Heckel A , Hasselbach K M
Published: August 2018

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:

1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.

2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.

3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.

The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.
REC. t-PA GENETICALLY MODIFIED AT THE CLEAVAGE SITE OF ONE-CHAIN TO TWO-CHAIN CONVERSION: ENZYMOLOGY AND DIAGNOSTIC APPLICATIONS
Authors: Rånby M , Brändstrôm A , Hansen L , Henson K , Larsen G
Published: August 2018

Native one-chain t-PA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys-. Variants of one-chain t-PA where the -Arg- was replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through genetic modification. The three mutants and the wild type were expressed in animal cells and purified in the one-chain form by affinity chromatography as was t-PA from Bowes melanoma cells. In contrast to wild type and melanoma t-PA the mutants reacted poorly with polyclonal antibodies raised against the peptide -Gln-Pro-Gln-Phe-Arg-Ile-Lys--Gly-Gly- indicating mutation in the sequence. Of these proteins only the Arg to Thr mutant was resistant to plasmin cleavage as evidenced by SDS-PAGE. t-PA antigen values (ELISA) and fibrinolytic activity values (fibrin clot lysis assay) yielded the following specific activities expressed in IU/|μg: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr), 810 (wild type) and 660 (melanoma t-PA). The amidolytic activities for the one-chain proteins against D-Ile-Pro-Arg-pNA at pH 9.0 and 37°C, expressed in mOD per minute at 1 M-g/mL of enzyme were: 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to Thr), 10.0 (wild type), 9.6 (melanoma t-PA) as compared to 55.2 for two-chain melanoma t-PA.

All mutants including the uncleavable Arg to Thr mutant could be used in determination of PAI activity in plasma samples. Only one-chain t-PA reacts selectively with PAI 1. Thus, use of the Arg to Thr mutant represents a theoretical advantage in PAI 1 activity determination since preparations of this mutant most likely is free of contaminating two-chain t-PA.

The plasminogen activation rate as measured in a coupled assay in the presence and absence of fibrin at 0.5 jiM plasminogen and 37°C was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant wich was considerably higher than that of melanoma one chain t-PA and the other mutants wich all were about 550 fold. The stimulation factor for melanoma two-chain t-PA was in the same experiment about 120 fold. The extra fibrin sensitivity of the Arg to Thr mutant resulted in improved soluble fibrin assay according to Wiman and Renby Thromb. Haemostas, (1986) 55:189-193.

In conclusion: the use of a plasmin insensitive protein-engineered mutant of t-PA gives advantages in assays for PAI 1 and soluble fibrin.
A MECHANISM FOR THE POTENTIATING EFFECT OF UROKINASE (UK) OR TISSUE PLASMINOGEN ACTIVATOR (t-PA) ON CLOT LYSIS BY PRO-UROKINASE (PRO-UK)
Authors: Gurewich V , Black J , Pannell R
Published: August 2018

Pro-UK has been shown to selectively activate fibrin-bound plasminogen (Blood 67:1215, '86). Synergism with t-PA has been postulated to be related to t-PA's rapid induction of plasmin generation which exposes new plasminogen binding sites on fibrin (JBC Harpel et al 260:4432, '85). As a result, more substrate is made available for pro-UK, thereby attenuating its lag phase and greatly potentiating its fibrinolytic effect (Thrombos. Res. 44:217, '86). This hypothesis was tested with UK, which has a rapid onset of action similar to t-PA. In vitro lysis (125I-clots suspended in citrate plasma), by UK (0.05-0.2 μg/ml) plus pro-UK (0.8-0.95 μg/ml) was compared with that of UK or pro-UK alone. Marked potentiation consistent with synergism by UK was found. For example, 0.1 μg/ml UK plus 0.9 μg/ml pro-UK induced clot lysis equivalent to that of 1.25 μg/ml pro-UK or 1.50 μg/ml UK alone using either 50% or 100% clot lysis as end points. Similar findings were obtained when clots were exposed to these concentrations of UK and pro-UK sequentially instead of simultaneously. For this, a clot suspended in plasma was incubated (37°C) for 1 h. with UK, then removed and suspended in fresh plasma (3 ml) for 1 h. after which pro-UK was added. In another set of experiments, a radiolabeled clot was incubated in buffer containing plasmin (2 μg/ml) for 20 min. ( <1% lysis), then transferred to plasma and incubated for 30 min., after which pro-UK (0.8-1.0 μg/ml) or t-PA (0.04 μg/ml) was added. Plasmin pretreatment of clots caused marked potentiation of lysis by pro-UK but did not potentiate t-PA. This potentiation of clot lysis by plasmin pretreatment was nullified by treatment of clots with carboxypeptidase-B, to remove lysine residues.

It was concluded that UK potentiates pro-UK induced clot lysis by exposing plasminogen binding sites on fibrin according to the mechanism postulated above for t-PA. The potentiating effect of UK on pro-UK appears to be analogous to that of t-PA and may also be defined as being synergistic.
ACTIVATION PATHWAYS OF PLASMINOGEN BY UROKINASE AND TISSUE PLASMINOGEN ACTIVATOR IN THE PLASMA OR CLOTTED PLASMA: COMAPRISON WITH PURIFIED SYSTEMS
Authors: Makino Y , Yoshida M , Takada Y , Takada A
Published: August 2018

The activation pathways of a native plasminogen (Glu-plg) were studied in the plasma and purified systems. When purified Glu-plg was activated by urokinase (UK) or tissue plasminogen activator (t-PA), the activation of Glu-plg proceeded very slowly in the presence of fibrinogen, but rapidly in the presence of non crosslinked or cross-linked fibrin. Little formation of modified pig (Lys-plg) was observed in the presence of fibrinogen, but significant amounts of Lys-plg were formed in the presence of fibrin clot. The addition of α2antiplasmin (α2AP) to the mixture of Glu-plg, UK or t-PA, and fibrinogen or fibrin resulted in little formation of Lys-plg in the presence of fibrinogen but significant formation of Lys-plg in the presence of non cross-linked or cross linked fibrin. Alphapantiplasmin did not prevent the formation of Lys-plg from Glu-plg by plasmin in the presence of fibrin regardless of possible cross-linkage of α2AP to fibrin. In contrast to purified system, the activation of Glu-plg by UK or t-PA in the presence of plasma clot resulted in little formation of Lys pig The addition of thrombin and Ca++ to the plasma activated by UK or t-PA resulted in little formation of Lys-plg. These results suggest that Glu-plg was mainly directly activated by UK or t-PA to Glu-plasmin (then Lys-plasmin) in the plasma or plasma clot (non cross-linked or cross-linked), and that some of Glu-plg was converted to Lys-plg by preformed plasmin after UK or t-PA induced activation of Glu-plg in the presence of fibrin. It should be stressed that not all the molecules of Glu-plg were converted to Lys-plg in the presence of fibrin before their activation by activators. Some amounts of Glu-plg were present throughout the activation process in its native form.
AMINO ACID CHANGES AT LYS-158 THAT ALTER THE SENSITIVITY OF SCUPA TO CLEAVAGE BY PLASMIN
Authors: VOVIS G F , MAO J , BROEZE R , ABERCROMBIE D , PUMA P , HSAIO K , ALMEDA S
Published: August 2018

Plasmin converts Scu-PA to two-chain urokinase by hydrolyzing the lys-158/ile-159 peptide bond. Using site directed mutagenesis, the codon at amino acid position 158 was changed from one that codes for lysine to one that codes for either alanine, glutamic acid, or methionine. These DNA constructions were expressed and amplified in Chinese hamster ovary cells. The resulting protein products were isolated and characterized ip vitro. Under conditions where Scu-PA is completely converted by plasmin to two chain urokinase, none of these derivatives were cleaved by plasmin. However, all of these molecules, including Scu-PA, were cleaved by thrombin. These derivatives exhibited low amidolytic activity as determined by using the chromogenic substrate S2444 and low fibrinolytic activity as determined by using fibrin plates. All three derivatives activated either glu-plasminogen or lys-plasminogen to plasmin ip vitro but at rates significantly slower than that seen with Scu-PA.
IN VITRO CLOT LYSIS INDUCED BY SINGLE-CHAIN UROKINASE (scu-PA) AND ACTIVATION OF THE CONTACT PHASE SYSTEM
Authors: Hauert J , Bachmann F
Published: August 2018

Scu-PA was purified from ccaiditioned medium of cell lines (HT 1080, MG 118) by immunoaffinity chrcxnatography on anti-u-PA Sepharose and passage through benzamidine-Sepharose to remove traces of two-chain u-PA (tcu-PA). The amidolytic activity determined with S-2444 was 20'000 u-PA IU/mg before and undetectable ( <700 IU/mg) after the benzamidine-Sepharose step. Pretreatment of scu-PA by plasmin yielded a specific activity of 100'000 IU/mg. We have studied, in vitro, the lysis of plasma clots containing 125I-fibrin as tracer. To obtain 50% lysis in 90 min, 130 IU/ml of scu-PA had to be added to plasma before clotting; an equivalent rate of lysis was obtained with 45 IU/ml of tcu-PA. When, after the addition of scu-PA, the contact phase system of plasma was activated using ellagic acid and phospholipids, lysis was complete within minutes following clotting; 7 IU/ml of scu-PA induced 50% lysis in 90 min. An equivalent concentration of tcu-PA or plasmin-treated scu-PA did not bring about clot lysis after 7 h. Using plasmas deficient in prekalli-krein or in high molecular weight kininogen no lysis occured in the presence of 7 IU/ ml of scu-PA and contact phase activation; lysis reached 15% after 7 h with FXII deficient plasma and was normal with FXI deficient plasma. Clot digestion was totally abolished in the presence of a kallikrein inhibitor (SBTI 0.2 mg/ml) or antibodies directed against prekallikrein or u-PA. Hie addition of FXII inhibitor (LBTI 0.2 mg/ml) or of antibodies against FXII reduced the rate, but did not totally block the lysis. Contact dependent fibrinolysis in the presence of 7 IU/ml of scu-PA was furthermore modulated by plasminogen activator inhibitor 1 (PAI-1). A positive exponential correlation between the level of PAI-1 activity and 50% lysis time was observed (r2=0.83, n=12).

We have previously reported that kallikrein efficiently converts scu-PA into tcu-PA. The present study shows that, in normal human plasma, the fibrinolytic effect of scu-PA is twenty fold potentiated through contact activation and suggests an important physiological role for scu-PA counteracting excessive clot formation in situations associated with contact activation of the coagulation system.
SENSITIVE AND QUANTITATIVE ASSAYS OF FUNCTIONALLY ACTIVE PLASMINOGEN ACTIVATORS IN PLASMA
Authors: Mahmoud M , Gaffney P J
Published: August 2018

A previously described (Mahmoud, M. and Gaffney, P.J., Thrombos.Haemostas. 53: 356-9, 1985) bioimmunoassay for tissue plasminogen activator (t-PA) has been modified in terms of catcher antibody concentration and conditions of incubation of various of the steps in this assay to achieve a sensitivity range of 5 - 500 x 10-3 iu/ml of t-PA. A similar assay has been developed for urokinase (UK) achieving a sensitivity range of 5500 x 10-4 lu/mi. The sensitive t-PA assay has been demonstrated to be a valuable replacement of the traditional euglobulin clot lysis time (ECLT) assay and has the added advantage of avoiding loss of t-PA during formation of a euglobulin fraction and of calibration in terms of an International Standard for t-PA. Using both these assays it has been demonstrated that the enhanced fibrinolytic activity observed following exercise is due to increased levels of t-PA while the level of UK in plasma remains unaltered. This was further confirmed by quenching experiments using specific antibodies to t-PA and UK. In contradiction to an earlier report from this laboratory, free t-PA (10 - 60 x 10-3 iu/ml) has been demonstrated in resting plasma, while the level or free UK activity was 50 - 100 x 10-3.

These sensitive t-PA and UK assays suggest that physiological and drug induced (heparin fraction CY 222 from Laboratoire Choay, Paris, and SP54 from Benechemie, Munich) enhancement of fibrinolytic potential m the human circulation is mainly mediated by t-PA. Venous occlusion of matched groups of subjects with and without deep venous thrombosis (DVT) showed a wide variation in response m t-PA levels in each group and no change in u-PA levels. There was no difference between the DVT and non DVT groups and this suggests that the notion of the 'non-responder' being more prone to thrombosis needs re-examination. Further experimentation with these sensitive assays may help to elaborate whether the levels of UK in plasma play any role in the haemostatic balance m man.
SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA
Authors: Grøndahl-HANSEN J , Agerlin N , Nielsen L S , Danø K
Published: August 2018

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.

The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.
INTRAVENOUS PRESSURE AND t-PA ANTIGEN RELEASE DURING VENOUS OCCLUSION OF UPPER AND LOWER LIMBS
Authors: Haaland K A , SkjØnsberg H O , Thaulow E , GjØnnes G , Godal C H
Published: August 2018

Venous occlusion by means of a sphygmomanometer cuff is a well-established part of the procedure for assessment of fibrinolytic capacity. It has been suggested that the resultant intravenous pressure is responsible for plasminogen activator release.

In this present study we wanted to compare i .v. pressure in the upper and lower limbs, during venous occlusion, and measure t-PA ag release. 8 male volunteers participated. A VenfIon(O.D. 1.2 mm) was inserted in a distal, superficial vein. Blood samples, discarding the first 2-3 ml. were drawn at 4 min. intervals and pressure recordings (Hewlett-Packard)made every min. during the first 8 min. of venous occlusion and thereafter at 4 min. intervals. The cuff was inflated to a pressure midway between systolic and diastolic values after separate measurements of blood pressures of the upper and lower extremities. The occlusion lasted for 20 min.

Blood pressure measurements in the lower limbs gave consistently higher values than for the upper limbs. Hence, the resultant cuff pressure was on the average 20 mmHg higher in the legs.

There was no significant difference in i.v. pressure, evaluated as total area beneath a pressure/time graph, between the upper and lower extremities. In the arms the i.v. pressure reached a plateau phase after 2-4 min. The pressure buildup was slower in the legs.

t-PA values for the upper limbs after 20 min. of venous occlusion: median 15 ng/ml (range 9.4-26.0) and for the lower limbs: 8.15 ng/ml (range 4.8-13.3). This difference is significant, p = 0.035. There was no sifnificant difference between resting t-PA ag' levels prior to occlusion.

We conclude that the fibrinolytic response, measured as t-PA ag, after venous occlusion, is twice as high in the upper as in the lower limbs and not 4-times as high as previously claimed. A possible explanation for the lower fibrinolytic response, could be the slower buildup of i.v. pressure during venous occlusion in the lower extremities.
ON DIFFERENT MOLECULAR EORMS OE PLASMINOGEN ACTIVATOR INHIBITOR
Authors: Carlsson I , Chmielewska J , Wiman B
Published: August 2018

The production of plasminogen activator inhibitor (PAI) by the human cell-lines Hep G2 and HT 1080 have been studied by immunochemical and functional methods. In conditioned medium collected after 2h, the PAI seemed to be almost fully active, but with increasing incubation time the activity was gradually lost, in spite of that the PAI-antigen content increased continously. The active PAI form can be separated from the inactive form by gel-filtration. The inactive form behaves as a low Mr (about 50,000) component in the absence and in the presence of sodium dodecyl-sulphate. In contrast, the active form of PAI behaves as a high Mr (>300,000) compound in the absence of sodium dodecylsulphate but as a low MT compound in its presence. The low M_r inactive PAI has been purified to homogeneity from HT 1080 conditioned medium, collected in the absence of fetal calf serum. This was achieved by chromatography on Concanavalin A-Sepharose, gel-filtration on Sephacryl S-300 and affinity chromatography on insolubilized monoclonal antibodies against PA-inhibitor. On treatment of this form of the inhibitor with 4 mol/L Guanidinium chloride, the activity was regained, but its gel-filtration behaviour was unchanged in the absence of serum/plasma (Mr about 50,000). Addition of plasma or serum prior to the gel-filtration, changed the elution pattern of PAI towards a high Mr form. The reason for this behaviour is not yet fully understood, but the most plausible explanation is the presence of a high Mr PAI-binding protein in plasma/serum. This hypothesis is presently being explored .
ASSOCIATION OF PLASMINOGEN ACTIVATOR INHIBITOR(PAI-1) WITH THE MEMBRANE AND EXTRACELLULAR MATRIX OF HUMAN ENDOTHELIAL CELLS
Authors: Levin G E , Santel L
Published: August 2018

The cytosol, membrane-containing, and extracellular matrix(ECM) fractions of cultured human endothelial cells were isolated and analyzed for the presence and levels of PAI-1. Cells(4×106) released intact from the substratum had 70% (2.85±0.16 units)of their total inhibitor activity in the soluble portion of cell homogenate with the remaining activity(1.26±0.30 units) in the 100,000xg pellet.The ECM contained over twice as much activity as the total cellular inhibitor(9.82±0.35 units).Similar results were obtained with ECM after cell removal by 0.5% Na deoxycholate and hypotonic buffer treatment. Analysis of all three samples by SDS-PAGE and reverse fibrin autography showed inhibitor activity at an identical position on the gel corresponding to Mr=46000.Immunoblot analysis demonstrated that this inhibitor activity represented PAI-1. Inhibitor was not removed from the membrane by treatment with high salt, EDTA,divalent cations, low ionic strength buffer or sonication but was dissociated in the presence of detergents, guanidine HC1, or high pH. In contrast, ECM-associated PAI-1 was not affected by any of these treatments. Pulse-chase experiments indicated that the inhibitor was associated only transiently with the ECM in the presence of cells. None of the PAI-1 deposited during the labeling period was observed after 24 hrs. Incubation of the isolated ECM with protolytically active tPA resulted in a decline in the intensity of the Mr=46000 inhibitor band and the appearance of a Mr=44000 band and a Mr=110000 tPA-inhibitor complex band. Both of these bands were also found in the supernatent. The Mr=44000 band represented PAI-1 as shown by immunoprecipitation studies, although it did not contain anti-tPA activity.Thus, ECM-bound PAI-1 is susceptible to cleavage and release by tPA but does not in all cases form a stable complex with the protease. Elastase had a similar effect: following incubation, all of the PAI-1 was released from the ECM into the supernatent and was converted to the Mr=44000 inhibitor form. Collagenase, heparinase, and chondroitinase ABC had no effect. Therefore, PAI-1 is susceptible to inactivation and release from ECM by several proteases.
THE EXTRACELLULAR MATRIX (ECM) OF CULTURED BOVINE AORTIC ENDOTHELIAL CELLS (BAEs) CONTAINS FUNCTIONALLY ACTIVE TYPE I PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1)
Authors: Mimuro Jun , Schleef Raymond R , Loskutoff David J
Published: August 2018

The ECM of cultured BAEs was analyzed by immunoblotting and reverse fibrin autography and shown to contain PAI-1. PAI-1 containing ECM was incubated with tissue plasminogen activator (tPA) to determine whether the bound PAI-1 was functionally active or latent. The majority of the detectable PAI-1 in the ECM formed complexes with tPA indicating that it was active. The resulting tPA/PAI-1 complexes were released from ECM and recovered in the reaction solution indicating that the PAI-1 in such complexes had lost its ECM binding site. The PAI-1 could not be removed from ECM by incubating it in high salt (2M NaCl), sugar (galactose, mannose, 1 M), heparin (10 mg/ml) or lysine (1 M). However, PAI-1 could be extracted from ECM by treatment with arginine (0.5 M), potassium thiocyanate (2 M), or by incubation at acid pH (2.5). These treatments did not remove appreciable amounts of fibronectin or von willebrand's factor from ECM. ECM depleted of PAI-1 by acid extraction was able to bind all forms of exogenously added PAI-1 including both the active and the latent molecules. The majority of the bound PAI-1 could not form complexes with exogenously added tPA suggesting that the latent form was not activated as a consequence of this interaction. Although the PAI-1 activity in conditioned medium was unstable and decayed with a half life of less than 3 hrs, the half-life of ECM associated PAI-1 was greater than 24 h. These data suggest that PAI-1 is produced by cultured BAEs in an active form, and either released into the medium where it is rapidly inactivated, or bound to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.
ISOLATION OF THE GENE CODING FOR HUMAN PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 (PAI-1)
Authors: Bosma P J , van den Berg E A , Kooistra T
Published: August 2018

A human placenta genomic DNA cosmid library was screened for the presence of the PAI-1 gene using a cDNA probe coding for PAI-1. Two overlapping recombinant cosmids were obtained that contain human DNA spanning 55 kb. The cosmids were mapped using 3' and 5' end probes isolated from an almost full-length cDNA clone of 2.5 kb. The two cosmids were found to contain the entire structural PAI-1 gene (approximately 15 kb) and also included 25 kb 5' flanking sequences. The transcription initiation site was identified by SI nuclease protection experiments and the promotor region was sequenced. Further experiments will be directed at characterizing the regulatory elements of the PAI-1 gene.

In order to determine the chromosomal localization of the PAI-1 gene we have hybridized our genomic clones in situ to metaphase chromosomes of a human blood cell culture. Preliminary experiments show a specific hybridization signal which will enable us to sublocalize the chromosomal position of the PAI-1 gene.
INTERACTION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA)-INHIBITOR COMPLEX WITH FIBRIN
Authors: Okada M , Sakata Y , Matsuda M
Published: August 2018

Tissue type plasminogen activator (tPA) binds to fibrin resulting in marked enhancement of its activity, but also binds to its some specific fast acting inhibitors (PAIs). The ternary interaction of tPA, PAI and fibrin has not been fully elucidated, however. We studied the interaction of the tPA-PAI complex with fibrin using PAI-1 derived from human umbilical vein endothelial cells (ECs), either as a denaturant activated purified form (dPAI) or a native form (nPAI).

When ECs were incubated with125I-tPA, nPAI formed a complex with 125I-tPA (125I-complex) in dose and time dependent manners. Resultant 125I-complex was immunopurified to give a single HOKd protein and examined for its binding to fibrin under various conditions. Mixtures of fibrinogen and 125I-complex were clotted with thrombin and aprotinin, and the clots formed were separated by synersis, washed with Tris-buffer containing albumin and aprotinin. The radioactivity was counted in the clots and the clots liquor, and the %binding of the 125I-complex was caluculated. We found that the 125 I-complex bound to fibrin and that the binding was inhibited by excess amounts of tPA competitively , but by neither purified latent PAI nor dPAI. Inthe presence of 50 mM EACA, 125I-comolex did not bind to fibrin. However, 50% of 125I-tPA bound to fibrin in the presence of excess amounts of EACA. Moreover, binding of 125I-complex to fibrin decreased in accordance with decreasing amounts of fibrinogen clotted. Since tPA has been reported to bind to fibrin through the finger domain at low concentrations of fibrinogen and also through kringle-2 domain at high concentrations of fibrinogen, respectively, our data indicate that the complex binds to fibrin rather specifically via tPA part of the complex, probably via its kringle-2 domain and the finger domain might be masked by PAI complexed with tPA.

As denaturant activated purified PAI a Tone found to bind to fibrin, the binding of tPA-dPAI complex may be rather differently including binding mediated by the dPAI part of the complex.
IMMUNOHISTOCHEMICAL LOCALIZATION OF PLASMINOGEN ACTIVATOR INHIBITOR (PAI) IN TISSUE
Authors: Risberg B , Hansson G K , Eriksson E , Wiman B
Published: August 2018

The origin of tissue plasminogen activator inhibitor (PAI) has not been fully elucidated. Platelets are rich in PAI and endothelial cells (EC) in culture produce the inhibitor (PAI 1), which seems to be a major secretory protein. Another inhibitor (PAI 2) has been demonstrated in the placenta. In the present study we localized PAI 1 in various human tissues using a polyclonal antibody against human PAI 1 and fluorescence technique. Tissue sections were incubated with a polyclonal rabbit-anti-human PAI antibody in various dilutions followed by incubation with biotinylated goat-anti-rabbit IgG and FITC-labelled Avidin. Positive identification using this technique was made in endothelium of liver sinusoids and in hepatocytes. Most vessels in systemic and pulmonary circulation showed positive fluorescence in the endothelial layer. No quantitative evaluation was possible with this technique. Synthesis of PAI in liver could provide an explanation for the efficient inactivation of tissue plasminogen activator (t-PA) during liver passage. Localization of PAI in vascular tissue corroborated studies from EC cultures.