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Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with Mr congruent to 70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with Mr congruent to 140,000 without plasminogen activator activity. Overnight incubation at 37 degrees C of postexercise plasma revealed a shift of the Mr congruent to 70,000 peak to the Mr congruent to 140,000 position, suggesting that the Mr congruent to 140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). alpha 2-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma 13 and is therefore most probably at least in part responsible for the generation of the Mr congruent to 140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of 125I-labeled extrinsic plasminogen activator in alpha 2-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-alpha 1-antitrypsin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with alpha 2-antiplasmin and alpha 1-antitrypsin in plasma was obtained from two-site immunoradiometric assays, in which solid-phase anti-inhibitor antibody bound the corresponding complex, which was then detected with radiolabeled, affinospecific antibody against extrinsic plasminogen activator. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with alpha 2-antiplasmin and alpha 2-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH 2Cl, at rest and after exercise and are therefore assumed to circulate in vivo.

Patients with hyperlipoproteinaemia or spontaneous thromboembolism, known to be poor responders to DDAVP or to venous occlusion with regard to the rise in fibrinolytic activity of the blood, appeared to show a normal increase in t-PA-antigen after the same procedure. In their plasma a higher than normal level of free, fast-acting t-PA-inhibitor was found as measured by titration with purified t-PA. This free t-PA-inhibitor level only decreased after the test, in contrast to its complete disappearance in normal responders. The same happened in a healthy volunteer who failed to exhibit a rise in fibrinolytic activity after exhaustive exercise. We suppose that the lack of response of the fibrinolytic activity in these cases is due to a high inhibitor level and not to impaired release of t-PA into the blood. In contrast, patients with terminal renal insufficiency showed only a slight increase in t-PA-antigen after DDAVP. The level of free fast-acting inhibitor was normal in most cases and did not change appreciably during DDAVP-infusion. In these patients, a true impairment of the release of t-PA appears to exist.

Human tissue-type plasminogen activator was produced in centigram quantities from 50-60 1 batches of conditioned medium of a human melanoma cell culture. The yields of the procedure remained essentially unchanged during 50 subsequent preparations. The final products were of high purity as assessed by SDS gel electrophoresis. These materials, after filtration on 0.22 microM Milliporefilters were sterile and free of viruses and pyrogens. Their stability was very good in the fluid form at temperatures up to 56 degrees C. Material obtained by the present procedure has been used to investigate the biological and thrombolytic properties of tissue-type plasminogen activator.

Plasminogen activator(PA)-tissue and urokinase type-and PA-inhibition in plasma were investigated in 52 consecutive cancer patients with a variety of tumors. At first patients were analyzed as one group. Secondly patients were subdivided into two groups, one with (n = 42) and one without (n = 10) metastasis. Our results show that tissue-type-PA antigen (t-PA-antigen) and PA-inhibition were both significantly increased irrespective of the presence or absence of tumor metastasis (p less than 0.001 compared to age matched healthy controls. In the group without metastasis a significantly decreased level of t-PA activity was found (p less than 0.001) but in the group with metastasis t-PA activity was normal. These data seem to reject the hypothesis that decreased plasma fibrinolytic activity is one of the prerequisites for tumor metastasis.

It is known that plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may continue during plasma handling, leading to artificially low fibrinogen (Fbg) and α2-antiplasmin (AP) values. Addition of D-Phe-Pro-Arg-CH2Cl or quenching antibodies against t-PA prevents this phenomenon, but these additions do not allow measurement of t-PA activity. The question of this study is, why the in vitro effects occur, even during freezing of the samples. Normal plasma was supplemented with various amounts of two-chain melanoma or recombinant t-PA and stored at -20°C, with and without a prior snap-freeze procedure. AP consumption (chromo-genic substrate assay) and Fbg degradation (Clauss method), measured in thawed samples, were most pronounced in the non snap-frozen samples. As it took a relatively long time before these samples were really frozen, the time course of the effects was studied at different temperatures. Plasma samples containing 1000 IU t-PA per ml were incubated at 37, 25,10, 0 and -8°C between 0 and 120 min. AP reduction was most rapid at 37°C (50% after 13 min), was less at 25°C (50% after 30 min), but did not further decrease at lower temperatures. The AP reduction at temperatures between 25 and -8°C corresponded to the effect of 40% t-PA activity at 37°C. The Fbg values gave a similar picture: the most rapid reduction occurred at 37°C, a slower reduction at 25°C, but no further reduction (even a small increase) was found from 25 to -8°C. The experiments were repeated in a purified system, consisting of t-PA, plasminogen, Fbg and AP. In contrast to the plasma system, AP reduction gradually decreased from 37 to 0°C. The apparent t-PA activity at 0°C was 6% of the activity at 37 °C.It is concluded that the in vitro effects in plasma samples containing t-PA can be, at least partially, explained by an abnormally strong plasminogen activation around 0°C. A normal temperature dependency in the purified system strongly suggests that unknown plasma factors enhance plasminogen activation at low temperatures.

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.

α2-Antiplasmin (AP) occurs in blood in two functional forms: a plasminogen binding (PB) and a non-PB (NPB) form with differences in the mechanism of plasmin inhibition. The two forms can be assayed using modified crossed immuno-electrophoresis with plasminogen incorporated in the first dimension gel. We studied the consumption of both AP-forms by activating plasminogen with t-PA: In vitro by addition of one-chain t-PA to plasma and in vivo in patients receiving two-chain rt-PA (BW-t-PA) during myocardial infarction. Results demonstrate that at various dosages of t-PA (up to 3 μg/ml plasma or 100 mg/90 min) after 90 min specifically the PB form was consumed (up to 95%), while the NPB form remains essentially unchanged (decrease ≤ 15%). Also during longer incubation the NPB form was not decreased. The consumption of the PB form was accompanied by the formation of plasmin-AP (PAP) complexes, which could be precipitated by antibodies to AP. SDS-PAGE followed by immunoblotting showed the complex and the post-complex peptide of about 14 kD immunochemic-ally related to AP. Also agarose-electrophoresis resulted in the separation of the peptide from the PAP complex. It showed a high electrophoretic mobility, which could be strongly reduced by the presence of plasminogen and it is therefore identified as the C-terminal part of AP, harbouring the plasminogen binding site of AP. Other experiments indicate that in plasma the C-peptide remains bound to the PAP complex: Upon gelfiltration the peptide co-chromatographs with the PAP complex and in vivo the peptide remains in circulation as long as the PAP complex is present in plasma. It is concluded that upon activation of plasminogen in vitro in plasma and in vivo during thrombolytic therapy with t-PA preferentially the PB-form of AP becomes consumed with the formation of a PAP complex. The PAP complex harbours a non covalently attached C-terminal peptide. Apparently the NPB form becomes not complexed to plasmin even during extensive systemic effects of t-PA in vivo.

A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 microliters containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator.

During thrombolytic therapy of myocardial infarction (MI) with urokinase or streptokinase (SK), levels of fibrin(ogen) degradation products in serum are often dramatically elevated as a result of a combination of systemic fibrinogenolysis and local thrombolysis. Others have measured increased levels of D-dimer in serum of MI patients after SK therapy and postulated that thrombolysis could be monitored during SK therapy by measuring D-dimer levels. In the present study rt-PA was infused into healthy volunteers to analyse if elevated FbDP levels in MI patients really reflect coronary thrombolysis or could be due to a systemic effect. Over a period of 60 min., three groups (n = 6 each) were given i.v. 0.23 mg rt-PA/'kg (group I), 0.50 mg rt-PA/kg (group II) and a placebo infusion (group III), respectively. Two blood samples were taken from an anticubital vein in the arm contralateral to the site of infusion (one on citrate/ aprotinin, the other on citrate alone) at different time points. Using a new enzyme immunoassay (EIA), based on monoclonals and development by us, we measured FbDP in plasma (not serum). Before infusion all volunteers had FbDP levels ≪ 0.5µg/ml. Upon infusion FbDP levels in groups I and II increased to average values of 1.0 ± 0.4/µg/ml and 0.8 ± 0.2/µg/ml, respectively, for the samples taken in citrate/aprotinin. The values in citrate alone did not differ significantly, and were 1.1 ± 0.5/µg/ml and 0.8 ± 0.3/µg/ml for groups I and II, respectively. FbDP levels in group III remained ≪ 0.5/µg/ml. The results show that FbDP levels increase upon rt-PA infusion, even in healthy volunteers. This suggests lysis of systemic fibrin. We conclude that lysis of systemic fibrin limits the value of FbDP levels as a measure for coronary thrombolysis.

The plasma activity level of the recently discovered fast-acting inhibitor of tissue-type plasminogen activator (t-PA) was found to be temporarily increased after surgery, myocardial infarction and severe trauma. Detailed analysis of the postoperative period revealed simultaneously increased t-PA antigen and inhibition and decreased t-PA activity only on the first postoperative day. These changes were more rapid than those in fibrinogen and C-reactive protein. It is concluded that t-PA inhibition shows the most rapidly changing pattern observed so far in response to trauma. The postoperative fibrinolytic shutdown in blood fibrinolytic activity can be ascribed to a primary increase in t-PA inhibitor levels. Chemicals/CAS: C reactive protein, 9007-41-4; fibrinogen, 9001-32-5; plasminogen activator, 9039-53-6; tissue plasminogen activator, 105913-11-9; Antigens; Tissue Plasminogen Activator, EC 3.4.21.68

alpha 2-Antiplasmin (alpha 2-AP) is a major fibrinolysis inhibitor, whose complete, congenital absence has been found to be associated with a distinct hemorrhagic diathesis. We studied a 15-yr-old male with a hemorrhagic diathesis after trauma from early childhood on. This bleeding tendency was associated with a minimal alpha 2-AP level recorded functionally in the immediate plasmin inhibition test: less than or equal to 4% of normal. However, a normal plasma concentration of alpha 2-AP antigen (83%) was found. His sister (5 yr old) showed similar results (2 and 92%). In their family, eight heterozygotes could be identified by half-normal activity results and normal antigen concentrations. The inheritance pattern is autosomal recessive. On analysis, the alpha 2-AP of the propositus was homogeneous in all respects tested, suggesting a homozygous defect. We designated the abnormal alpha 2-AP as alpha 2-AP Enschede. alpha 2-AP Enschede showed the following characteristics: (a) complete immunological identity with normal alpha 2-AP; (b) normal molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) normal alpha-electrophoretic mobility; (d) presence in plasma of both molecular forms excluding an excessive conversion to the less reactive non-plasminogen-binding form; (e) quantitatively normal binding to lys-plasminogen and to immobilized plasminogen kringle 1-3; and (f) normal Factor XIII-mediated binding to fibrin. Functional abnormalities were found in: (i) no inhibition of amidolytic activities of plasmin and trypsin, even on prolonged incubation; (ii) no formation of plasmin-antiplasmin complexes in plasma with plasmin added in excess; and (iii) no inhibition of fibrinolysis by fibrin-bound alpha 2-AP. In the heterozygotes, the presence of abnormal alpha 2-AP did not interfere with several functions of the residual normal alpha 2-AP. One-dimensional peptide mapping showed an abnormal pattern of papain digestion. We conclude that in this family, abnormal antiplasmin molecules, defective in plasmin inhibition but with normal plasminogen-binding properties, have been inherited. The residual plasminogen-binding properties do not protect against a hemorrhagic diathesis.

Fibrinolytic factors were measured before and after DDAVP-infusion in 18 patients on chronic, regular haemodialysis, 11 of whom underwent bilateral nephrectomy, and in 7 patients in whom non-functioning kidneys were still present. Baseline fibrinolytic activity was normal or high in all but two cases. Before haemodialysis, the response to DDAVP-infusion was greatly reduced in the majority of patients as compared with healthy controls, irrespective of the baseline level. This was in accordance with mean t-PA-antigen levels which increased only slightly after DDAVP. When DDAVP was given after haemodialysis, previous non-responders showed a normal increase in fibrinolytic activity. The level of free t-PA-inhibitors was normal in most cases as were levels of alpha 2-antiplasmin and alpha 2-macroglobulin.

Pharmacokinetics of recombinant tissue plasminogen activator (rt-PA, large-scale process) were determined based on antigen and activity after infusion of 0.25 mg/kg in 8 healthy volunteers. Plasma antigen was measured using enzyme-linked immunosorbent assay (ELISA) with and without treatment of blood at collection with D-Phe-Pro-Arg-CH2Cl (P PACK); activity was quantified in acidified plasma both on fibrin plates and in a chromogenic assay. Highest rt-PA concentrations were measured in ELISA with P PACK-treated samples, yielding the following pharmacokinetic parameters (2-compartment model, mean +/- S.D.): Cmax = 973 +/- 133 ng/ml, CL = 687 ml/min, dominant half-life t1/2 alpha = 3.3 +/- 0.4 min, t1/2 beta = 26 +/- 12 min, V1 = 3.9 +/- 0.6 l and Vss = 7.2 +/- 1.0 l. The other assays yielded lower rt-PA concentrations, which affected clearance and volume parameters but not t1/2 beta and t1/2 beta. Linear regressions of the fibrin plate and chromogenic assay results vs. ELISA yielded excellent correlations (R greater than 0.96, n = 55-57) but slopes of 0.76 and 0.64, respectively. This indicates that about 25-35% of rt-PA antigen in thawed plasma samples are not detected in activity assays, due at least partially to in vitro binding of rt-PA by proteinase inhibitors.

Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.

The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 microgram purified material was obtained from 11 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agents showed one main band with Mr of about 60,000, and a minor band with Mr about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.

The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, two-chain t-PA was immobilized onto microtitration plates and incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with an enzyme immunoassay. Hardly any binding to t-PA was observed with fibrinogen, fragments X, Y and E. A moderate binding was observed with fragments D(cate) and D(EGTA) and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 65 nM). Results of control experiments, in which the binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured, using the same assay, were in line with literature data: hardly any binding was found with fibrinogen, fragments X and Y. A moderate binding was observed with fragments D and E and a strong binding with FCB-2 (Kd apparent = 100 nM). The stimulatory capacity of the various fragments on the Lys-plasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y and D, and high for FCB-2. It is concluded that the t-PA binding site in the fibrin (ogen) molecule resides in the distal domains from which fragments D and FCB-2 are derived. The site is apparently hidden in fibrinogen and early fibrinogen degradation products. Binding of both plasminogen and t-PA is required for stimulation of the plasminogen activation, as illustrated by fragment E which binds plasminogen and no t-PA, and has no stimulatory capacity.

The relative fibrinogenolytic, fibrinolytic and thrombolytic properties of human tissue plasminogen activator and human urokinase were compared in purified systems, in whole human plasma and in a system composed of a radioactive human blood clot (125I-fibrinogen) hanging in circulating human plasma. The human tissue plasminogen activator was highly purified from the culture fluid of a human melanoma cell line. In purified systems composed of fibrinogen of fibrin, plasminogen and alpha 2-antiplasmin as well as in whole plasma, tissue plasminogen activator digested fibrin without degrading fibrinogen significantly. Urokinase did not have this specific fibrinolytic effect. In the circulating plasma system, the degree of fibrinolysis was proportional to the amount of activator added, tissue plasminogen activator being about 10 times more efficient than urokinase. In addition, tissue plasminogen activator appeared to cause negligible fibrinogen degradation. Tissue plasminogen activator still induced significant thrombolysis at a concentration of 10 IU per ml whereas no effect of urokinase was observed at 20 IU per ml. Infusion of 100 IU (1 microgram) of tissue plasminogen activator per ml resulted in moderate activation of the fibrinolytic system as judged from a decrease of plasminogen and alpha 2-antiplasmin to 40-50 percent. Nevertheless, extensive fibrinolysis (50 to 80 percent of radioactivity released after 12 hrs) and only very limited fibrinogenolysis were observed. An equivalent amount of urokinase (100 IU per ml) only induced approximately 15 percent lysis in 12 hrs. At higher concentrations of urokinase (260 IU per ml or more) extensive activation of the fibrinolytic system was obtained as evidenced by a depletion of plasminogen, alpha 2-antiplasmin and fibrinogen. This was associated with extensive fibrinolysis (approximately 60 percent after 12 hrs). It is concluded that human tissue plasminogen activator is a more specific and effective fibrinolytic-thrombolytic agent than human urokinase.

In this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4 degrees C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37 degrees C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (M(r) ??? 38,000) and a light chain (M(r) ??? 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity. Chemicals/CAS: plasminogen activator, 9039-53-6; Amino Acids; Cyanogen Bromide, 506-68-3; Fibrinogen, 9001-32-5; Iodoacetamide, 144-48-9; Mercaptoethanol, 60-24-2; Tissue Plasminogen Activator, EC 3.4.21.68

The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.

This mini-review deals with structure-function relationships of human tissue-type plasminogen activator. The enzyme consists of a single polypeptide chain of 527 amino acids. A two-chain form is produced by proteolytic cleavage of the Arg 275-Ile 276 peptide bond. The aminoterminal heavy or A-chain consists of a finger domain, a growth factor domain and two kringle domains. The carboxyterminal light or B-chain contains the active site and is homologous to the catalytic chains of other serine proteases. The light chain is able to activate plasminogen, but requires the heavy chain for fibrin-binding and fibrin-stimulation. Particularly, the finger domain and kringle 2 of the heavy chain are involved in the interaction with fibrin. Other specific properties of the plasminogen activator, such as its rapid hepatic clearance and its inhibition by plasminogen activator inhibitors have not yet been related to specific domains in the protein structure.

Fibrinolytic activity in plasma euglobulin fractions can be increased by oral administration of stanozolol. This increase is not caused by increased synthesis or release of tissue-type plasminogen activator. A decreased level of fast acting t-PA inhibition is very probably the cause of the higher activity. These results suggest that this inhibition has a regulatory role on fibrinolysis in vivo.

The in vitro interaction of rt-PA with the coagulation and fibrinolytic system was investigated. After addition of rt-PA to citrated plasma prolonged clotting times for prothrombin time, partial thromboplastin time and thrombin time, decreased activities of individual clotting factors and decreases of alpha 2-antiplasmin, plasminogen and fibrinogen were measured. The alterations were dependent on incubation times and rt-PA concentrations and were mainly observed at rt-PA concentrations exceeding therapeutic levels. These in vitro phenomena could be prevented to different degrees using various inhibitors. Because of interferences with some fibrinolytic and coagulation assays aprotinin was only of limited utility as an inhibitor. PPACK lengthened clotting time assays based on generation of endogenous thrombin, but did not affect fibrinolytic assays. The most versatile inhibitor was a specific anti-rt-PA antibody which enabled correct measurements of all fibrinolytic and most coagulation assays. It is concluded that high rt-PA levels in samples taken from patients during fibrinolytic therapy induce in vitro artefacts which can be prevented by the use of suitable inhibitors. In 8 healthy male volunteers aged 32 +/- 7 years pharmacokinetic investigations were done. After infusion of 0.25 mg rt-PA/kg bw mean maximal plasma levels were 970 +/- 130 ng/ml. The elimination of rt-PA from plasma was fitted to a two compartment model and was characterized by two half lives of t1/2 alpha = 3.3 and t1/2 beta = 26 minutes.

In a search for compounds that can enhance tissue-type plasminogen activator (t-PA) synthesis in cultured human endothelial cells we found that dibutyryl cyclic AMP (at a concentration of 2 mM) led to a several-fold increase in t-PA production by endothelial cells over a 24 h incubation period. Further researchshowed that this stimulating effectcould be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but notby addition of 8-bromo cyclic AMP. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused anincreasingly stimulatory effect, reaching a plateau with 5 mM butyrate. The relative increase in t-PAproduction in thempresence of 5 mM butyrate varied among different endothelial cell cultures from 6 to 25-fold in 24 h CM. The butyrate-induced increase in t-PA production wasaccompanied by increased t-PA mRNAlevels.Analysis of radiolabelled CM andcell extracts by SDS-PAGE and autoradiography indicated that the potent action of butyrate is restricted to a limited group of proteins. We found that theaccumulation of PA-inhibitor activity in CM from butyrate-treated cells increased only moderately.In our study of the relationshipbetween structure and stimulatory activity we found that butyrate wasby far the most effective inducer oft-PA synthesis. Shortening or lengthening the carbon chain by one carbon atom decreased the stimulatory effect after 24 h of incubation by 50-70%. Further changes in the length of the carbon chain almost completely suppressed the stimulatory activity. Similarly, alterations of the aliphatic chain by introduction of functional groups, a double bond or a branched structure rendered butyrate ineffective in increasing t-PA synthesis. Thus, a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxyl moiety at the other seems to be required for the rather specific induction of t-PA synthesis in cultured human endothelialcells.

In a randomised study, thirty patients with myocardial infarction received either 20, 50 or 100 mg recombinant BW-t-PA in 90 minutes. Blood samples were taken just before the infusion, and after 90 min. In vitro effects of t-PA were prevented by addition of 250 μg/ml polyclonal t-PA antibodies, which were shown to block in vitro actions of up to 5 μg t-PA/ml during plasma handling.There were dose related (MIU/kg/90 min) decreases of α2 -antiplas-min (AP, functional assay), plasminogen (PLG, streptokinase assay) and fibrinogen (FBG, Clauss) at 90 min (lineair regression: r = 0.67; 0.55 and 0.40, n = 30, all p < 0.05, resp.). Decreases for the 100 mg dose were for the respective components: 70 ± 10 (SD)%, 28 ± 7% and 33 ± 17% of pretreatment levels. The extent of the AP changes appeared to be the most sensitive parameters and these changes correlated with those in PLG and FBG. Pretreatment values of FBG were elevated (137 ± 37%) and at 90 minutes were 100 ± 23% of pooled plasma. Decreases in FBG were significantly (p < 0.001) correlated with absolute pretreatment levels, indicating preferential reductions of elevated levels of fibrinogen.Using our new enzymimmunoassays based on monoclonals for degradation products of fibrinogen (FDP), fibrin (FbDP) and the total (TDP), these products were measured in plasma (not serum). Before infusion 14 patients showed TDP levels (range: 0.5 - 2.3 μg/ml); at 90 min 29 patients (0.6 - 13 μg/ml. The increase in TDP accounted for only 0.75% (range up to 5.3%) of the apparent FBG reduction (Clauss) at 90 min. Detectable FbDP were generated in 24 patients at 90 min. For the two highest dosages the level (median 2.0 μg/ml) was significantly higher than at the dosage of 20 mg (median 0.85 μg/ml), indicating weaker fibrinolytic effect of t-PA at the lowest dosage. To evaluate the fibrinolytic/fibrinogenolytic effects of the t-PA the ratio FbDP/FDP was calculated. For the 50 mg dosage all ratios were ≥ 1; for 100 mg only 6 patients showed a ratio ≥ 1 at 90 min. This may indicate relative specific increase of fibrinogenolysis at higher dosages.

SummaryThe plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop.Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 μg purified material was obtained from 1 1 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacryl amide gel electrophoresis in the presence of reducing agents showed one main band with M r of about 60,000, and a minor band with M r about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.

In nine patients with non-malignant diseases undergoing major upper abdominal surgery, the mechanism of the postoperative fibrinolytic shut-down was investigated because of its potential significance for postoperative deep vein thrombosis by employing new and specific methods for assessing and stimulating the fibrinolytic system. The shut-down was found to result from an impairment of the balance between tissue-type plasminogen activator, t-PA, and its recently discovered fast-acting inhibitor. In this balance, the t-PA antigen concentrations both in resting conditions and after stimulation evoked by desamino-D-arginine vasopressin (DDAVP) were found to be unchanged by surgery. However, there was a significant postoperative increase in t-PA inhibitor levels. The release of t-PA under the stimulus of DDAVP infusion overcame the postoperative shut-down of t-PA activity. However, DDAVP infusion was associated with potentially unfavourable increases in the Factor VIII/von Willebrand factor complex. The discovery of increased t-PA inhibitor in the postoperative period opens new possibilities for a rational approach to reduce or abolish the postoperative fibrinolytic shut-down. Chemicals/CAS: blood clotting factor 8, 9001-27-8; desmopressin, 16679-58-6; plasminogen activator inhibitor, 105844-41-5; tissue plasminogen activator, 105913-11-9; Desmopressin, 16679-58-6; Factor VIII, 9001-27-8; Glycoproteins; Plasminogen Inactivators; Tissue Plasminogen Activator, EC 3.4.21.68; von Willebrand Factor

SummaryFibrinolytic activity in plasma euglobulin fractions can be increased by oral administration of stanozolol. This increase is not caused by increased synthesis or release of tissue-type plasminogen activator. A decreased level of fast acting t-PA inhibition is very probably the cause of the higher activity. These results suggest that this inhibition has a regulatory role on fibrinolysis in vivo.

Thromboembolism is a frequent and often lethal complication of many medical diseases and surgical procedures. Streptokinase, a bacterial non-enzymatic protein, and urokinase, an enzyme obtained from human urine, are both potent activators of the human fibrinolytic system, and have been extensively investigated as a means of medical treatment of this condition. Although it is well established that these agents can clear obstructed vessels in roughly 50% of cases, the fact that they induce relatively extensive systemic fibrinogen breakdown and a serious haemorrhagic diathesis has prevented their general application for thrombolysis. The physiological plasminogen activator in blood (blood activator), which is probably released from the vascular wall (vascular activator) and is identical or at least very similar to the activator extracted from human organs (tissue activator), differs from urokinase (for references see ref. 1). This activator has a markedly higher fibrinolytic to fibrinogenolytic ratio than urokinase in vitro1 and might therefore constitute a better thrombolytic agent. We have developed a purification method for tissue plasminogen activator from a human melanoma cell line which enables us to produce milligramme quantities on a laboratory bench scale2 and we have now compared its thrombolytic effect with that of urokinase in an experimental animal model. We have found that tissue plasminogen activator causes thrombolysis at lower doses than urokinase, without extensive plasminogen activation in the circulating blood and without haemostatic breakdown.