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Plasminogen activators and plasminogen activator inhibitors: biochemical aspects.

Although this chapter does not represent a historical review, it will be clear how the biochemistry of t-PA, u-PA, PAI-1 and PAI-2 has evolved and where we stand in 1994. While the functional activities of the proteins were recognized at least three to four decades ago, highly purified preparations became available around 1980. In the mid-eighties the cDNAs of the proteins were cloned, representing a major breakthrough in the biochemistry of the four proteins. Amino acid sequences were derived from the nucleotide sequences, homologies with other proteins were recognized and larger amounts of (recombinant) proteins became available for research. In addition, mutant proteins were prepared by recombinant DNA technology, enabling investigation of structure-function relationships. This report is mainly based on the latter studies. Detailed information about three-dimensional structures of the proteins and the mode of interaction with other macromolecules is still lacking. To obtain this information will be the goal for biochemists in the coming years.

Amino Acid Sequence↗

Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.

Animals↗

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states.

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy.

Glycoproteins↗

Relationship of urokinase type plasminogen activator, plasminogen activator inhibitor type 1 and activated protein C in fibrinolysis of human placenta.

Suppression of the fibrinolytic activity plays an important role in the prevention of hemorrhage during pregnancy and labor. A hypofibrinolytic and hypercoagulable state may be established in the placenta during pregnancy. However, little infraction is present in the normal placenta. This evidence shows that placenta maintains the fibrinolytic activity in spite of hypercoagulable state. As there is a high amount of APC in the placenta, APC is thought to be involved in fibrinolysis of placenta. Thus, we studied the role of APC on fibrinolysis in placenta. (1) uPA activity of cell membrane reappears after incubation with uPA/PAI-1 complex and a large amount of APC by flow cytometry, (2) APC was made PAI-1/APC complex after incubation of uPA/PAI-1 complex with APC. Our results suggest that APC is the important substance for fibrinolysis in the placenta by decreasing of PAI activity.

Blotting, Western↗

Expression of urokinase plasminogen activator and the urokinase plasminogen activator receptor in myeloma cells.

Binding of urokinase (uPA) to its receptor (uPAR; CD87) focuses proteolytic activity on the cell surface and this system is of importance in malignant matrix degradation and tumour invasion. By immunocytochemistry and flow cytometry, we found that primary myeloma cells and myeloma cell lines expressed uPA and uPAR. Soluble uPA was present in cell line supernatants and lysates in low concentrations. In cell lines, uPA and uPAR were located both on the cell surface and intracellularly, but the expression of both proteins was low. Higher levels of uPAR was detected on the cell surface of primary myeloma cells. When primary myeloma cells were gated by CD45 expression, stronger expression was found on immature CD45+ cells than on mature CD45-/dim cells. Finally, both myeloma cell lines and primary cells were able to cleave a uPA-specific substrate showing that the uPA system is functionally active. We conclude that myeloma cells are able to produce uPA and uPAR. This opens up a possible role of the uPA system in myeloma cell invasion and in the proteolytic digestion of bone matrix.

Flow Cytometry↗

Synergistic effect on thrombolysis of sequential infusion of tissue-type plasminogen activator (t-PA) single-chain urokinase-type plasminogen activator (scu-PA) and urokinase in the rabbit jugular vein thrombosis model.

In a quantitative model of thrombolysis, consisting of rabbits with a 125I-fibrin labeled blood clot in the jugular vein, simultaneous intravenous infusion over 4 hours of t-PA and scu-PA or of t-PA and urokinase had a significantly greater (p less than 0.01) thrombolytic effect than could be anticipated on the basis of the added effects of each agent alone. In order to further investigate the mechanism of this in vivo synergism, recombinant t-PA (rt-PA) and scu-PA in synergistic amounts were infused: 1) simultaneously over 4 hours, 2) rt-PA over 1 hour, then 15 min later scu-PA over 2 hours and 3) scu-PA over 1 hour, than 15 min later rt-PA over 2 hours. Simultaneous infusion of 0.1 mg/kg rt-PA and 0.2 mg/kg scu-PA gave 48 +/- 2 percent thrombolysis (mean +/- SEM, n = 5) and of 0.2 mg/kg rt-PA and 0.4 mg/kg scu-PA 67 +/- 5 percent (n = 5). When these infusions were given sequentially, rt-PA followed by scu-PA gave 32 +/- 5 (n = 4) and 49 +/- 8 (n = 4) percent lysis, but scu-PA followed by rt-PA yielded only 14 +/- 1 (n = 4) and 21 +/- 1 (n = 4) percent lysis, indicating that synergism occurs when rt-PA is followed by scu-PA but not when scu-PA is followed by rt-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗