Hydrolysis Of Plasminogen Research Articles

Plasmodium falciparum proteases hydrolyze plasminogen, generating angiostatin-like fragments

Malaria is a disease caused by Plasmodium parasites and remains one of the most prevalent and persistent maladies, affecting hundreds of millions of people. In the present work, we evaluated the capability of Plasmodium falciparum proteases to hydrolyze the multifunctional protein plasminogen, which is implicated in angiogenesis and coagulation processes by the generation of angiostatin and plasmin, respectively. Using fluorescence microscopy, we visualized the internalization of FITC-labeled plasminogen in erythrocytes infected by P. falciparum and showed that the parasites are able to hydrolyze the protein. The cleavage of plasminogen by the P. falciparum proteases was also observed by SDS-PAGE, followed by immunoblotting with anti-angiostatin antibody. N-terminal sequencing of the main generated fragments indicated that they are comprised in the five plasminogen kringle domains, suggesting as being angiostatin-like peptides. This assumption was reinforced by the demonstration that the products of plasminogen processing mimic angiostatin functions, including the capability to inhibit angiogenesis and to stimulate calcium response in endothelial cells in vitro. However, no plasmin activity was detected after plasminogen hydrolysis by P. falciparum. Nonetheless, exogenous tissue plasminogen activator (tPA) activated plasmin in infected erythrocytes, suggesting that the uptake of plasminogen by P. falciparum may be modulated by the vertebrate host. Taken together, the data presented here provide evidence for the processing of host plasminogen by malaria parasites to generate active fragments that may modulate host physiology events during malaria infection.

Activity of Bacillus polymyxa protease on components of the plasmin system in milk

Abstract The activity of a metalloprotease produced by Bacillus polymyxa toward the plasmin system was studied in both buffer and milk. Chromogenic assays were carried out to monitor effects of the protease on plasmin activity in a buffer system. Among the plasmin system components, plasminogen was most affected by the protease. Hydrolysis of plasminogen was identified and visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and casein–SDS–PAGE, respectively. Results confirmed that B. polymyxa protease cleaved bovine plasminogen, generating a plasmin-like activity. Kinetic parameters of the protease showed that its activity on plasminogen can be physiologically relevant where significant impact on plasminogen activation in milk can occur. This finding was further confirmed when, during refrigerated storage, a significant increase ( P 0.01 ) in plasmin-like activity was observed in protease-treated pasteurized milk. Results of this study confirmed the interaction of the B. polymyxa protease with the plasmin system by acting as a plasminogen activator.

Heparin modulation of human plasma kallikrein on different substrates and inhibitors

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

カラゲニン誘発炎症に伴って増強されるマウスひのプラスミノゲン分解活性

The variation of spleen plasminogenolytic activity was investigated using a carrageenan-induced inflammation in mice. Immuno-blotting method using antiserum for human plasminogen was adopted for the observation of plasminogenolysis by the spleen extract with 2M KSCN. The plasminogenolytic activity was detected even in the normal spleen extract. The remarkable enhancement of it in the spleen from the 6th to the 10th day after the carrageenan injection followed the increases of plasma fibrinogen and serum FD (g) P levels. The hydrolysis of plasminogen by the spleen extract made fragments of 55kDa, 35kDa and 28kDa, further the 55kDa fragment changed to the small one. These plasminogenolytic activities were completely inhibited with diisopropyl fluorophosphate, soybean trypsin inhibitor or α1-trypsin inhibitor, but not inhibited with aprotinin. The variation of the spleen plasminogenolytic activity after the carrageenan injection and these effects of proteinase inhibitors for the activity were similar to the fibrinolytic activity in the spleen. These data show that the plasminogenolytic activity in the spleen was derived from a same proteinase as fibrinolytic activity.

A plasminogen activator from benign ovarian cystadenoma: Partial purification and characterization

A plasminogen activator has been partially purified from benign serous cystadenomas by a combination of Sephadex G-200 gel filtration, CM-Sephadex ion exchange, Concanavalin A-Sepharose and arginine-Sepharose affinity chromatographies. Its apparent size was very large and could not penetrate Sepharose 6B or 5% SDS polyacrylamide gel. It hydrolyzed plasminogen in a manner similar to that of urokinase in terms of their apparent Michaelis constants, although a one-minute lag period had to be allowed for this activation before the hydrolysis of plasminogen. It was very sensitive to reducing agent such that 5 mM dithiothreitol could completely destroy its activity. The activator crossreacted with anti-uterine plasminogen activator IgG, but did not react with anti-urokinase at all. It also bound fibrin well. In the absence of plasminogen, the activator was devoid of amidolytic activity towards S-2251, S-2302 and S-2288 but had a small but measurable activity against S-2444.

ACTIVE, SOLUBLE PRODUCT OBTAINED BY ENZYMATIC HYDROLYSIS OF PLASMINOGEN.

SummaryInsoluble plasminogen when treated with a certain carboxypeptidase preparation after destruction of CP activity, or with α-chymotrypsin, produced a soluble, active material with all the biochemical activities of the original substance. The identity of the hydrolyzing enzyme has not been established. Evidence is presented that the product obtained is partially hydrolyzed plasminogen (PHP).