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.

Elevated levels of angiostatin in effusions from patients with malignant disease.

The aim of this study was to determine the presence of angiostatin in ascitic and pleural effusions from cancer patients, as well as of metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA), both involved in angiostatin generation in in vitro models. Ascitic fluids, pleural exudates, and sera from 21 cancer patients were analyzed for the presence of angiostatin by western blot, whereas gelatinases MMP-2 and MMP-9, and uPA were evaluated by zymography. Our study revealed elevated levels of angiostatin in effusions of cancer patients, contrasting with mostly intermediate levels in less than half of their sera, and undetectable levels in normal sera. Despite the observation of enhanced levels of HMW-uPA and MMP-2 in malignant effusions from cancer patients, their analysis in individual samples showed no association between angiostatin presence and the enzymes, suggesting that the latter would not play an unimportant role, if any, in in vivo generation of angiostatin.