PLATELET-MEDIATED PLASMINOGEN PROCESSING PRODUCES ANGIOSTATINS: AN IMMUNOCHEMICAL STUDY

Abstract

The study of reciprocal interactions between the plasminogen/plasmin system and the platelet componentof hemostasis is necessary both for understanding the biochemical mechanisms regulating the processes of thrombosis and thrombolysis and for elucidating the role of platelets in angiogenesis. Aim. The study aimed to investigate the peculiarities of plasminogen processing by cytosolic and plasma membrane-associated proteases of platelets. Methods. Gel-permeation filtration was used for the isolation of platelets from the donor’s blood plasma. Plasminogen was purified from Cohn’s fraction III2,3 of human blood plasma by affinity chromatography on lysine-Sepharose. The viability of washed platelets and their response to an agonist were assessed by optical aggregometry. The processing of plasminogen on platelets was induced by stimulating the cells with thrombin (1 NIH/ml) after pre-incubation with 0.25 μM Pg for 30, 60, or 120 min. Plasminogen and its fragments were detected by immunoblot with the use of previously obtained polyclonal antibodies to plasminogen kringles (K1-3 and K5). Results. It was established that exogenous plasminogen is adsorbed onto the plasma membrane of platelets, converted into the Lys-form, and further fragmented into angiostatins and mini-plasminogen. This indicates the involvement of various platelet proteases in plasminogen cleavage. It was shown that platelets are capable of internalizing exogenous plasminogen in its Glu-form, while formed angiostatins are not internalized by the cells. It has been determined that internalized Glu-plasminogen (0.25 μM) may change its conformation to a Lys-like form within  120 minutes of incubation with platelets, as immunochemically detected with the use of antibodies against K5 plasminogen fragment. Conclusion. The obtained results provide new insights into the mechanisms by which platelets may regulate the functioning of the plasminogen/plasmin system. This regulation occurs through their ability to generate plasminogen fragments (angiostatins) and having the potential for internalization and further secretion of the formed angiostatins by both native and activated platelets.

Keywords

• plasminogen
• kringle-containing fragments
• angiostatins
• platelets
• limited proteolysis
• immunoblot
• antibodies.