Tissue Plasminogen Activator in Human Megakaryocytes and Platelets: Immunocytochemical Localization, Immunoblotting and Zymographic Analysis

Summary

Two approaches were used to identify and characterize the presence of tissue plasminogen activator (t-PA) in megakaryocytes and platelets. We investigated the fibrinolytic activity of human megakaryocytes (MK) and platelets. The presence of t-PA antigen in megakaryocytes and platelets was demonstrated using immunocytochemical techniques and polyclonal or monoclonal antibodies specific for t-PA. When cells were applied to fibrin plates, lysis zones developed around isolated human megakaryocytes, whereas no fibrinolytic activity appeared when either intact washed platelets or platelet lysate were deposited. After SDS-PAGE of platelet and MK extracts (Triton X-100) immunoblotting and peroxidase staining identified t-PA antigen in several bands. Zymographic analysis of SDS-PAGE carried out on fibrin film overlays identified one or two zones corresponding to free or complexed t-PA. These results indicate that t-PA is present in platelets as well as in the precursor cells, however, in platelets, t-PA may not be immediately available for plasminogen activation and fibrin degradation. From our findings and from previous work of others, it appears that platelets may either activate or inhibit the fibrinolytic system. Therefore the conditions of plasminogen activation by platelet t-PA and plasmin inhibition by platelet α₂-antiplasmin or other inhibitors have to be precised before the role of platelets in clot dissolution is understood.

The physiological role of platelets in fibrinolysis and clot dissolution remains unclear. In 1953, the antifibrinolytic activity of blood platelets was demonstrated (1) and in the early 1960’s a fibrinolytic activity, increasing with platelet concentration in the experimental system, was shown (2, 3). In 1979, it was demonstrated that metabolically active platelets were necessary for platelets to play a role in the fibrinolytic system (4). More recently it was established by Plow and Collen (5) that the specific plasmin inhibitor, α₂-antiplasmin is a constituent of platelet α-granules.

In the present study, we investigated the fibrinolytic components and activity of human megakaryocytes and platelets, using zymographic and immunochemical techniques. We report here our observations that human megakaryocytes and platelets contain tissue plasminogen (t-PA) which possesses fibrinolytic activity.

Keywords

Fibrinolysis - Immunoperoxidase - Megakaryocytes - Platelets - Immunoblotting

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