title: Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.
abstract: The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.
authors: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Tomasz
authors: T
authors: Brzoska
authors: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan; Department of Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Aki
authors: A
authors: Tanaka-Murakami
authors: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Yuko
authors: Y
authors: Suzuki
authors: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Hideto
authors: H
authors: Sano
authors: Department of Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Naohiro
authors: N
authors: Kanayama
authors: Department of Medical Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors: Tetsumei
authors: T
authors: Urano
mesh_terms: D000818:Animals; D005341:Fibrinolysin / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010958:Plasminogen / Q000378:metabolism*; D013927:Thrombosis / Q000188:drug therapy / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D049452:Green Fluorescent Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin