2014 Fiscal Year Research-status Report
Imaging analyses for activated platelets' surface to initiate blood coagulation both in in-vivo and in-vitro systems.
| Project/Area Number |
26860146
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | fibrinolysis / coagulation / plasminogen / platelets |
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| Outline of Annual Research Achievements |
Blood platelets through the exposure of anionic phospholipids, mainly phosphatidylserine (PS), on their surface amplify and promote coagulation. Previously we have reported that both exposing PS platelets and generated fibrin were localized only in the core of the intravascular thrombus (Hayashi et al., Pflugers Arch 2008). During pursuing our original project aimed to investigate the expression of procoagulant activity for tissue factor-factor VIIa complex on anionic phospholipids, we also observed the accumulation of exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) in the center of the microthrombus. This finding prompted us to further investigate the mechanism of plasminogen accumulation and fibrinolysis initiation. With the employment of confocal imaging techniques we have found that 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 were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Moreover, aprotinin reduced the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were ultimately lysed. These results successfully demonstrated that fibrinolysis is endogenously activated at a site of platelet activation at an early phase of thrombus formation (published: Brzoska et al., PloS ONE 2015).
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In-vivo study of the microcirculation of a living green fluorescent protein-expressing transgenic mice (GFP mice) or Wild-type (WT) mice was performed with an employment of the intravital fluorescence confocal microscopy. Mesenteric venules were identified and endothelial injury was induced by laser irradiation. The kinetic and distribution of exogenously infused Glu-plg within a thrombus has been analyzed. In order to elucidate the mechanism of Glu-plg accumulation ε-aminocaproic acid, carboxypeptidase B, aprotinin, and mini-plasminogen were adequately used. To determine whether the microthrombus was sensitive to fibrinolysis, tPA was given intravenously 40 minutes after thrombus formation.
For in-vitro experiments blood samples were collected through the inferior vena cava of anesthetized GFP mice. Platelet rich plasma and the suspension of washed platelets were prepared. Confocal laser scanning microscope was employed to analyze platelet Glu-plg binding both in the presence and absence of fibrin network.
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| Strategy for Future Research Activity |
Employing intra-vital confocal microscopy, we reported that the process of platelet PS exposure, fibrin formation and lysine binding site-dependent Glu-pl accumulation took place only in the center of the thrombus but not in its periphery. These suggest that coagulation and fibrinolysis are closely related and finely regulated. To analyze in-vitro the spatiotemporal regulatory mechanisms of coagulation and fibrinolysis we designed the experiment, with the employment of diluted platelet-rich plasma supplemented with fluorescently labeled coagulation- and fibrinolytic- factors using Confocal Laser Scanning Microscopy, where fibrin network formation and its following lysis can be analyzed.
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| Causes of Carryover |
Some of the chemical reagent purchases were postponed to the next fiscal year. These chemicals are going to be used in the future planned experiments.
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| Expenditure Plan for Carryover Budget |
To analyze in-vitro the spatiotemporal regulatory mechanisms of coagulation and fibrinolysis we designed the experiment, with the employment of diluted platelet-rich plasma supplemented with fluorescently labeled coagulation- and fibrinolytic- factors using Confocal Laser Scanning Microscopy, where fibrin network formation and its following lysis can be analyzed. Remaining founds are going to be spent on the fluorescent Alexa Fluor Dyes.
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