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Recombinant murine-activated protein C is neuroprotective in a murine ischemic stroke model
José A Fernández1, Xiao Xu, Dong Liu
1Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Abstract:
Recombinant mouse protein C was cloned, expressed, purified, and activated by Protac or thrombin. The anticoagulant activities of mouse and human activated protein C (APC) were compared using mouse and human plasma and the neuroprotective properties of murine APC were studied in an ischemic stroke model. Both human APC and mouse APC prolonged the activated partial thromboplastin time in a dose-dependent manner, but mouse APC was sixfold more effective than human APC as an anticoagulant in mouse plasma. Human protein S enhanced prolongation of the APTT clotting time of human plasma by human APC, but not by mouse APC. Hydrolysis of the S-2366 chromogenic substrate by murine APC was essentially identical to human APC. Mouse plasma contains 75 nM protein C. In a murine ischemic stroke model based on middle cerebral artery occlusion, murine APC was highly neuroprotective. The results show that recombinant murine APC is functionally similar to human APC both in vitro and in vivo and that it displays significant species specificity. The results imply that murine APC is notably superior to human APC for studies of murine disease models, including thrombosis and ischemic brain injury.
Insights
Recombinant mouse activated protein C (APC) shows potent anticoagulant and neuroprotective effects in mouse models. Murine APC is significantly more effective than human APC in mouse plasma, highlighting its superiority for preclinical studies.
Area of Science:
- Biochemistry
- Hematology
- Neuroscience
Background:
- Activated protein C (APC) is a crucial anticoagulant and cytoprotective enzyme.
- Understanding species-specific activity is vital for preclinical research.
- Recombinant murine APC offers a potential tool for studying thrombosis and neuroprotection in mice.
Purpose of the Study:
- To characterize the anticoagulant and neuroprotective properties of recombinant mouse APC.
- To compare the efficacy of murine APC with human APC in vitro and in vivo.
- To evaluate the species specificity of APC activity.
Main Methods:
- Cloning, expression, and purification of recombinant mouse protein C.
- Activation of protein C using Protac or thrombin.
- Assays for anticoagulant activity using activated partial thromboplastin time (APTT) in mouse and human plasma.
- Chromogenic substrate hydrolysis assay.
- Evaluation of neuroprotection in a murine ischemic stroke model (middle cerebral artery occlusion).
Main Results:
- Mouse APC demonstrated significantly higher anticoagulant activity (sixfold) in mouse plasma compared to human APC.
- Human protein S enhanced the anticoagulant effect of human APC but not mouse APC in human plasma.
- Murine APC exhibited potent neuroprotective effects in a middle cerebral artery occlusion stroke model.
- Functional assays showed murine APC is similar to human APC in substrate hydrolysis, indicating conserved enzymatic activity.
Conclusions:
- Recombinant murine APC is functionally comparable to human APC in vitro enzymatic activity but exhibits significant species-specific anticoagulant superiority in mouse plasma.
- Murine APC is highly effective in preventing neuroprotection in a murine ischemic stroke model.
- Murine APC is a superior agent for investigating thrombosis and ischemic brain injury in mouse models.