M-AAA-thrombin: potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.
BACKGROUND
Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus, several antithrombotic agents targeting thrombin have been developed. We previously reported on a thrombin derivative prepared by dual chemical modifications designated as 'M-anhydrothrombin', which possessed both anticoagulant and antiplatelet properties. In order to obtain a more potent antithrombotic thrombin derivative, we prepared a recombinant thrombin mutant and its chemically-modified derivative, and examined their antithrombotic efficacies.


RESULTS
We prepared a thrombin mutant, 65A43A205A-Th (designated as "AAA-Th") in which Lys65(70)(1), and His43(57) and Ser205(195) were replaced by Ala, and its chemically-modified derivative at the carboxyl groups (designated as "M-AAA-Th"). M-AAA-Th possessed no enzymatic activity, but retained high affinity and specificity for factor VIII, and prolonged the APTT with a slight effect on PT and no effect on TT. Platelet aggregation induced by PAR1 activation was also suppressed by M-AAA-Th. In contrast, conventional thrombin inhibitors, argatroban and hirulog, substantially prolonged the TT rather than the APTT and the PT. In thromboelastgraph assays, M-AAA-Th suppressed whole blood clotting in a dose-dependent manner, and its effect was synergistically enhanced in the presence of soluble thrombomodulin (s-TM). M-AAA-Th also demonstrated a potent antithrombotic property in the FeCl(3)-induced carotid arterial thrombosis model in guinea pigs with minimum effects on the APTT and PT and no prolongation of the TT.


CONCLUSIONS
M-AAA-Th may be a potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.
Hosokawa
Kazuya
K
Department of Life Science, Graduate School of Life Science, University of Hyogo, Hyogo, Japan.
Matsuda
Hiroyuki
H
Ohnishi
Tomoko
T
Gonda
Maki
M
Koide
Takehiko
T
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D005169:Factor VIII / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013927:Thrombosis / Q000097:blood / Q000188:drug therapy*
D000925:Anticoagulants; D010975:Platelet Aggregation Inhibitors; D044463:Receptor, PAR-1; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin