Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis.
Novel antithrombin molecules were identified from the ixodidae tick, Haemaphysalis longicornis. These molecules, named madanin 1 and 2, are 7-kDa proteins and show no significant similarities to any previously identified proteins. Assays using human plasma showed that madanin 1 and 2 dose-dependently prolonged both activated partial thromboplastin time and prothrombin time, indicating that they inhibit both the intrinsic and extrinsic pathways. Direct binding assay by surface plasmon resonance measurement demonstrated that madanin 1 and 2 specifically interacted with thrombin. Furthermore, it was clearly shown that madanin 1 and 2 inhibited conversion of fibrinogen into fibrin by thrombin, thrombin-catalyzed activation of factor V and factor VIII, and thrombin-induced aggregation of platelets without affecting thrombin amidolytic activity. These results suggest that madanin 1 and 2 bind to the anion-binding exosite 1 on the thrombin molecule, but not to the active cleft, and interfere with the association of fibrinogen, factor V, factor VIII and thrombin receptor on platelets with an anion-binding exosite 1. They appear to be exosite 1-directed competitive inhibitors.
Iwanaga
Shiroh
S
Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Japan. iwanaga@ans.kobe-u.ac.jp
Okada
Masakazu
M
Isawa
Haruhiko
H
Morita
Akihiro
A
Yuda
Masao
M
Chinzei
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002417:Cattle; D003001:Cloning, Molecular; D005165:Factor V / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D026863:Ixodidae / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism
D000925:Anticoagulants; D000991:Antithrombins; D019476:Insect Proteins; D012471:Salivary Proteins and Peptides; C475280:madanin 1 protein, Haemaphysalis longicornis; C475281:madanin 2 protein, Haemaphysalis longicornis; D005165:Factor V; D005169:Factor VIII; D005337:Fibrin; D005340:Fibrinogen; D013925:Thromboplastin; D013917:Thrombin