Amplified anticoagulant activity of tissue factor-targeted thrombomodulin: in-vivo validation of a tissue factor-neutralizing antibody fused to soluble thrombomodulin.
Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in approximately 60% of the animals and a clinical score of approximately 2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neitherAb(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED(50), nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10- to 100-fold enhancement of the anticoagulant potency, relative to the ED(50) in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.
Wang
Yi-Xin
YX
Berlex Biosciences, Richmond, California, USA. jim_wang@berlex.com
Wu
Chenliang
C
Vincelette
Jon
J
Martin-McNulty
Baby
B
Alexander
Serene
S
Larsen
Brent
B
Light
David R
DR
McLean
Kirk
K
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology*; D001777:Blood Coagulation; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013923:Thromboembolism; D018180:Thrombomodulin / Q000097:blood* / Q000737:chemistry; D013925:Thromboplastin / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
D000925:Anticoagulants; D011993:Recombinant Fusion Proteins; D018180:Thrombomodulin; D013925:Thromboplastin