Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood.
Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.
Orvim
U
U
Nycomed Imaging AS, Oslo, Norway.
Barstad
R M
RM
Orning
L
L
Petersen
L B
LB
Ezban
M
M
Hedner
U
U
Sakariassen
K S
KS
D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects*; D002863:Chromogenic Compounds / Q000378:metabolism; D003619:Dansyl Compounds / Q000494:pharmacology*; D015942:Factor VIIa / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D018056:Hemorheology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010973:Platelet Adhesiveness / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism
D002863:Chromogenic Compounds; D003619:Dansyl Compounds; D005343:Fibrinolytic Agents; D009842:Oligopeptides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; C109749:dansyl-glutamyl-glycyl-arginyl-factor VIIa; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D005337:Fibrin; D013925:Thromboplastin; D015942:Factor VIIa