Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonist.
BACKGROUND
Activation of platelets with a combination of collagen and thrombin generates a subpopulation of highly procoagulant 'coated' platelets characterized by high surface expression of fibrinogen and other procoagulant proteins.


OBJECTIVE
To analyze the interaction of recombinant factor VIIa (rFVIIa) with coated platelets.


RESULTS
rFVIIa localized to the coated platelets in flow cytometry experiments, while minimal rFVIIa was found on platelets activated with adenosine diphosphate, thrombin or via glycoprotein VI individually, and essentially no rFVIIa was found on non-stimulated platelets. Removal of the gamma-carboxyglutamic acid (Gla) domain of rFVIIa, and addition of EDTA, annexin V or excess prothrombin inhibited rFVIIa localization to the coated platelets, indicating that the interaction was mediated by the calcium-dependent conformation of the Gla domain and platelet exposure of negatively charged phospholipids. A reduced level of platelet fibrinogen exposure was observed at hemophilia A-like conditions in a model system of cell-based coagulation, indicating that coated platelet formation in hemophilia may be diminished. Addition of rFVIIa dose-dependently enhanced thrombin generation and partly restored platelet fibrinogen exposure.


CONCLUSIONS
The data suggest that rFVIIa localized preferentially on platelets activated with dual agonists, thereby ensuring enhanced thrombin generation localized at the site of injury where both collagen and tissue factor are exposed, the latter ensuring the formation of thrombin necessary for coated platelet formation.
Kjalke
M
M
Haemostasis Biology, Novo Nordisk A/S, Måløv, Denmark. mkja@novonordisk.com
Kjellev
S
S
Rojkjaer
R
R
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D001665:Binding Sites; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism*; D003029:Coagulants / Q000737:chemistry*; D003094:Collagen / Q000378:metabolism; D015942:Factor VIIa / Q000096:biosynthesis*; D005434:Flow Cytometry; D006467:Hemophilia A / Q000473:pathology; D006801:Humans; D010743:Phospholipids / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000378:metabolism / Q000502:physiology*; D013925:Thromboplastin / Q000378:metabolism
D003029:Coagulants; D010743:Phospholipids; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D015055:1-Carboxyglutamic Acid; D003094:Collagen; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin