Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model.
The role of tissue factor/factor VIIa (FVIIa) and factor VIIIa/factor IXa (FVIIIa/FIXa) complexes in thrombus formation was examined in a human ex vivo blood flow system by use of active site-blocked FVIIa (FVIIai) and FIXa (FIXai) as selective inhibitors. Blood was drawn directly from the veins of volunteers into a mixing device where FVIIai and FIXai were mixed with flowing blood. The blood then entered parallel-plate chambers containing coverslips coated with human fibrillar collagen or tissue factor-expressing cell layers of tumor necrosis factor-alpha-stimulated human endothelial cells, human smooth muscle cells, and J82 cells. Exposure of stimulated endothelial cells to blood flowing at a venous shear rate of 65/s led to fibrin deposition, which was inhibited by infusion of FVIIai (IC50, 3 nmol/L), as quantified by micro-densitometry of fibrin-stained coverslips. Whereas FIXai (600 nmol/L) was only a weak inhibitor, FVIIai (60 nmol/L) reduced fibrinopeptide A (FPA) plasma levels from 504 +/- 79 to 171 +/- 27 ng/mL and concomitantly inhibited platelet thrombus deposition. Similarly, experiments with smooth muscle cells and J82 cells showed that FVIIai but not FIXai efficiently reduced FPA levels. Conversely, with tissue factor-free collagen, ,hich induces platelet-dependent fibrin formation, infusion of FIXai but not of FVIIai inhibited fibrin deposition (IC50, 8 nmol/L) and reduced FPA levels from 55 +/- 8 to 9 +/- 5 ng/mL. However, FIXai did not affect the number of platelet thrombi deposited on collagen.(ABSTRACT TRUNCATED AT 250 WORDS)
Kirchhofer
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Pharma Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Tschopp
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Baumgartner
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HR
D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation; D001792:Blood Platelets / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors*; D015945:Factor XIa / Q000037:antagonists & inhibitors*; D005337:Fibrin / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D015539:Platelet Activation; D012212:Rheology; D013927:Thrombosis / Q000201:enzymology*; D013997:Time Factors; D001620:beta-Thromboglobulin / Q000378:metabolism
D010455:Peptides; D001620:beta-Thromboglobulin; D005344:Fibrinopeptide A; D005337:Fibrin; D003094:Collagen; D015942:Factor VIIa; D015945:Factor XIa