Abnormal hemostasis in a knock-in mouse carrying a variant of factor IX with impaired binding to collagen type IV.
BACKGROUND
Factor IX binds to collagen type IV, but this binding has no known consequence.


OBJECTIVE
To determine the effect of reduced binding of FIX to collagen IV.


METHODS
We constructed and characterized 'knock-in' mice containing the mutation lysine 5 to alanine (K5A) in the Gla domain of their FIX. The K5A mutation dramatically reduced the affinity of FIX for collagen type IV, but had no measurable effect on platelet binding, phospholipid binding, or in vitro clotting activity. However, K5AFIX mice had a mild bleeding tendency, despite their in vitro clotting activity being normal. Hemostatic protection from delayed rebleeding was intermediate between wild-type and hemophilia B mice (which had no detectable clotting activity); moreover, survival of K5A FIX mice after nascent clot removal was dramatically improved as compared with hemophilia B mice. Importantly, there was no detectable difference between K5AFIX and wild-type mice in either a laser-induced thrombosis model or the chromogenic FIX activity assay. In contrast, after ferric chloride injury, which exposes collagen IV as well as other basement membrane proteins, intravital microscopy revealed that vessel occlusion was significantly slower in K5AFIX mice than in wild-type mice.


CONCLUSIONS
Our results indicate that the FIX molecule with decreased affinity for collagen IV has altered hemostatic properties in vivo and that the binding of FIX to collagen IV probably plays a significant functional role in hemostasis.
Gui
T
T
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Reheman
A
A
Ni
H
H
Gross
P L
PL
Yin
F
F
Monroe
D
D
Monahan
P E
PE
Stafford
D W
DW
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D024141:Collagen Type IV / Q000378:metabolism*; D005164:Factor IX / Q000032:analysis / Q000235:genetics*; D055879:Gene Knock-In Techniques; D014644:Genetic Variation; D002836:Hemophilia B; D006470:Hemorrhage; D006487:Hemostasis / Q000235:genetics*; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D013927:Thrombosis
D024141:Collagen Type IV; D005164:Factor IX