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Stabilization of thrombin-activated porcine factor VIII:C by factor IXa
phospholipid
P Lollar, GJ Knutson and DN Fass
The activation of porcine factor X by an enzymatic complex consisting of
activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor
VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of
an irreversible inhibitor of factor Xa, 5-dimethylamino-
naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone (
DEGR -CK). The formation of factor Xa was measured continuously by
monitoring the increase in solution fluorescence intensity that occurs upon
formation of DEGR -factor Xa. Omission of any component from the enzymatic
complex reduced the reaction rate to a negligible level. In the presence of
fixed excess factor IXa, the velocity of factor X activation was linearly
dependent on the concentration of factor VIII:C, and thus, provided a
plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH
U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and
calcium, followed at variable time intervals by the addition of factor X
and DEGR -CK, was complete within 5 min, as judged by the fluorometric
assay, and resulted in little or no loss of factor VIII:C activity over a
period of 20 min; whereas, activation in the absence of either IXa or
phospholipid vesicles decreased the half-life of factor VIII:C to
approximately 5 min. Analysis of 125I-factor VIII:C-derived activation
peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis
revealed identical results, regardless of whether factor IXa and/or
phospholipid vesicles were included in the activation, suggesting that the
lability of factor VIII:Ca is not due to a major alteration of its primary
structure. We conclude that the activated porcine factor VIII:C molecule is
stabilized markedly because of its interaction with factor IXa and
phospholipid.
Volume 63,
Issue 6,
pp. 1303-1308,
06/01/1984
Copyright © 1984 by The American Society of Hematology
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