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JBC, Vol. 250, Issue 6, 2150-2156, Mar, 1975
S. P. Bajaj, R. J. Butkowski and K. G. Mann
The binding of Ca2+ to prothrombin and the intermediates of prothrombin
activation was investigated by equilibrium dialysis using 45Ca2+ as the
ligand. Scatchard plots of these data indicate that prothrombin (Mr =
70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in
terms of their binding affinity. Six of these Ca2+ binding sites have log
Kassoc = 3.5 and all are found intact in the NH2-terminal segment
(activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four
or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7
present in prothrombin are found intact in the remaining COOH-segment
(activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon
further activation the Ca2+ binding sites residing in intermediate 1 are
found intact in activation intermediate 4 (which constitutes the
NH2-terminal segment of the intermediate 1 molecule). The remaining
COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the
intermediate 1 molecule has no affinity for Ca2+. The activation of
prothrombin and activation intermediates 1 and 2 was studied using these
activators: Factor Xa alone, Factor Xa-Ca+, AND Factor
Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was
progressively increased as Ca2+ and phospholipid were added to the system,
whereas no significant increase in the rates of activation of intermediate
1 and 2 was observed. When Factor V was added to the Factor
Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was
greatly enhanced. In the absence of Ca2+, Factor V had no effect on the
rate of thrombin formation from intermediate 1. Factor V had no stimulatory
effects on the rate of intermediate 2 activation. However, in the presence
of an equimolar amount of intermediate 4, Factor V accelerated the
conversion of intermediate 2 to thrombin. These studies indicate that the
Ca2+ binding sites of the prothrombin molecule are contained in the "pro"
fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate
1 and intermediate 2, both of which lack the strong Ca2+ binding sites of
prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid
complex activation when compared to prothrombin. The addition of Factor V
to the catalyst results in acceleration of the activation rate of
intermediate 1 and an equimolar mixture if intermediates 2 and 4. These
results lead us to conclude that the strong Ca2+ binding sites are the
sites of phospholipid binding (intermediate 3), whereas the seak binding
sites are the sites of Factor V binding (intermediate 4).
Prothrombin fragments. Ca2+ binding and activation kinetics
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