Factor V is activated and cleaved by platelet calpain: comparison with
thrombin proteolysis
HN Bradford, A Annamalai, K Doshi and RW Colman
Department of Medicine, Temple University School of Medicine, Philadelphia,
PA 19140.
Platelets are known to process human factor V during secretion and/or
membrane binding. We studied the functional and structural changes produced
in human factor V by purified human platelet calpain (calcium- activated
thiol protease) and compared the alterations with those induced by
thrombin. A maximum increase in coagulant activity of 2.5- fold was
observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03
U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for
alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to
reactions initiated by calpain resulted in further activation comparable to
that of thrombin alone, whereas the subsequent addition of calpain had no
effect on the extent or pattern of the activation of factor V by thrombin.
The cleavage pattern of factor V produced by these two enzymes are
distinctly different. Although thrombin activation eventually results in
four final components designated C1 (150 kd), D (105 kd), E (71 kd), and
F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd
within one minute. Further digestion of the 200 kd species by calpain gives
rise first to a polypeptide of 160 kd that is converted to a 140 kd and a
120 kd species by two minutes with an increase in coagulant activity.
Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10
directed to factor V and the thrombin-generated C1 fragment yields results
demonstrating a common epitope in these calpain-generated components of
200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide
gives rise to polypeptides of 100 and 65 kd, both undetectable on
immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of
less certain origin. Thus, platelet calpain generates a complex but
reproducible cleavage pattern different from thrombin that may explain the
partial activation observed. Nevertheless, calpain processing may play a
role in early hemostatic reactions involving platelets before the
appearance of the first thrombin molecule.
Volume 71,
Issue 2,
pp. 388-394,
02/01/1988
Copyright © 1988 by The American Society of Hematology
