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Figure 4.
Figure 4. Platelet adhesion to mutant vWF A1 domain fragments.
Amino acid substitutions are indicated with the one letter code
of the native and mutant residues. Single substitutions with Ala
caused essentially complete loss of function at the following
positions: Glu 557 in strand  2;
His 559 in loop 2-
3;
Tyr 565 in strand 3;
Lys 572 in loop 3-
 2;
Glu 596 and Lys 599 in helix 3.
The G561S substitution in the 2-
3
loop impaired the function of the wild type A1 domain as well as
of the I546V mutant. In contrast, control mutations of Lys 585
in loop 2-
3,
Arg 632 in helix 4,
Lys 644 in loop 4-
5,
and His 656 in loop 5-
5
had minimal or no effect on function. The number of surface
interacting platelets was counted between 1 and 4 min from the
beginning of flow; the wall shear rate was 1,500 s^-1. The
results of mutant fragments are expressed as a percentage of
those obtained with a wild type control tested on the same
experimental day. The average number of platelets interacting
with wild type vWF fragment was 57 (n = 11). The results
represent the mean with standard error of the mean of two to
four separate experiments performed with each mutant fragment.
Video clips can be viewed on the following web
sites:http://www.scripps.edu/mem/biochem/KI and
http://www.scripps.edu/mem/eht/ruggeri. These clips show the
rolling of platelets, seen as white round objects, tethered to
immobilized wild type A1 domain (top half of the screen) or
I546V mutant A1 domain (lower half of the screen). The wall
shear rate was 1500 s^ -1. Platelets interacting with the mutant
A1 domain roll with considerably lower velocity (median = 4.2
 m
s^-1) than those interacting with the wild type control (median
= 44.7 m
s^ -1). The few larger objects that appear transiently in both
screens and move rapidly are leukocytes that interact briefly
with activated platelets on the surface.
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