kd
kd
Table 1. Parameters for fVIII interaction with PS-containing surfaces formed on the HPA
and L1 chips. The parameters are derived from kinetic curves (shown on Fig. 1 and 2)
using BIACORE Software.
PAGE 30
BIAJOURNAL – N O 1 – 2000
SCIENTIFIC REPORT
Coagulation Factor VIII Binding Properties
1
2
1
Andrey Sarafanov
, Evan Behre
and Evgueni Saenko
2
Holland Laboratory, American Red Cross, Rockville, Maryland 20855,
Biacore
USA.Inc, Piscataway, NJ, USA
1
That factor VIII, a vital component in the blood coagulation cascade, relies not
only on the chemical composition but also on the retention of the three-dimensional structure of a lipid bilayer to functionally bind membrane-anchored substrates is illustrated here using two different lipophilic BIACORE sensor chips.
F
Factor VIII (fVIII) is an essential component of the intrinsic pathway of blood
coagulation. Activated fVIII functions as
a cofactor for the serine protease factor, IXa; the
subsequent membrane-bound complex (factor
Xase) activates factor X to factor Xa, which participates in the activation of prothrombin to
thrombin, the key enzyme of the coagulation
cascade.
The major role of the phospholipid surface is
to reduce interactions between the components
of the factor Xase complex from a three- to a
two-dimensional space, which dramatically decreases the Km for factor X. Deficiency or defects
in fVIII result in a severe bleeding disorder,
hemophilia A, a genetic disease that occurs in 1
male in 5000.
Synthetic membranes of phosphatidylcholine
(PC) require the inclusion of at least 4% phosphatidylserine (PS) to form binding sites for fVIII
(1). Formation of additional fVIII binding sites
on the intact synthetic lipid vesicles at physiological PS concentration (≤ 5%) is mediated by the
addition of phosphatidylethanolamine (PE).
We compared the binding properties of 10 nM
fVIII lipid surfaces containing 4% PS, formed on
biosensor chips with different surface chemistries,
the properties of which induced phospholipid
surfaces of a different structure. When phospholipid vesicles are immobilized on the hydrophobic
alkanethiol-coated surface of the HPA chip, a flat
rigid lipid monolayer is formed (Figure 1A). In
contrast, vesicles immobilized on the L1 chip,
coated with lipophilically modified dextrane,
form a flexible lipid bilayer resembling a physiological membrane (Figure 1B).
Sensorgrams describing the interactions of
fVIII with vesicles with different PS content,
immobilized on an HPA chip and an L1 chip are
shown in Figures 2A and 2B, respectively. The
kinetic parameters (kon, koff and Kd) determined
for the lipid surfaces formed on HPA and L1
chips with a 10 % and 25% PS content are similar. In contrast, at physiological concentration of
PS (4%) the signal produced by fVIII binding to
the surface formed on the HPA chip was close to
baseline, whereas the use of L1 chip provided
BIAJOURNAL – N O 1 – 2000
reliable measurements of association and dissociation of fVIII (Figure 2 and Table 1).
Since it has been previously shown that the
presence of PE in membranes of vesicles containing 4% PS induces the formation of binding
sites for fVIII, we examined the effect of PEinclusion into lipid surfaces that were formed on
HPA and L1 chips. A 5.5-fold increase of the
number of fVIII binding sites was observed only
using the L1 chip (Table 1). The magnitude of
this effect was similar to that previously observed for intact lipid vesicles in solution.
At least three possible explanations for the PE
effect on fVIII binding to surfaces with a low PS
content have been proposed (2): (i) bulky PC hinders the access of fVIII to PS, whereas smaller PE
molecules do not; (ii) PE induces clustering of PS
whereby they may function as fVIII binding sites;
(iii) fVIII recognizes binding sites formed by PtdL-Ser and the adjacent PE moiety; if this is true,
the PE moiety may be provided by either PS
(containing a PE moiety) or PE itself.
Based on the data summarised here, we have
made the following conclusions:
• The L1 chip is superior to the HPA chip for
measurements of fVIII interaction with lipid
surfaces containing physiological concentrations of PS
• The fVIII binding properties of lipid surfaces
containing low PS concentration formed on L1
chips but not on HPA chips resemble those of
intact vesicles of similar composition; this may
be due to the greater mechanical flexibility of
the lipid surface formed on the L1 chip,
endowing the membrane with properties similar to those of intact vesicles and possibly of
physiological membranes
• The inclusion of physiological concentrations of PE in lipid surfaces formed on L1
chips, but not on HPA chips, induces the formation of additional fVIII binding sites, a phenomenon similar to that observed for lipospheres in solution.
References
1 Saenko, E.L. et al. J.Biol.Chem. 273: 27918 (1998).
2 Gilbert, G.E. and Arena, A.A. J.Biol.Chem.
270:18500 (1995)
Figure 1. Curves 1,2 and 3 correspond to 25%, 10 % and 4% of PS
in the PSPC surface
Figure 2. 1: fVIII binding to surfaces containing 4% PS and 96%
PC. and 2: to surfaces containing
4% PS, 76% PC, and 20% PE.
PAGE 31