Abstract
Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.
Highlights
factor VIII (FVIII) in the circulation forms a tight, noncovalent complex with von Willebrand factor (vWF), and both the amino-terminal and the carboxylterminal regions of FVIII are involved in this interaction [6, 7]
To characterize the bound FVIII and FVIIIa structurally, platelets were incubated with thrombin (0.1 u/ml), CaCl2 (5 mM), EGRFIXa (45 nM), and factor X (FX) (1.5 M) for 20 min at 37 °C in the presence of either 125I-FVIII or 125I-FVIIIa and centrifuged through 20% sucrose to separate the bound from free ligand as described under “Experimental Procedures.”
Previous studies from our laboratory have demonstrated that platelets possess specific, high affinity, and saturable receptors for factor IXa (FIXa) [21] and FX [23], and Nesheim et al [22] have shown that FVIII binds to platelets in a saturable and specific manner
Summary
Reagents—Phenalanyl-prolyl-arginine chloromethyl ketone (PPACK) and calcium ionophore A23187 were from Calbiochem. We had success in developing a highly reproducible method utilizing the Bolton-Hunter reagent [36] to radiolabel FVIII to high specific radioactivity while retaining virtually 100% of its specific biological activity In this procedure, 20 –50 g of rFVIII was precipitated with 25% PEG 8000, washed, and resuspended in 50 l of 0.5 M NaCl, 20 mM HEPPS, pH 8.0, 5 mM CaCl2, 0.1% Tween 80 just before use.
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