Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding

Abstract

The importance of factor VIII (FVIII) 1 and FIX in hemostatic reactions is evident by the fact that hemophilia A (FVIII deficiency) and hemophilia B (FIX deficiency) are the two most serious congenital coagulation defects, both producing severe, life-threatening and life-long hemorrhagic disease. FVIII is synthesized as a single polypeptide chain containing 2,351 amino acids (1) and shows a discrete domain structure, A1-a1-A2-a2-B-a3-A3-C1-C2; the domains are separated by short spacers (a1, a2, and a3) composed of acidic regions that contain clusters of Asp and Glu residues (2). The A domains display ϳ30% homology to each other, and they also share sequence homology with ceruloplasmin and FV. The B domain is unique to FVIII. The C domains share homology with FV and discoidin 1, a lectin (1). During proteolytic activation of pro-cofactor (FVIII), the B domain that does not contribute to cofactor function is excised. The heavy chain (HC) and light chain (LC) remain noncovalently associated through the A1 and A3 domains in a metal ion-dependent manner and participate as an active cofactor in tenase complex formation (1). The following terminology, suggested by Lenting et al. (2), for FVIII subunits was used: A1 ϭ HC-derived fragment containing residues 1-372; A2 ϭ HC-derived fragment containing residues 373-740; A1-A2 ϭ HC subunit of FVIII containing residues 1-740 (without the B domain); B ϭ HC subunit residues 741-1648; LC ϭ LC of FVIII; A3-C1-C2 ϭ LC-derived fragment arising from the cleavage at position 1689. Thrombin cleaves FVIII at one specific site within the LC, Arg 1689 , and at two sites in the HC, Arg 372 and Arg 750 , resulting in a heterodimer of 50 kDa (A1 domain), 43 kDa (A2 domain), and 73 kDa (A3-C1-C2 dimer); all of these subunits are required for the procoagulant activity (3). More specifically, the cleavages at Arg 372 and Arg 1689 are required to exert full cofactor activity (4, 5), whereas cleavage of the LC at Arg 1689 is also responsible for dissociation of FVIII from von Willebrand factor (vWF) (6).FVIII in the circulation forms a tight, noncovalent complex with vWF, and both the amino-terminal and the carboxylterminal regions of FVIII are involved in this interaction (6, 7). The formation of the FVIII-vWF complex is essential for the survival of FVIII in the circulation. Another functional consequence of FVIII-vWF complex formation is to prevent binding of FVIII to components of the FX-activating complex, e.g. the binding of FVIII LC to FIXa has been shown to be inhibited by vWF (8). Furthermore, FVIII-vWF complex is less susceptible to proteolytic degradation by proteases like activated protein C (9) and FXa (10). vWF also prevents FVIII binding to phospholipids (11) and platelets (12). In contrast, vWF does not protect FVIII from thrombin cleavage (13,14). Recently, it has been shown (15) that FIXa displays similar affinity for pro-cofactor (FVIII) and active cofactor (FVIIIa), but as mentioned above, the FIXa-binding site is not accessible when FVIII is in com-

The factor VIII C1 domain contributes to platelet binding

“…This C1C2 binding was only partially competed by FVIII or FVIIIa. Fluorescein-labeled FVIII and FVIIIa did not bind to platelets before activation, as reported by others, 19,36,37 so this observed C1C2 binding is unlikely to be physiologically relevant. However, Nesheim et al 38 reported 450 binding sites per activated platelet for 35 S-Met-labeled FVIII and approximately 20 FVIII molecules bound per nonactivated platelet.…”

“…42 The estimate of 7000 C1C2-binding sites per activated platelet ( Figure 7) was 4-to 5-fold greater than the 1400 sites estimated for C2. Previous estimates of FVIIIa molecules bound to activated platelets have ranged from 200 to 2000 molecules bound per platelet, 10,19,37,38,43 although higher estimates of up to approximately 25 000 sites per activated platelet have also been reported. [42][43][44][45] Different methods of activating the platelets might account for some of this variation, and heterogeneity in the activated platelet population can also complicate analysis of binding.…”

“…Further studies are needed to distinguish direct versus indirect effects of the C1 domain in binding of FVIII and FVIIIa in contributing to the binding interactions. The physiological attachment of FVIIIa and FIXa to the platelet surface likely involves interactions with other membrane components besides phospholipids 19,38 and is influenced by various agonists (eg, fibrin). 37 The orientation of C1 with respect to the membrane surface and the roles of specific residues in membrane binding are the subjects of current investigations.…”

“…19 For direct fluorescein labeling, a second recombinant FVIII (Kogenate FS) was purchased from Bayer Pharmaceuticals (Elkhart, IN), labeled with fluorescein (above) and thrombin activated as with the other FVIII preparation. FVIII clotting activity was assessed using a 1-stage, semiautomated assay system essentially as described for factor IX activity, 20 but using commercial factor VIII-deficient plasma.…”

PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

“…FVIII (K d ~3.0 nM) (4,9,10) and FVIIIa (K d ~0.8 nM) (4) also bind to specific, high-affinity sites on activated platelets. Although FX and prothrombin occupy a high-capacity, low-affinity (K d ~300 nM) shared site (5), FX also occupies a specific binding site (K d ~5 nM), consisting of bound FVIIIa (4,6,11).…”