Review
Role of Activation of the Coagulation Factor VIII in Interaction with vWf, Phospholipid, and Functioning within the Factor Xase Complex

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Abstract

Blood coagulation factor VIII (fVIII) in its nonactivated form circulates in plasma in a complex with von Willebrand factor (vWf). Upon activation by thrombin- or factor Xa-mediated site-specific proteolysis, activated fVIII (fVIIIa) serves as a cofactor for factor IXa. This protein complex assembled on a phospholipid surface (factor Xase) activates factor X. This complex plays the key role in the intrinsic pathway of blood coagulation. We reviewed the molecular events triggered by fVIII activation, which are required for the assembly and functioning of the Xase complex, including fVIIIa dissociation from vWf and a significant increase of fVIII affinity for binding to the phospholipid surface. Both events are mediated by activation-related cleavage within fVIII light chain (LCh), releasing the 40 amino-acid N-terminal LCh peptide, which is followed by a conformational change within the C2 domain. The conformational change within LCh is also required for the optimal fVIII cofactor functioning within the factor Xase complex, exerted via fVIIIa interactions with phospholipid, factor IXa, and factor X. Since factor IXa not only stabilizes but also proteolytically inactivates fVIIIa within the factor Xase complex, the stability of the membrane-bound fVIIIa in the presence and absence of factor IXa is discussed. In conclusion, we outline some new possible directions of the research. One of them arises from the recently demonstrated ability of plasma lipoproteins to provide a phospholipid surface for the assembly of the factor Xase complex in vitro. This finding raises a possibility that lipoproteins participate in factor Xase functioning in vivo and suggests a direct link between elevated levels of lipoproteins associated with atherosclerosis and increased thrombogenicity associated with this disease.

Section snippets

Function and Key Interactions of fVIII in the Intrinsic Coagulation Cascade

Factor VIII is an essential component of the intrinsic pathway of blood coagulation. In this pathway, activated fVIII functions as a cofactor for the serine protease factor IXa, and their membrane-bound complex (factor Xase) activates factor X to factor Xa (van Dieijen et al. 1981). Consequently, factor Xa participates in activation of prothrombin into thrombin, the key enzyme of the coagulation cascade. Assembly of the factor Xase complex occurs on membranes of activated platelets at the sites

Activation of fVIII

Thrombin and factor Xa are known to be the proteases responsible for activation of fVIII Vehar et al. 1984, Lollar et al. 1985, Hill-Eubanks and Lollar 1990. Both of them cleave the fVIII molecule at Arg372 and Arg740 within the HCh and at Arg1689 in the LCh, producing the A1, A2, and A3-C1-C2 fragments, which compose the heterotrimeric activated fVIII. Factor Xa, however, has one more additional cleavage site at Arg1721, which is also associated with fVIII activation. Although activation by

Dissociation of Activated fVIII from vWf

Removal of the LCh acidic region by cleavage at Arg1689 (or Arg1721) is an ultimate requirement for the release of activated fVIII from vWf and its functioning within the Xase complex. The importance of this cleavage was demonstrated by the following observations. Hemophilia A-associated or artificially introduced point mutations at the position 1689, making this site resistant to proteolysis, prevent fVIII dissociation from vWf and subsequent exertion of its cofactor activity Kamisue et al.

Activation of fVIII Leads to Conformational Changes within the C2 Domain, Which Are Required for the fVIII Release from vWf

The acidic region of the LCh not only directly participates in vWf binding, but it is also required for maintaining normal conformation of the C2 domain binding site for vWf. The possibility that the conformational change within the C2 domain occurs upon removal of the acidic region was tested with the use of a monoclonal antibody with the epitope within the C2 domain residues 2170–2327. The reduced affinity of the thrombin-cleaved LCh for this antibody compared to that of the intact LCh

Activation of fVIII Increases Its Affinity for Phospholipids

FVIII binding to phospholipid is mediated by the LCh (Arai et al. 1989), which is entirely responsible for the high affinity of this interaction Saenko et al. 1998, Spaargaren et al. 1995. Similar kinetic parameters for the LCh and fVIII binding to phosphatidyl serine (PS)-containing membranes and platelets indicate that the HCh does not have any contribution to the LCh binding to phospholipids (Saenko et al. 1998), which is in contrast to the stabilizing role of HCh in fVIII/vWf interaction

Stability of the Membrane-Bound fVIIIa

Binding of fVIIIa to phospholipid does not change the rate of its inactivation resulting from dissociation of the A2 subunit Fay et al. 1996, Fay and Smudzin 1992, Lollar et al. 1992. The rate constants kon and koff for equilibrium fVIIIa-PL ↔ A2+A1/A3-C1-C2-PL describing inactivation of the phospholipid-bound fVIIIa are 1.3 × 106 M−1min−1 and 0.32 min−1 (Lollar et al. 1992), respectively, and the Kd is 260 nM (Fay and Smudzin 1992). The half-life of 2.1 min for the membrane-bound fVIIIa

Effect of the LCh Conformational Changes Occurring upon Activation on Interaction of fVIIIa with Other Components of Xase Complex

It was proposed (Fay 1999) that the A3 domain of membrane-bound fVIIIa involved in the high-affinity binding with factor IXa is positioned closer to the membrane than the A2 subunit involved in modulation of the factor IXa active site. It is relevant to propose that the conformational change occurring within the LCh upon removal of the acidic region provides such orientation of fVIIIa, in which the A2 domain gets closer to the factor IXa active site. This assumption was supported by

Major Questions to Be Addressed and New Directions of Research

Three-dimensional structures of the A domains of fVIII were predicted based on the sequence homology with ceruloplasmin (Pemberton et al. 1997). The structure of the isolated C2 domain has also been recently reported (Pratt et al. 1999). However, the actual structures of the intact and the cleaved LCh remain to be elucidated. This will help to localize the residues within the intact and activated LCh involved in the interactions with vWf and phospholipid and will also enable us to understand

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