Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 5

In the presence of anionic phospholipids and Ca2+, activated protein C inactivates factors Va and VIIIa. MLID92275243  86 Factor Va is cleaved by activated protein C after residues 506 and 1,765, although cleavage after residue 506 is likely responsible for the loss in cofactor activity. MLID87280215  87 Similarly, activated protein C cleaves factor VIII and factor VIIIa after multiple residues 336, 562, and 740. MLID92041838  88 Inactivation is more closely associated with cleavage after residue 562, the site homologous to residue 506 in factor V. Binding sites for activated protein C were localized to the light chains at residues 1,865–1,874 in factor Va and 2,009–2,010 in factor VIII, which are both localized to a homologous region in the COOH-terminal end of the A3-domain. MLID90110208  89

Binding to Phospholipids

Phospholipids interact with substrates, enzymes, and cofactors to play a critical role in the assembly and functional activity of the coagulation protease complexes. Negatively charged phospholipids are required for factor VIIIa-mediated enhancement of the activation of factor X. MLID81142359 MLID90110140 MLID93069110  7,90,91 In vivo, the negatively charged phospholipids are likely provided by activated platelets and damaged endothelial cells. Factors VIII and V bind phosphatidylserine by both hydrophobic and electrostatic interactions. MLID82108388 MLID83231443 MLID85122693 MLID87157601 MLID87137403 MLID92382588  92–98 However, factor V does not efficiently compete with the binding of factor VIII to phospholipid vesicles composed of 15% phosphatidyl-L-serine. MLID90110140  90 Under equilibrium conditions, factor VIII can bind phospholipid vesicles containing 15–25% phosphatidylserine with an apparent Kd of 2–4 nM. MLID90110140 MLID92381007 MLID92348453  90,99,100 Saturation occurs between 170 and 385 mol phospholipid/mol factor VIII, MLID90110140  90 although the process involves both rapid and slow interactions. MLID93232040  101 Factor VIII binding to phospholipid involves stereoselective recognition of the O-phospho-L-serine moiety of phosphatidylserine. MLID93385096  102 Factor V displays a similar affinity to phospholipids but has a lower requirement for phosphatidylserine. Since the composition of phosphatidylserine exposed on the platelet membrane surface can increase from 2% to 13% after stimulation, MLID84080433  103 the increase in phosphatidylserine content could account for the ability of factor VIII to bind the surface of thrombin-activated platelets specifically.

Addition of negatively charged phospholipids to the factor VIII/vWF complex dissociates factor VIII from vWF. MLID82134732  55,56 Interestingly, thrombin-treated factor VIII does not bind vWF with high affinity MLID87304256 MLID87246689 MLID88273151  53,62,65 but does retain phospholipid binding properties. 56 The phospholipid-binding domain within factor VIII is localized to the light chain, MLID88178572  104,105 and antibody inhibition studies suggest that the phospholipid binding site likely occurs in the C2 domain. MLID89255956  106 Peptides corresponding to the COOH terminus of factor VIII (residues 2,303–2,332) inhibit the interaction of factor VIII with phospholipid. MLID90248570  69 In addition, deletion analysis suggests that a phospholipid-binding domain resides in the factor V C2 domain. MLID92156167  107 By contrast, a proteolytic fragment of the factor V A3 domain inhibits factor V binding to phospholipid, suggesting that a phospholipid-binding site resides in the NH2-terminal end of the factor Va light chain. MLID91072354  108 Thus, at present it is unclear whether the phospholipid-binding sites of factors V and VIII occur at the same positions within the proteins.

Regulation of Factor VIII Activity

Activation of Factor VIII

On treatment of intact factor VIII with thrombin, a rapid 30-fold increase and subsequent first-order decay of procoagulant activity occurs. The activation coincides with proteolysis of both the heavy and light chains of factor VIII (Fig. 105-1). Cleavage within the heavy chain after arginine residue 740 generates a 90,000 MW polypeptide that is subsequently cleaved after arginine residue 372 to generate polypeptides of 50,000 MW and 43,000 MW. 109 Concomitantly, the 80,000 MW light chain is cleaved after arginine residue 1,689 to generate a 73,000 MW polypeptide. 109 Each thrombin cleavage site is bordered by a region rich in acidic amino acids that also contains the post-translationally modified amino acid tyrosine sulfate. MLID92207952  25 The tyrosine sulfate residues enhance thrombin cleavage at adjacent sites, 70 suggesting that these regions interact with the anion binding exosite in thrombin, similar to the thrombin interaction with the COOH-terminal end of hirudin that has acidic amino acids and also contains tyrosine sulfate. MLID90327074 MLID90264426  110,111