Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin.

Abstract

Blood coagulation factor VIII (fVI) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothropsjararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by lightchain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

“…Cleavage at Arg 1689 is responsible for the dissociation of fVIII from vWf (11-13), which is a requisite step during fVIII activation because fVIII bound to vWf cannot bind to phospholipid membranes (14, 15). However, in the absence of vWf, cleavage at Arg 1689 is not required to produce a fVIIIa molecule with substantial activity (12,13,16).…”

“…Cleavage at Arg 1689 is responsible for the dissociation of fVIII from vWf (11-13), which is a requisite step during fVIII activation because fVIII bound to vWf cannot bind to phospholipid membranes (14, 15). However, in the absence of vWf, cleavage at Arg 1689 is not required to produce a fVIIIa molecule with substantial activity (12,13,16).At the plasma concentration of fVIII (ϳ1 nM) at physiological pH, human fVIIIa activity spontaneously decays to undetectable levels with a half-life of 2 min at 23°C (17-19) as a result of A2 subunit dissociation (4,5,20). A2 subunit dissociation is a reversible process, and active, heterotrimeric fVIIIa can be reconstituted using sufficiently high concentrations of purified A2 subunit and A1/A3-C1-C2 dimer (5).…”

The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

“…Activated fVIII (fVIIIa) is a heterodimer of 50-, 43-, and 73-kDa subunits, all of which are required for procoagulant activity (12). Cleavage of the LCh at Arg 1689 , which releases the acidic region (fVIII residues 1649 -1689), is responsible for dissociation of fVIIIa from vWf (8,13). The importance of the LCh acidic region for fVIII binding to vWf was suggested by the observations that several anti-acidic region monoclonal antibodies (mAbs) with epitopes within residues 1670 -1689 (14 -17) inhibit fVIII binding to vWf, as does complete deletion of the acidic region (18).…”

Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor.

“…A central question is whether light chain cleavage is essential only for the dissociation ofFactor VIII from vWf, or ifit is an intrinsic requirement for FVIII activity. This issue has been studied by Hill-Eubanks, Parker, and Lollar, using a snake venom protease that generates a thrombin-like cleavage ofthe porcine Factor VIII heavy chain, but that does not cleave the light chain (16). These investigators showed that porcine Factor VIII procoagulant activity can be generated by heavy chain cleavage alone if vWf is removed.…”

“…Consistent with that observation, porcine vWf inhibited the VIII:C function of venom enzyme-activated porcine Factor VIII, but it had no effect on thrombin-activated Factor VIII. For this reason, Hill-Eubanks et al suggested that the essential role of light chain cleavage is the dissociation of the vWf/Factor VIII complexes (16).…”