Cysteamine enhances the procoagulant activity of Factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine).

Abstract

IntroductionWe have recently identified the molecular defect responsible for cross-reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East HartfordlFVIII-EHI) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose-and time-dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII-EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.(J. Clin. Invest. 1992. 89:1375-1381

Cysteamine revisited: repair of arginine to cysteine mutations

“…A similar reduction in activity has been observed in FVIII wild-type plasma protein incubated at 37°C for 1 h with cysteamine (1-100 mM) (Aly et al 1992). Some reasons argued by the authors are: the accessibility of natural cysteine residues in the ASL protein by free cysteamine, the possible impact of cysteamine in the wild-type protein by acting as a mild competitive inhibitor of ASA and the potential of cysteamine to modify cysteines in partner proteins and alter the 3D protein configuration, the protein process and the trafficking of ASL and other proteins.…”

“…A 18% decrease in the enzymatic activity of wild-type CBS was documented after 8 h of incubation with cysteamine (Mendes et al 2015). A similar effect was also observed with ApoE3 (Weisgraber 1990) and factor VIII (Aly et al 1992). In addition, cysteamine failed to target CBS p.Arg336Cys in cultured lymphoblast of Qatari homocystinuria patients (Blom et al, unpublished results) and in cultured 293T cells containing Arg to Cys mutations in the expressed ASL protein (Inauen et al 2016).…”

“…This variant abolishes thrombin cleavage site and thereby blocks FVIII function. The possibility that FVIII may be susceptible to chemical modification by cysteamine was considered after the successful results shown for ApoE2 (Aly et al 1992, Gahl et al 1985). The incubation of plasma of FVIII hemophilic patients with cysteamine at micro- to millimolar levels increased FVIII activity 14fold and restored light chain cleavage by thrombin.…”

“…The incubation of plasma of FVIII hemophilic patients with cysteamine at micro- to millimolar levels increased FVIII activity 14fold and restored light chain cleavage by thrombin. Aly et al, suggested that Cys 1689 is converted by cysteamine treatment to a positively charged lysine analogue recovering the thrombin cleavage site (Aly et al 1992). …”

The diversity of the immune response to the A2 domain of human factor VIII

“…40,41 Furthermore, although mutations at Arg1689 cause hemophilia A, dissociation of Arg1689 mutant fVIII/VWF complexes with disulfide-bond reducing agents can produce fVIII procoagulant activity in vitro. 42,43 Thus, cleavage at Arg1689, while necessary for dissociation of fVIII from VWF, is not necessary for significant fVIIIa activation per se. These results suggest that B66 and 1D4 possess both VWF-dependent and VWF-independent inhibitory properties.…”