Abstract
Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Factor VIII contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 that are modified by post-translational sulfation. This modification is required for full factor VIII procoagulant activity. We have employed site-directed mutagenesis to identify the individual sulfated tyrosines within factor VIII that influence activity. The molecules were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [35S]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for full activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr346-->Phe and Tyr1664-->Phe mutants displayed delayed thrombin activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, and 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr1680-->Phe mutant factor VIII displayed a 5-fold reduced affinity for von Willebrand factor. The results demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sites within factor VIII, two of which are required for proteolytic activation. The results demonstrate that the six sites of tyrosine sulfation modulate factor VIII activity through different mechanisms.
Highlights
Homologous C domains(Fig. 1) (4, 5 )
The results demonstrate that1) sulfation at tyrosine residues 346 and 1664 increases factor VI11 activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF
The factor VI11 activity in conditioned me- In particular,tyrosine sulfation a t residues 346,1664, and1680 dium from cells transfected with thecombined mutant, Y-F1-6, were preferentially required for activity in thefactor VI11 clotwas reduced to 44% of wild-type factor VI11 in the absence of ting assaycompared to the factor Xa generation assay
Summary
In the presence of increasing concentrations of reduced to 67% in the factor VI11 clotting assay These data sodium chlorate, the activity of wild-type factor VI11 was re- demonstrated that sulfatioonf specific tyrosine residues within duced to approximately 40%, correlating withreduced [35Slsu1- the acidic regions of factor VI11 influences factor VI11 activity. Protein concentrations were deter- mutant, Y-F‘, and wild-typefactor VI11 prepared from cclls mined by enzyme-linked immunosorbentassay as described under “Ex- treated with sodium chlorateboth boundvWF to a similar perimentalProcedures.”Thespecificactivity results (unitslmg) are from oneset of experimentaldata, and the percentagesof wild-type are extent, but half-maximal complex formation occurred a t apaveragedfrom triplicate assays with the standard error (S.E.) pre- proximately a5-fold greater concentrationof factor VIII. Wild-type factor VIII.These results demonstrate thsaut lfation of TyrI6*”increases theaffinity for vWFbinding by 5-fold
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
