Interaction of the 268-282 region of glycoprotein Ibalpha with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII.
Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII.
De Cristofaro
R
R
Department of Internal Medicine, Hemostasis Research Center, Catholic University School of Medicine, 00168 Rome, Italy. rdecristofaro@rm.unicatt.it
De Filippis
V
V
D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005169:Factor VIII / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014443:Tyrosine; D005169:Factor VIII; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa