Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is essential.
Human coagulation factor XI (FXI) is a plasma serine protease composed of 2 identical 80-kd polypeptides connected by a disulfide bond. This dimeric structure is unique among blood coagulation enzymes. The hypothesis was tested that dimeric conformation is required for normal FXI function by generating a monomeric version of FXI (FXI/PKA4) and comparing it to wild-type FXI in assays requiring factor IX activation by activated FXI (FXIa). FXI/PKA4 was made by replacing the FXI A4 domain with the A4 domain from prekallikrein (PK). A dimeric version of FXI/PKA4 (FXI/PKA4-Gly326) was prepared as a control. Activated FXI/PKA4 and FXI/PKA4-Gly326 activate factor IX with kinetic parameters similar to those of FXIa. In kaolin-triggered plasma clotting assays containing purified phospholipid, FXI/PKA4 and FXI/PKA4-Gly326 have coagulant activity similar to FXI. The surface of activated platelets is likely to be a physiologic site for reactions involving FXI/FXIa. In competition binding assays FXI/PKA4, FXI/PKA4-Gly326, and FXI have similar affinities for activated platelets (K(i) = 12-16 nM). In clotting assays in which phospholipid is replaced by activated platelets, the dimeric proteins FXI and FXI/PKA4-Gly326 promote coagulation similarly; however, monomeric FXI/PKA4 has greatly reduced activity. Western immunoblot analysis confirmed that activated monomeric FXI/PKA4 activates factor IX poorly in the presence of activated platelets. These findings demonstrate the importance of the dimeric state to FXI activity and suggest a novel model for factor IX activation in which FXIa binds to activated platelets by one chain of the dimer, while binding to factor IX through the other.
Gailani
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Department of Pathology, Vanderbilt University, Nashville, TN, USA. dave.gailani@mcmail.vanderbilt.edu
Ho
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Sun
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MF
Cheng
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Walsh
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PN
D001777:Blood Coagulation; D001792:Blood Platelets / Q000737:chemistry* / Q000502:physiology; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D019281:Dimerization; D004789:Enzyme Activation; D005164:Factor IX / Q000502:physiology*; D015949:Factor IXa / Q000502:physiology; D005172:Factor XI / Q000502:physiology; D015945:Factor XIa / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D015539:Platelet Activation; D011288:Prekallikrein / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins
D011993:Recombinant Fusion Proteins; D005164:Factor IX; D005172:Factor XI; D011288:Prekallikrein; D015949:Factor IXa; D015945:Factor XIa