The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface.

Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we showed that, in an ELISA setup, a purified extracellular fragment of GPIbalpha bound to immobilized rFVIIa. Surface plasmon resonance established a affinity constant (K(d)) of approximately 20 nM for this interaction. In addition, CHO cells transfected with the GPIb-IX-V complex could adhere to immobilized rFVIIa, whereas wild-type CHO cells could not. Furthermore, platelets sti-mulated with a combination of collagen and thrombin adhered to immobilized rFVIIa under static conditions. Platelet adhesion was inhibited by treatment with O-sialoglycoprotein endopeptidase, which specifically cleaves GPIbalpha from the platelet surface. In addition, rFVIIa-mediated thrombin generation on the activated platelet surface was inhibited by cleaving GPIbalpha from its surface. In summary, 3 lines of evidence showed that rFVIIa interacts with the GPIb-IX-V complex, and this interaction enhanced tissue factor-independent thrombin generation mediated by rFVIIa on the activated platelet surface. The rFVIIa-GPIbalpha interaction could contribute to cessation of bleeding after administration of rFVIIa to patients with bleeding disorders.

[1]  P. D. de Groot,et al.  Mechanism of action of recombinant factor VIIa , 2003, Journal of thrombosis and haemostasis : JTH.

[2]  D. Monroe,et al.  Human monocytes support factor X activation by factor VIIa, independent of tissue factor: implications for the therapeutic mechanism of high-dose factor VIIa in hemophilia. , 1994, Blood.

[3]  J. Oliver,et al.  A possible mechanism of action of activated factor VII independent of tissue factor. , 1998, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[4]  K. Fukudome,et al.  Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity , 2007, Journal of thrombosis and haemostasis : JTH.

[5]  B. Dahlbäck,et al.  Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain* , 2006, Journal of Biological Chemistry.

[6]  P. D. de Groot,et al.  Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation. , 2003, Blood.

[7]  M. Blombäck,et al.  The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX , 2003, Journal of thrombosis and haemostasis : JTH.

[8]  G. Kenet,et al.  A new approach to treatment of bleeding episodes in young hemophilia patients: a single bolus megadose of recombinant activated factor VII (NovoSeven®) , 2003, Journal of thrombosis and haemostasis : JTH.

[9]  José A López,et al.  Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. , 2005, Blood.

[10]  A. Kaplan,et al.  Platelet Glycoprotein Ib: A Zinc-Dependent Binding Protein for the Heavy Chain of High-Molecular-Weight Kininogen , 1999, Molecular medicine.

[11]  J. Oliver,et al.  Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system , 2004, Journal of thrombosis and haemostasis : JTH.

[12]  K. Mann,et al.  How factor VIIa works in hemophilia , 2003, Journal of thrombosis and haemostasis : JTH.

[13]  K. Mann,et al.  The Regulation of the Factor VII-Dependent Coagulation Pathway: Rationale for the Effectiveness of Recombinant Factor VIIa in Refractory Bleeding Disorders , 2000, Seminars in thrombosis and hemostasis.

[14]  R. Colman,et al.  Human Factor XII Binding to the Glycoprotein Ib-IX-V Complex Inhibits Thrombin-induced Platelet Aggregation* , 2000, The Journal of Biological Chemistry.

[15]  E. Persson,et al.  Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A. , 2003, Blood.

[16]  C. Sáez,et al.  Human platelets synthesize and express functional tissue factor. , 2007, Blood.

[17]  E. Santagostino,et al.  A prospective randomized trial of high and standard dosages of recombinant factor VIIa for treatment of hemarthroses in hemophiliacs with inhibitors , 2006, Journal of thrombosis and haemostasis : JTH.

[18]  R. Rojkjaer,et al.  Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonist , 2007, Journal of thrombosis and haemostasis : JTH.

[19]  D. Monroe,et al.  High‐dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia‐like conditions in a cell‐based model system , 2001, British journal of haematology.

[20]  Nigel Mackman,et al.  Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenecity of human platelets , 2006, The Journal of experimental medicine.

[21]  P. D. de Groot,et al.  Enhanced in vitro procoagulant and antifibrinolytic potential of superactive variants of recombinant factor VIIa in severe hemophilia A , 2003, Journal of thrombosis and haemostasis : JTH.

[22]  J. Sixma,et al.  Structures of Glycoprotein Ibα and Its Complex with von Willebrand Factor A1 Domain , 2002, Science.

[23]  E. Erhardtsen,et al.  Potential role for rFVIIa in transfusion medicine , 2002, Transfusion.

[24]  C. Esmon,et al.  Endothelial Cell Protein C Receptor Acts as a Cellular Receptor for Factor VIIa on Endothelium* , 2007, Journal of Biological Chemistry.

[25]  J. V. van Mourik,et al.  The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein* , 1999, The Journal of Biological Chemistry.

[26]  P. D. de Groot,et al.  Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A. , 2002, Blood.

[27]  K. Mann,et al.  Mechanism of factor VIIa-dependent coagulation in hemophilia blood. , 2002, Blood.

[28]  C. C. Reynolds,et al.  Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. , 1992, The Journal of biological chemistry.

[29]  J. Badimón,et al.  Blood-borne tissue factor: another view of thrombosis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  U. Hedner.  Recombinant activated factor VII as a universal haemostatic agent. , 1998, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[31]  C. van ′t Veer,et al.  Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa. , 1994, The Journal of biological chemistry.

[32]  G. Jamieson,et al.  The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets. , 1986, The Journal of biological chemistry.

[33]  H. Roberts,et al.  Rationale for the use of high dose rFVIIa in a high‐titre inhibitor patient with haemophilia B during major orthopaedic procedures , 2001, Haemophilia : the official journal of the World Federation of Hemophilia.

[34]  P. D. de Groot,et al.  Glycoprotein Ibα–Mediated Platelet Adhesion and Aggregation to Immobilized Thrombin Under Conditions of Flow , 2005, Arteriosclerosis, thrombosis, and vascular biology.

[35]  W. Ruf,et al.  Orchestration of coagulation protease signaling by tissue factor. , 2002, Trends in cardiovascular medicine.

[36]  J. Oliver,et al.  Platelet activity of high‐dose factor VIIa is independent of tissue factor , 1997, British journal of haematology.

[37]  Dougald M Monroe,et al.  The use of recombinant factor VIIa in the treatment of bleeding disorders. , 2004, Blood.