The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam, J. A.; Hamer, R.J.; Beeser‐Visser, Nel H.; Bouma, Bonno N.; Sixma, Jan J.

doi:10.1111/j.1432-1033.1990.tb15481.x

Cited by 87 publications

(75 citation statements)

References 35 publications

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“…On the other hand, FXa cleaves only the free form of FVIII, and FXa-catalyzed FVIII activation is completely prevented by the formation of the FVIII⅐vWF complex (17). Therefore, thrombin cleavage at Arg 1689 regulates the procoagulant pathway by limiting the association between FVIII and vWF.…”

Section: Blood Clotting Factor VIII (Fviii)mentioning

confidence: 99%

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Nogami¹,

Shima²,

Hosokawa³

et al. 2000

Journal of Biological Chemistry

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Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg 1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Blood clotting factor VIII (FVIII) 1 is a crucial glycoprotein that accelerates the intrinsic coagulation cascade by acting as a cofactor of factor IXa in the tenase complex (1), and a deficiency of FVIII results in the common hereditary bleeding disorder, hemophilia A. FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (vWF) that stabilizes the synthesis and cofactor activity of FVIII (2-4). Mature FVIII is synthesized as a single chain polypeptide of approximately 300 kDa consisting of 2,332 amino acid residues with a mosaic domain structure arranged in the order of A1-A2-B-A3-C1-C2 (5-7) and secreted into plasma as variable series of heterodimers consisting of the heavy chain (HCh) and the light chain (LCh). The HCh is composed of the A1, A2, and parts of the B domain and exhibits size heterogeneity (90 -210 kDa) due to proteolysis within the B domain. The 90-kDa HCh reflects the absence of the B domain, whereas the 210-kDa species includes the intact B domain. The LCh (80 kDa) corresponds to the A3, C1, and C2 domains. The two chains are noncovalently linked by metal ions between the A1 and A3 domains (6, 7).FVIII is transformed into an active form by limited proteolysis by two essential serine proteases, thrombin and FXa (8, 9). This procoagulant activity is more pronounced after thrombin activation than after FXa activation (10). Cleavage at Arg 740 removes the size-heterogeneous B domain from the HCh, producing a 90-kDa HCh fragment, and further cleavage of the 90-kDa HCh fragment (A1-A2) at Arg 372 between the A1 and A2 domains produces 54-(A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg 1689 between the B and A3 domains removes 40 amino-terminal acidic peptides from the A3 domain and produces a 72-kDa fragment. A unique cleavage by FXa at Arg 1721 produces a 67-kDa LCh fragment. The active form of FVIII is a metal-linked heterotrimer consisting of the A1/A2/A3-C1-C2 domains, lacking the middle B domain (6). Cleavage at Arg 740 between the A2 and B domains does not contribute to the generation of the FVIII activity. In contrast, cleavages at Arg 372 and Arg 1689 are essential for optimal FVIII activity (11-13). Activation of human FVIII at its physiological concentration and at pH 7.4 is followed by a reduction in activity (14). This loss of activity is not caused by further proteolysis but by dissociation of the A2 domain from the active form of FVIII heterotrime...

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Section: Blood Clotting Factor VIII (Fviii)mentioning

confidence: 99%

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Nogami¹,

Shima²,

Hosokawa³

et al. 2000

Journal of Biological Chemistry

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“…The procoagulant activity of FVIIIa produced by FXa is 4-fold lower and is more stable than that generated by thrombin (22). Furthermore, the presence of vWF moderates the activation of FVIII by FXa but not by thrombin (23). Recently, Lapan and Fay (24) localized a factor X (FX) binding site within the A1 domain.…”

mentioning

confidence: 99%

Role of Factor VIII C2 Domain in Factor VIII Binding to Factor Xa

Nogami

Shima

Hosokawa³

et al. 1999

Journal of Biological Chemistry

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Factor VIII (FVIII) 1 is a glycoprotein cofactor that accelerates the generation of factor Xa (FXa) by factor IXa in the presence of Ca 2ϩ and negatively charged phospholipid (PL) expressed on a membrane surface (1). Quantitative and qualitative deficiencies of FVIII result in the congenital bleeding disorder, hemophilia A. FVIII is noncovalently bound to von Willebrand factor (vWF) in plasma. vWF regulates the synthesis, the cofactor activity, and the transport of FVIII to the site of vascular injury (2-4). Mature FVIII is synthesized as a single chain polypeptide consisting of 2332 amino acid residues (5, 6). Based on internal homologies of the amino acid sequence, FVIII has three types of domains arranged in the order of A1-A2-B-A3-C1-C2 (7). FVIII circulates in the plasma as a heterodimer of a heavy chain (HCh) consisting of the A1, A2, and heterogeneous fragments of partially proteolyzed B domains, together with a light chain (LCh) consisting of A3, C1, and C2 domains (6, 7).Several findings have indicated that the structure and function of the C2 domain is important for the expression and regulation of FVIII. The C2 domain contains a PL binding site (8, 9) and a vWF binding site (10 -12) together with a common epitope for FVIII inhibitor alloantibodies, which develop in patients with severe hemophilia A (13). Furthermore, residues Val 2248 -Gly 2285 within the C2 domain contain the epitope for a monoclonal antibody ESH8, which reduces the rate of FVIII/ vWF dissociation after thrombin activation of FVIII (14).FVIII is transformed into an active form (FVIIIa) by limited proteolysis by two serine proteases, thrombin and FXa (15,16 (18 -20). FXa-dependent FVIII activation is different from thrombin-dependent FVIII activation in several ways (21,22). The procoagulant activity of FVIIIa produced by FXa is 4-fold lower and is more stable than that generated by thrombin (22). Furthermore, the presence of vWF moderates the activation of FVIII by FXa but not by thrombin (23). Recently, Lapan and Fay (24) localized a factor X (FX) binding site within the A1 domain. The role of FXa-dependent FVIII activation in vivo is still uncertain, however.In the present study, we demonstrated that an anti-C2 monoclonal antibody, containing an epitope within residues Val 2248 -Gly 2285 , inhibited FXa cleavage of FVIII in the absence of PL. Furthermore, the C2 domain competed with FVIII for FXa cleavage of the LCh and bound directly to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine, indicating that the C2 domain contains a FXa binding site. EXPERIMENTAL PROCEDURES ProteinsFVIII was affinity-purified using monoclonal antibody NMC-VIII/10, recognizing the FVIII A3 domain. Elution from the monoclonal antibody column was performed with 1 M KI and 40% ethylene glycol as described previously (25). The specific activity of the purified FVIII was 2700 units/mg. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the purified FVIII was free of vWF anti...

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“…In plasma, FVIII circulates in an inactive form bound to von Willebrand factor (vWF) through noncovalent interactions and requires proteolytic cleavage by thrombin or factor Xa for activation (4)(5)(6)(7)(8). Upon proteolytic cleavage by thrombin, activated FVIII (FVIIIa) is released from vWF and functions to increase the V max of factor IXa proteolytic activation of factor X by at least 4 orders of magnitude in the presence of phospholipid and calcium (9,10).…”

mentioning

confidence: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

Proc. Natl. Acad. Sci. U.S.A.

126

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Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII), necessitating exogenous replacement by either plasma-or recombinant-derived FVIII preparations. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-aa single-chain glycoprotein of 280 kDa from which a signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (1-3). Whereas the A and C domains exhibit 35-40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein. Intracellular proteolytic processing within the B domain after residue Arg-1648 generates an 80-kDa light chain (domains A3-C1-C2) and a heterogeneous-sized heavy chain of 90-200 kDa (domains A1-A2-B). The heavy and light chains are associated as a heterodimer through a divalent metal-ion-dependent linkage between the A1 and A3 domains.In plasma, FVIII circulates in an inacti...

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The effect of von Willebrand factor on activation of factor VIII by factor Xa

Cited by 87 publications

References 35 publications

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Role of Factor VIII C2 Domain in Factor VIII Binding to Factor Xa

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

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