The effect of thrombin on the complex between factor VIII and von Willebrand factor

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

doi:10.1111/j.1432-1033.1987.tb13331.x

Cited by 61 publications

(40 citation statements)

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“…5). This is in contrast to our earlier observation [23] that the 42-kDa fragment generated after cleavage of factor VIII with thrombin remained bound to von Willebrand factor. In the present study, we find that the thrombin-generated fragment is only partially bound to von Willebrand factor, while the majority is released into the supernatant (Fig.…”

Section: Discussioncontrasting

confidence: 56%

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

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

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Section: Discussioncontrasting

confidence: 56%

“…In a previous paper [23], we followed the proteolytic steps involved in activation of factor VIII by thrombin and the Correspondence to J . J. Sixma Department of Haematology, University Hospital Utrecht, P.O.…”

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confidence: 99%

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

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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“…The change of the global charge of activated factor VIII upon activation, as observed by its shift in affinity on the Mono-Q column and coelution with von Willebrand factor, sustains this hypothesis. After activation, the decrease in von Willebrand factor activity relative to factor-VIII activity confirms that von Willebrand factor is complexed with normal factor VIII and is released after proteolysis of the SO-kDa chain by thrombin [27].…”

Section: Discussionmentioning

confidence: 70%

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor

Bihoreau

Sauger

Yon

et al. 1989

European Journal of Biochemistry

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Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VI11 forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg.At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the SO-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal f-actor VIII, the ratio of von Willebrand activity to factor-Vlll activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin.In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45 -50 ~ 70 kDa).The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.Factor VIII is a glycoprotein present in the plasma at a very low concentration (0.1 pg/ml). Its absence or deficiency characterizes hemophilia A. It drives, as a cofactor, the activation of factor X mediated by factor IXa in the presence of phospholipids and calcium, in the blood coagulation intrinsic pathway [l]. The primary structure of f k t o r VIII contains 2332 amino acids and exhibits a triplicated region of 330 amino acids (A domains), a unique region of 983 amino acids (B domain), and a carboxy-terminal duplicated region of 150 amino acids (C domain), that are arranged in the order AlFactor VIII is susceptible to proteolysis by thrombin, plasmin and other serine proteases [3, 41. Factor VIII purified from plasma fractions of intermediate purity, consists of variable active forms with molecular masses ranging over 290-170 kDa, though a single chain precursor of 330 kDa has been reported [5]. Continuous proteolysis of the 210-kDa proteins yields a series of polypeptides of 210-90 kDa [6, 71. The polypeptides of 210 kDa and 80 kDa represent the N-terminal and C-terminal parts of factor VIII, respectively [2,8, 91. Fay et al. [lo] have identified the heterodimeric structure of the active factor VIII as the association, by a metal ion, of lightCorrespondence to H. Van de Pol, Centre National de Transfusion Sanguine...

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“…Thrombin cleavage at Arg 1689 dissociates FVIII from its complex with vWF (16), and subsequently free FVIII promotes tenase activity on phospholipid (PL) micelles. 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).…”

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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The effect of thrombin on the complex between factor VIII and von Willebrand factor

Cited by 61 publications

References 33 publications

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

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

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor

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

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