Role of human factor VIII in factor X activation.

SummaryCoagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine. The mutants were expressed in COS-1 cells and their antigen levels and procoagulant activities were measured. The residues were classified in three categories: those with a non-detrimental alteration of their activities (activity >50 % of control FVIII; n=98), those with a moderate alteration (15 %<activity<50%; n=45) and those that were severely affected (activity<15%; n=63). The mutants sensitive to mutation were retrieved in the HAMSTeRS database with a higher percentage than those that were not affected (58.8% vs. 9.2%). The results revealed the existence of clusters of residues that are sensitive (Arg418-Phe436, Thr459-Ile475, Ser535-Gly549, Asn618-Ala635) or not (Leu398-Arg418, Pro485-Asp500, Gly506-Gly520, Pro596-Asp605) to mutations. The stretches of residues sensitive to mutations were buried within the molecule suggesting that these amino acids participate in the maintenance of the A2 domain structure. In contrast, residues resistant to mutations formed external loops without well- defined structures suggesting that these loops were not crucial for the process of factor X activation. This study provided a detailed map of the FVIII A2 domain between residues 371 and 649, identifying residues crucial for maintaining FVIII function and residues that can be mutated without jeopardising the coagulant activity.

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Inhibition of Human Platelet Aggregation by the Proteolytic Effect of Streptokinase Role of the Human Factor-VIII-Related Protein

Pathophysiology of Haemostasis and Thrombosis ◽

10.1159/000214149 ◽

1976 ◽

Vol 5 (5) ◽

pp. 306-317

Author(s):

Ricardo Castillo ◽

Santiago Maragall ◽

Javier Alvarez Guisasola ◽

Francisco Casals ◽

Juan Profitós ◽

...

Keyword(s):

Platelet Aggregation ◽

Factor Viii ◽

Human Factor ◽

Human Platelet ◽

Related Protein ◽

Human Platelet Aggregation ◽

Human Factor Viii

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The role of the first growth factor domain of human factor IXa in binding to platelets and in factor X activation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42481-7 ◽

1992 ◽

Vol 267 (12) ◽

pp. 8571-8576

Author(s):

S.S. Ahmad ◽

R Rawala-Sheikh ◽

W.F. Cheung ◽

D.W. Stafford ◽

P.N. Walsh

Keyword(s):

Growth Factor ◽

Human Factor ◽

Factor X ◽

Factor Ixa ◽

Factor X Activation

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Aggregation of Human Platelets by Bovine or Human Factor VIII: Role of Carbohydrate Side Chains

Nature ◽

10.1038/244167a0 ◽

1973 ◽

Vol 244 (5412) ◽

pp. 167-168 ◽

Cited By ~ 40

Author(s):

J. VERMYLEN ◽

M. B. DONATI ◽

G. DE GAETANO ◽

M. VERSTRAETE

Keyword(s):

Factor Viii ◽

Human Factor ◽

Human Platelets ◽

Side Chains ◽

Human Factor Viii

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A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X

Blood ◽

10.1182/blood.v72.5.1761.bloodjournal7251761 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1761-1770

Author(s):

P Neuenschwander ◽

J Jesty

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Ionophore A23187 ◽

Factor X ◽

Apparent Dissociation Constant ◽

Factor Ixa ◽

Activated Platelets ◽

Factor Viiia ◽

Human Factor Viii ◽

Factor X Activation

Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

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Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII

Blood ◽

10.1182/blood.v52.5.928.bloodjournal525928 ◽

1978 ◽

Vol 52 (5) ◽

pp. 928-940

Author(s):

MB Hultin ◽

Y Nemerson

Keyword(s):

Factor Viii ◽

Factor Ix ◽

Constant Level ◽

Specific Factor ◽

Factor X ◽

Intrinsic Pathway ◽

Factor Ixa ◽

Factor X Activation ◽

Benzamidine Hydrochloride

We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.

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Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII

Blood ◽

10.1182/blood.v52.5.928.928 ◽

1978 ◽

Vol 52 (5) ◽

pp. 928-940 ◽

Cited By ~ 57

Author(s):

MB Hultin ◽

Y Nemerson

Keyword(s):

Factor Viii ◽

Factor Ix ◽

Constant Level ◽

Specific Factor ◽

Factor X ◽

Intrinsic Pathway ◽

Factor Ixa ◽

Factor X Activation ◽

Benzamidine Hydrochloride

Abstract We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.

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Role of the Activation Peptide Domain in Human Factor X Activation by the Extrinsic Xase Complex

Journal of Biological Chemistry ◽

10.1074/jbc.271.27.16126 ◽

1996 ◽

Vol 271 (27) ◽

pp. 16126-16134 ◽

Cited By ~ 42

Author(s):

Robert J. Baugh ◽

Sriram Krishnaswamy

Keyword(s):

Human Factor ◽

Factor X ◽

Activation Peptide ◽

Factor X Activation

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A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X

Blood ◽

10.1182/blood.v72.5.1761.1761 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1761-1770 ◽

Cited By ~ 49

Author(s):

P Neuenschwander ◽

J Jesty

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Ionophore A23187 ◽

Factor X ◽

Apparent Dissociation Constant ◽

Factor Ixa ◽

Activated Platelets ◽

Factor Viiia ◽

Human Factor Viii ◽

Factor X Activation

Abstract Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

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