Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.

SummaryFactor X (FX) is a vitamin K-dependent coagulation zymogen, which upon activation to factor Xa assembles into the prothrombinase complex to activate prothrombin to thrombin. FX can be activated by either factor VIIa-tissue factor or factor IXa-factor VIIIa in extrinsic and intrinsic pathways, respectively. In this study, we identified a bleeding patient with moderate FX deficiency who exhibits a clotting defect only in the intrinsic pathway. Exome sequencing revealed that the patient carries a novel homozygous missense mutation that results in substitution of Thr211 with Pro in the activation peptide of FX. Thr211 is the site of an O-linked glycosylation in the activation peptide of FX. We postulated that the lack of this post-translational modification specifically impacts the activation of FX by intrinsic Xase, thereby impairing thrombin generation in the subject. To test this hypothesis, we expressed both wild-type FX and FX containing this mutation in mammalian cells and following the purification of the zymogens to homogeneity characterized their properties in both purified and plasma-based assay systems. Analysis of the results suggests that Thr211 to Pro substitution renders the FX mutant a poor substrate for both physiological activators, however, at physiological concentration of the substrate, the clotting defect manifest itself only in the intrinsic pathway, thus explaining the bleeding phenotype for the patient carrying this mutation.

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The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin

Biochemical Journal ◽

10.1042/bj2430579 ◽

1987 ◽

Vol 243 (2) ◽

pp. 579-588 ◽

Cited By ~ 75

Author(s):

F A Ofosu ◽

P Sie ◽

G J Modi ◽

F Fernandez ◽

M R Buchanan ◽

...

Keyword(s):

Antithrombin Iii ◽

Anticoagulant Activity ◽

Factor Xa ◽

Factor V ◽

Factor X ◽

Intrinsic Pathway ◽

Sulphated Polysaccharide ◽

Pathway Activation ◽

Pentosan Polysulphate ◽

Inhibition Of Thrombin

Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of prothrombinase complex, the physiological activator of prothrombin.

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Immunological Studies on the Blood Coagulation Factor X and Its Warfarin-induced Precursor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649144 ◽

1974 ◽

Vol 31 (01) ◽

pp. 040-051 ◽

Cited By ~ 5

Author(s):

Gustav Gaudernack ◽

Åse Gladhaug Berre ◽

Bjarne Østerud ◽

Hans Prydz

Keyword(s):

Coagulation Factor ◽

Factor X ◽

Intrinsic Pathway ◽

Coagulation Activity ◽

Factor X Deficiency ◽

Warfarin Treatment ◽

The Cross ◽

Monospecific Antisera ◽

Purified Antigen ◽

Congenital Factor

SummaryMonospecific antisera against the human coagulation factor X have been raised in rabbits by injections of purified antigen. Such antiserum was used to study the cross-reacting material without factor X activity which is present in the blood of warfarin-treated patients and animals as well as to study the changes in factor X during coagulation. One patient with congenital factor X deficiency was also studied.A complete identity was found between factor X in Macaca mulatta and human blood. During warfarin treatment antigenically cross-reacting material appeared in plasma. This was not adsorbed on BaSO4, and inhibited the coagulation activity of normal factor X.Both this material, normal factor X and the cross-reacting material in plasma from a patient congenitally deficient in factor X gave rise to split products during coagulation by the intrinsic pathway, i. e. all of them served as substrates for the intrinsic activator of factor X.

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Bovine Factors X1And X2: Activation Peptide Based Chromatographic Differences

10.1055/s-0038-1665665 ◽

1979 ◽

Author(s):

Takashi Morita ◽

Craig Jackson

Keyword(s):

Amino Acid ◽

Anion Exchange ◽

Gel Filtration ◽

Heart Association ◽

Personal Communication ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Factor X ◽

Activation Peptide ◽

Activation Peptides

Bovine Factor X is eluted in two forms (X1and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglutamic acid residues have been shown to be identical in X1 and X2(H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by anion-exchange chromatography. The activation peptide (AP1) which eluted first was shown to be derived from Factor X1. while the activation peptiae (AP2) which eluted second was shown to be derived from X2 on the basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.M.J. is an Established Investigator of the American Heart Association).

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Two residues in the activation peptide domain contribute to the half-life of factor X in vivo

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2010.03905.x ◽

2010 ◽

Vol 8 (7) ◽

pp. 1651-1653 ◽

Cited By ~ 6

Author(s):

P. GUÉGUEN ◽

G. CHEREL ◽

I. BADIROU ◽

C. V. DENIS ◽

O. D. CHRISTOPHE

Keyword(s):

Half Life ◽

Factor X ◽

Activation Peptide

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Bovine Factors X1 and X2: Activation Peptide Based Chromatographic Differences

10.1055/s-0039-1684394 ◽

1979 ◽

Author(s):

Takashi Morita ◽

Craig M. Jackson

Keyword(s):

Amino Acid ◽

Gel Filtration ◽

Factor Xa ◽

Heart Association ◽

Personal Communication ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Factor X ◽

Activation Peptide ◽

Activation Peptides

Bovine Factor X is eluted in two forms (X1 and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglu-tamic acid residues have been shown to be identical in X1 and X2, (H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by an ion-exchange chromatography. The activation peptide AP1) which eluted first was shown to be derived from Factor X1 while the activation peptide (AP2) which eluted second was shown to be derived from X2 on basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.H.J. is an Established Investigator of the American Heart Association).

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A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0039-1697662 ◽

2019 ◽

Vol 119 (12) ◽

pp. 1981-1993 ◽

Cited By ~ 2

Author(s):

Vincent Muczynski ◽

Sebastien Verhenne ◽

Caterina Casari ◽

Ghislaine Chérel ◽

Laurence Panicot-Dubois ◽

...

Keyword(s):

Hemophilia A ◽

Thrombus Formation ◽

In Vitro Assays ◽

Factor X ◽

Injury Model ◽

Inhibitory Antibodies ◽

Activation Peptide ◽

Similar Survival ◽

Deficient Mice

AbstractEngineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

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Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex

Thrombosis and Haemostasis ◽

10.1055/s-0040-1715441 ◽

2020 ◽

Vol 120 (11) ◽

pp. 1512-1523

Author(s):

Mark Schreuder ◽

Geraldine Poenou ◽

Viola J. F. Strijbis ◽

Ka Lei Cheung ◽

Pieter H. Reitsma ◽

...

Keyword(s):

Factor Viia ◽

Intrinsic Factor ◽

Salt Bridge ◽

Original Form ◽

Factor V ◽

Factor X ◽

Prothrombinase Complex ◽

Factor Viiia ◽

Protease Activation ◽

Activation Peptide

AbstractThe venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa–factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor–factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16–Asp194 salt bridge via modification of the activation peptide.

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The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽

10.1182/blood.v65.5.1226.1226 ◽

1985 ◽

Vol 65 (5) ◽

pp. 1226-1231 ◽

Cited By ~ 16

Author(s):

TB McNeely ◽

MJ Griffith

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Coagulation Factors ◽

Factor Ix ◽

Factor X ◽

Clotting Time ◽

Intrinsic Pathway ◽

Blood Coagulation Factors ◽

Factor Ixa ◽

Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

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