Structural and Functional Characteristics of Proteolytically Modified Bovine Factor X and Xa (Des Light Chain Residues 1-41)

1979 ◽  
Author(s):  
T. Morita ◽  
C.M. Jackson
Keyword(s):  
Light Chain ◽  
Specific Activity ◽  
Factor Xa ◽  
Peptide Bond ◽  
Amino Acid Sequences ◽  
Structural Domain ◽  
Factor X ◽  
One Stage ◽  
The One ◽  
Bovine Factor

Incubation of bovine Factor X with α-chymotrypsin produces a peptide, residues 1-41 of the light chain and a modified Factor X designated “headless Factor X”. Clotting activity of “headless Factor X” is virtually zero by one stage assay. Composition, chromatographic elution characteristics and molecular weight estimates by SDS gel electrophoresis form the bases for the aforedesignated structure of “headless Factor X”. Activation of “headless Factor X” by the Russell’s viper venom activator requires Ca2+ as does normal Factor X, however, the rate is much slower. After full activation the specific activity of “headless Factor Xa” and normal Factor Xa differed by less than 20% using BOC-L-Val-L-Leu-Gly-L-Arg-pNA as substrate. However, clotting specific activity is less than 0.002% of normal Factor Xa in the one stage clotting assay. The activation peptides released from “headless Factor X” (residues 1-51 and 291-307) of the heavy chain were identical to those released from normal Factor X. Similar rapid, highly selective chymotryptic cleavage of Prothrombin Fragment 1, and the similarity in the amino acid sequences of the light chain of Factor X and Fragment 1 suggests that the region around the susceptible peptide bond must lie on the surface of both these molecules and perhaps exist in a “hinge” region connecting the Gla containing domain and the reamining structural domain of these portions of prothrombin and Factor X. (Supported by HL12820).

scholarly journals A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Manjunath Goolyam Basavaraj ◽  
Sriram Krishnaswamy
Keyword(s):  
Factor Viii ◽  
Western Blotting ◽  
Light Chain ◽  
Specific Activity ◽  
Factor X ◽  
Physiological Concentration ◽  
Proteolytic Activation ◽  
Linker Sequence ◽  
One Stage ◽  
Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg372-Ser373, Arg740-Ser741, and Arg1689-Ser1690. Cleavage at Arg740 removes the B-domain, and cleavage at Arg1689 removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg372 separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg372 might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln372 backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIIIWT), FVIII372Q, and FVIII372Q followed by a rigid (Ala-Pro)5 linker sequence (FVIII372Q-AP5). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIIIWT, FVIII372Q and FVIII372Q-AP5 were completely resistant to cleavage at Gln372 by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIIIWT and FVIII372Q-AP5 displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIIIWT and FVIII372Q-AP5 displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII372Q was severely impaired. However, despite lack of cleavage at Gln372 in FVIII372Q-AP5, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIaWT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa372Q-AP5 was within a factor of 2 of that observed with FVIIIaWT while the rate observed with FVIIIa372Q was >10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa372Q-AP5(t1/2 ~ 10 min) compared to FVIIIaWT (t1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg372 for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa372Q-AP5 suggests that cleavage at Arg372 does not solely explain the spontaneous loss of FVIIIa cofactor function. Disclosures Krishnaswamy: Bayer: Research Funding.

The Formation of Intermediate Product I in a Purified System The Role of Factor IX or of its Precursor and of a Hageman Factor-PTA Fraction

1961 ◽  
Vol 6 (02) ◽  
pp. 224-234 ◽  
Author(s):  
E. T Yin ◽  
F Duckert
Keyword(s):  
Intermediate Product ◽  
Factor Ix ◽  
Assay Method ◽  
Lag Phase ◽  
Factor X ◽  
Haemophilia B ◽  
Hageman Factor ◽  
One Stage ◽  
The One

Summary1. The role of two clot promoting fractions isolated from either plasma or serum is studied in a purified system for the generation of intermediate product I in which the serum is replaced by factor X and the investigated fractions.2. Optimal generation of intermediate product I is possible in the purified system utilizing fractions devoid of factor IX one-stage activity. Prothrombin and thrombin are not necessary in this system.3. The fraction containing factor IX or its precursor, no measurable activity by the one-stage assay method, controls the yield of intermediate product I. No similar fraction can be isolated from haemophilia B plasma or serum.4. The Hageman factor — PTA fraction shortens the lag phase of intermediate product I formation and has no influence on the yield. This fraction can also be prepared from haemophilia B plasma or serum.

Bovine Factors X1 and X2: Activation Peptide Based Chromatographic Differences

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).

Preparation of Highly Purified Human Thrombin and Factor Xa in a Simultainous Procedure

1975 ◽  
Author(s):  
M. Miller-Andersson
Keyword(s):  
Specific Activity ◽  
Factor Xa ◽  
Fresh Frozen Plasma ◽  
Factor X ◽  
Serum Factors ◽  
Human Thrombin ◽  
Fresh Frozen ◽  
Brain Material ◽  
The Brain ◽  
Chromatography Step

A preparation procedure that purifies Thrombin and activated factor X in a simultainous process has been worked out. Prothrombin complex was first prepared from fresh frozen plasma by chromatography on DEAE-Sephadex. Prothrombin activator was prepared from human brain thromboplastin and bovine serum factors. The activation was performed at 20° C and followed by assaying thrombin activity. After activation the brain material was removed by centrifugation. The protein mixture was immediately adsorbed on to Amberlite IRC-50 and both enzymes were fully adsorbed to the resin. They were eluted in a CaCl2 gradient and the two enzymes eluted in two slightly owerlapping peaks.The thrombin obtained in this procedure was highly purified with a specific activity of 2100 U per mg protein. The activated factor X contained traces of thrombin. It was fully suitable for anti Xa assays of heparin. When higher degree of purity was needed the activated factor X was further purified in an iron-exchange or affinity chromatography step.

The Determination of Factor X Using an Automated Amidolytic Technique

1979 ◽  
Author(s):  
E.M. van Wijk ◽  
L.H. Kahlé ◽  
J.W. ten Cate
Keyword(s):  
Antithrombin Iii ◽  
Oral Anticoagulant Therapy ◽  
Close Correlation ◽  
Bleeding Disorders ◽  
Factor X ◽  
Amidolytic Activity ◽  
One Stage ◽  
Automated Method ◽  
The One

An automated method for the determination of factor X has been developed using a procoagulant fraction from Russell’s Viper Venom (RW), which activates FX within 4 minutes at pH 8.6, resulting in a Xa activity that is assayed using the chromogenic substrate S 2222. The amidolytic activity generated by sufficient RW is proportional to the FX level of the plasma and is not influenced by antithrombin III within the time studied. By using polybrene, the influence of heparin (up to 10 U/ml) can be eliminated. The method has been adapted for performance automatically on an Automated Kinetic Enzyme and Substrate analyser (Vitatron), Comparison of FX activities of 30 normals and 41 patients with bleeding disorders revealed a close correlation of this method with the one stage clotting assay using thromboplastin (r=0.93). Values obtained in a group of 83 patients on oral anticoagulant therapy showed good correlation (r=0.87) but tended to be about 8% higher compared to the one stage method. No significant differences were found when RW was used in the clotting assay (r-0.91). The values obtained by the amidolytic assay correlated well with thrombotest (r=0.87).

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

scholarly journals Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation

1996 ◽  
Vol 319 (3) ◽  
pp. 873-879 ◽  
Author(s):  
Janet PLESCIA ◽  
Dario C ALTIERI
Keyword(s):  
Factor Viia ◽  
Alternative Pathway ◽  
Limited Proteolysis ◽  
Factor Xa ◽  
Peptide Bond ◽  
Cathepsin G ◽  
Activate Factor ◽  
Factor X ◽  
Proteolytic Activation

Leucocyte initiation of coagulation preserves the haemostatic balance and may aberrantly contribute to vascular injury. In addition to the extrinsic activation mediated by tissue factor: factor VIIa, monocytes express an alternative procoagulant response after binding of the zymogen factor X to the integrin Mac-1 (CD11b/CD18). Here, factor X-activating activity was found in purified monocyte granules, and coincided with size-chromatographed fractions containing cathepsin G. In contrast, elastase-containing granule fractions did not activate factor X. In the presence of Ca2+ ions, purified cathepsin G, but not elastase, cleaved factor X to a ∼ 54 kDa catalytically active derivative, structurally indistinguishable from the procoagulant product generated on monocytes after binding to Mac-1. Factor X activation by purified cathepsin G involved limited proteolysis of a novel Leu177-Leu178 peptide bond in the zymogen's activation peptide. Cathepsin G activation of factor X was completely inhibited by α1 antitrypsin, α1 antichymotrypsin, or soybean trypsin inhibitor, or by a neutralizing antiserum to cathepsin G, while eglin, or an anti-elastase antibody, were ineffective. Affinity chromatography on active-site-dependent inhibitors Glu-Gly-Arg-chloromethyl ketone or benzamidine completely abolished factor Xa activity generated by cathepsin G. Cathepsin G was not constitutively detected on the monocyte surface by flow cytometry. However, inflammatory stimuli, including formyl peptide or phorbol ester, or Mac-1 engagement with its ligands fibrinogen, factor X or serum-opsonized zymosan, triggered monocyte degranulation and cathepsin G activation of factor X. These findings demonstrate that monocytes can alternatively initiate coagulation in a sequential three-step cascade, including (i) binding of factor X to Mac-1, (ii) discharge of azurophil granules, and (iii) limited proteolytic activation of membrane-bound factor X by cathepsin G. By rapidly forming thrombin and factor Xa in a protected membrane microenvironment, this pathway may contribute a ‘priming’ signal for clotting, anticoagulation and vascular cell signal transduction, in vivo.

The Clotting of a Snake (Crotalus Viridis Helleri) Plasma and its Interaction with Various Snake Venoms

1976 ◽  
Vol 35 (02) ◽  
pp. 314-323 ◽  
Author(s):  
K.W.E Denson
Keyword(s):  
Human Plasma ◽  
Factor Xa ◽  
Factor X ◽  
Snake Venoms ◽  
Crotalus Viridis ◽  
Fibrin Monomer ◽  
Russell’S Viper ◽  
Marked Specificity ◽  
Bovine Factor

SummaryThe clotting of C. V. Helleri plasma is not accelerated by the factor X activator or throm-bin-like enzymes from its own venom. Clotting of the plasma is accelerated by the factor X activator from Russell’s viper venom, but not by the thrombin-like enzyme from Agkistrodon Rhodostoma venom (“Arvin”).The prothrombin activator from the Taipan venom clots C. V. Helleri plasma equally well as human plasma, but the thrombin which is produced has a marked specificity for its own fibrinogen, and clots bovine fibrinogen more slowly.C. V. Helleri plasma contains an inhibitor which progressively inactivates bovine factor Xa and thrombin, but the inhibitor is not potentiated by heparin. The slow, protracted clotting of the snake plasma either alone or when mixed with human plasma or bovine fibrinogen suggests that this inhibitor may interfere with the polymerisation of fibrin monomer.

Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

1990 ◽  
Vol 64 (02) ◽  
pp. 251-255 ◽  
Author(s):  
Claudine Mazurier ◽  
Armelle Parquet-Gernez ◽  
Maurice Goudemand
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Assay Method ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulant Activity ◽  
The One ◽  
In Vitro Data

SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

The Function of the Platelet Surface in Coagulation

1974 ◽  
Vol 32 (02/03) ◽  
pp. 492-501 ◽  
Author(s):  
J Burnham King
Keyword(s):  
Catalytic Activity ◽  
Density Gradient ◽  
Sole Source ◽  
Assay System ◽  
Factor X ◽  
Platelet Surface ◽  
Size And Shape ◽  
One Stage ◽  
Gradient Technique ◽  
The One

SummaryPlatelets washed by the albumen density gradient technique were used as the sole source of thromboplastin in a new test, and also as the sole source of factor X in a modification of the one-stage factor X assay test of Denson. These platelets were shown to be capable of generating considerable amounts of thrombin during periods of preincubation of up to 8 minutes. For at least 3 minutes of preincubation their size and shape remained invariant, as measured by counting and by sizing on a Coulter channelyser. It is suggested that this test measures a function of the platelet surface, rather than of the platelet after viscous metamorphosis. This function is tentatively named platelet catalytic activity (PCA). Support for this concept was given by the fact that aspirin and phenylbutazone did not affect the test. The factor X assay system confirmed the presence of a small amount of mostly inactive factor X on the surface of the platelet. This factor X could be activated by Russell viper venom.

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