The factor VIII protein and its function.

Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. Human factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1-C2. The protein undergoes processing prior to secretion into blood resulting in a heavy chain of 200 kDa (A1-A2-B) and a light chain of 80 kDa (A3-C1-C2) linked by metal ions. The role of factor VIII is to increase the catalytic efficiency of factor IXa in the activation of factor X. Variants of these factors lead frequently also to severe bleeding disorders.

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Sequence of the rat factor VIII cDNA

Thrombosis and Haemostasis ◽

10.1160/th03-06-0336 ◽

2004 ◽

Vol 91 (01) ◽

pp. 38-42 ◽

Cited By ~ 6

Author(s):

Christof Geisen ◽

Erhard Seifried ◽

Johannes Oldenburg ◽

Matthias Watzka

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Factor Viii ◽

Posttranslational Modifications ◽

Function Analysis ◽

Amino Acid Level ◽

Coagulation System ◽

Cleavage Sites ◽

Human Factor Viii ◽

Exon 20

SummaryFactorVIII acts as an essential compound of the tenase complex of the coagulation system. Herein we report the cDNA of the rat factor VIII. The rat cDNA comprises 6777 nucleotides and encodes a protein of 2258 amino acids, 61 amino acids less than mouse and 92 amino acids less than human factor VIII. The overall identity compared to human cDNA is 61% on the cDNA and 51% on the amino acid level. In cDNA, highest levels of sequence identity can be observed in the A and C domains (ranging between 68% and 73%), whereas B domain and the small acidic regions are more divergent (34%-49%). Compared to mouse and human most sites for posttranslational modifications such as sulfatation and glycosylation as well as thrombin and protein C cleavage sites are conserved in rat. Alternative transcripts lacking exon 17 and/or comprising additional 26 bp due to alternative splicing of exon 20 were found. Furthermore, 13 polymorphisms (seven in exon 14, one in exon 20, 23, 24, and 25, two in the 3’UTR) three of which lead to an amino acid exchange could be detected. Our findings might provide new insights into the structure-function analysis of the factor VIII protein and might prove useful for future animal models addressing the function of factor VIII.

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The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

Biochemical Journal ◽

10.1042/bj2650327 ◽

1990 ◽

Vol 265 (2) ◽

pp. 327-336 ◽

Cited By ~ 141

Author(s):

V J J Bom ◽

R M Bertina

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Reaction Rate ◽

Factor Viia ◽

Coagulation Factor ◽

Fluid Phase ◽

Fold Increase ◽

Factor Vii ◽

Factor X ◽

Extrinsic Pathway

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

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THE INTERACTION OF PURIFIED FACTOR VIII WITH PLATELETS

10.1055/s-0038-1643616 ◽

1987 ◽

Author(s):

P R GanZ ◽

E S Tackberry ◽

G Rock

Keyword(s):

Factor Viii ◽

Specific Activity ◽

Membrane Surface ◽

Platelet Membrane ◽

Factor X ◽

Single Chain ◽

Physiological Concentration ◽

High Molecular Weight Species ◽

Polyethylene Glycol Precipitation ◽

Kinetics Of

Factor VIII is known to interact with Factors IXa and X to generate activated Factor X. A requirement for phospholipid in this reaction suggests that this "tenase" protein complex is assembled on a membrane surface. As a first step in studying the involvement of Factor VIII in this process, we wished to determine whether purified Factor VIII could interact directly with platelets. Factor VIII utilized in these experiments was purified from heparinized blood by a six-stage procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single-chain high molecular weight species of approximately 260,000 (specific activity 5,200 units/mg). This homogeneous protein was then radiolabelled with Na125I by a procedure which allowed the retention of approximately 60-80% of the procoagulant activity of Factor VIII. The kinetics of binding of 125I-Factor VIII to washed platelets at physiological concentration (approximately 3xl08/mL) was examined. Our results showed that for Factor VIII concentrations between 0.38 and 3.0 ng/mL there was a linear uptake of radiolabelled Factor VIII, whereas for concentrations above 10ng/mL only a slight increase in uptake occurred. To further define the association of purified Factor VIII with the platelet membrane, we also labelled Factor VIII with a bifunctional, photoactivatable cross-linking reagent, N-[4-(p-azido-m-[125]iodophenylazo)benzoyl]3-aminopropyl-N1 -oxysuccinimide ester. Analysis by PAGE showed thatthis reagent reacts predominantly with residues in the light chain or neahe C-terminal portion of Factor VIII. When mixed with thrombin-stimulated platelets, the cross-linked Factor VIII molecule was shown to transfer greater than 80% of the 125I label to a polypeptide of M.W. 80,000-90,000 isolated from platelet lysates. Autoradiographs of the labelled platelet preparations demonstrated that other minor polypeptides were radiolabelled. These experiments suggest that Factor VIII interacts closely with a platelet membrane protein which could represent a binding site for Factor VIII

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The Interface between the EGF2 Domain and the Protease Domain in Blood Coagulation Factor IX Contributes to Factor VIII Binding and Factor X Activation

Biochemistry ◽

10.1021/bi060451h ◽

2006 ◽

Vol 45 (35) ◽

pp. 10777-10785 ◽

Cited By ~ 16

Author(s):

Caroline Fribourg ◽

Alexander B. Meijer ◽

Koen Mertens

Keyword(s):

Factor Viii ◽

Blood Coagulation ◽

Coagulation Factor ◽

Factor Ix ◽

Factor X ◽

Protease Domain ◽

Coagulation Factor Ix ◽

Factor X Activation

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Human factor VIII from heparinized plasma. Purification and characterization of a single-chain form

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1988.tb13731.x ◽

1988 ◽

Vol 170 (3) ◽

pp. 521-528 ◽

Cited By ~ 8

Author(s):

Peter R. GANZ ◽

Eilleen S. TACKABERRY ◽

Douglas S. PALMER ◽

Gail ROCK

Keyword(s):

Factor Viii ◽

Human Factor ◽

Single Chain ◽

Purification And Characterization ◽

Human Factor Viii ◽

Chain Form

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Evaluation of a potential redox switch in blood coagulation tenase

10.1101/515718 ◽

2019 ◽

Author(s):

Kristina M. Cook ◽

Diego Butera ◽

Philip J. Hogg

Keyword(s):

Blood Coagulation ◽

Disulfide Bonds ◽

Redox State ◽

Coagulation Factor ◽

Catalytic Efficiency ◽

Factor X ◽

Active Peptides ◽

Redox Active ◽

Fviii Activity ◽

Redox Switch

AbstractBlood coagulation factor IXa (FIXa) activates factor X that leads to thrombin and fibrin formation and a stable thrombus. FIXa catalytic efficiency is markedly enhanced when bound to the co-factor, factor VIII (FVIII), and a negatively charged phospholipid surface in the tenase complex. Small redox active peptides and protein oxidoreductases have been shown previously to have some FIXa co-factor activity and thiol modifying agents have been reported to influence FVIII activity. These observations suggested that FIXa might contain an allosteric disulfide that is regulated by FVIII. This idea was tested by measuring the influence of FVIII on the redox state of FIXa disulfide bonds and the effect of plasma oxidoreductases on FIXa activity. The redox state of nine of the 11 disulfide bonds in FIXa was measured using differential cysteine labelling and mass spectrometry and all were oxidized in the protein, and this did not change upon binding of the enzyme to FVIII. All eight disulfide bonds in FVIII were also predominantly oxidized and this did not appreciably change upon FIXa binding. In addition, relevant protein reductants in the circulation inhibited rather than activated FIXa activity. In conclusion, we found no evidence that the co-factor function of FVIII involves a change in the redox state of one or more FIXa disulfide bonds.

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THE EFFECT OF CALCIUM ON THE STABILITY OF PURIFIED FACTOR VIII

10.1055/s-0038-1644037 ◽

1987 ◽

Author(s):

P R Qanz ◽

E S Tackaberry ◽

D S Palmer ◽

B Malchy ◽

G Rock

Keyword(s):

Factor Viii ◽

Specific Activity ◽

Procoagulant Activity ◽

Factor X ◽

Single Chain ◽

Rate Of Decay ◽

The Stability ◽

The One ◽

Polyethylene Glycol Precipitation ◽

And Function

The involvement of calcium and phospholipid in the activation of Factor X to Xa by Factor IXa and Factor VIII has been well documented. Although we and others have shown that maintenance of physiological concentrations of calcium has a positive effect on the stability of Factor VIII in plasma, calcium’s role in the structure and function of Factor VIII remains to be fully elucidated. To this end, we examined the effect of calcium on the stability of highly purified Factor VIII. Homogeneous Factor VIII (specific activity approximately 5,200 U/mg) was prepared from heparinized blood using a six-step purification procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single chain high molecular weight species of approximately 260,000. The protein was tested for stability using the one stage assay over 6h of incubation at 4°C in buffers containing 0 mM, 5 mM, and 10 mM CaCl2. Addition of 5 mM and 10 mM CaCl2 to desalted, purified Factor VIII resulted in an immediate 12% (for 5 mM CaCl2) and 23% (for 10 mM CaCl2), enhancement of procoagulant activity compared to samples containing no added calcium. The calculated half-life (T1/2) of activity of Factor VIII in buffers containing no added calcium was 3.8h, whereas the Tl/2 for preparations incubated in the presence of 5 mM and 10 mM CaCl2 were increased to 5h and 5.5h respectively. Although the addition of calcium improved the recovery of activity over the first 0.5h of incubation, at later times the rate of decay in the calcium containing preparations was similar to Factor VIII preparations without added calcium. Our results suggest that removal of calcium from the microenvironment of purified Factor VIII by desalting, results in an immediate loss of procoagulant activity, which can be partially restored within the first 0.5h following readdition of calcium. The decay in Factor VIII activity observed at later times in the 0 mM, 5 mM and 10 mM CaCl2 containing buffers likely reflects calcium-independent denaturation of the protein.

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Abstract 350: Influence of HIV-1 Infection and Antiretroviral Therapy on the Coagulation System

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a350 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Kevin Fasing ◽

Brian R Weil ◽

Kyle J Diehl ◽

Jared J Greiner ◽

Brian L Stauffer ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Factor Viii ◽

Tissue Factor ◽

Plasma Concentrations ◽

Tissue Type ◽

Factor Vii ◽

Coagulation System ◽

Factor X ◽

Thrombotic Risk ◽

Hiv 1

HIV-1-infected individuals have a two- to four-fold greater incidence of cardiovascular disease and atherothrombotic events compared with the general population. The mechanisms responsible for this heightened cardiovascular risk are not fully understood. We have previously reported that the capacity of the endothelium to release tissue-type plasminogen activator (t-PA), the primary activator of fibrinolysis and a key endogenous defense mechanism against intravascular fibrin deposition and thrombosis, is impaired in HIV-1-seropositive adults. Whether diminished fibrinolytic activity is coupled with a hypercoagulative state in this population is unknown. Elevations in specific coagulation markers such as tissue factor and Factor VII are associated with increased thrombotic risk. The experimental aim of this study was to determine the influence of HIV-1 infection and antiretroviral therapy (ART) on markers of coagulation. To address this aim we studied 33 men: 16 HIV-1-seronegative (age: 41±3 yr); 7 HIV-1-seropositive treatment-naïve (34±2 yr); and 10 HIV-1-seropositive receiving ART (42±3 yr; efavirenz-based regimen). All subjects were non-obese, normotensive and free of overt cardiometabolic disease. Circulating concentrations of tissue factor, Factor VII, Factor VIII and Factor X were determined by immunoassay. There were no significant differences in plasma concentrations of tissue factor (32+3 vs 40+3 pg/mL), Factor VII (103+9 vs 100+5 %), Factor VIII (111+13 vs 117+8 %) and Factor X (89+5 vs 91+2 %) between HIV-1-seropositive treatment-naïve and healthy men. Moreover, there was no influence of ART on these circulating markers. Plasma tissue factor (41+5 pg/mL), Factor VII (107+6 %), Factor VIII (103+7 %) and Factor X (90+3%) were similar in the HIV-1-seropositive receiving ART compared with HIV-1-seropositive treatment-naïve and seronegative groups. These data suggest that neither HIV-1 infection per se nor ART are associated with unfavorable changes in specific coagulation markers. Thus, changes in the coagulation system that have been linked to increased thrombotic burden are not apparent in HIV-1-seropositive adults.

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