THE EFFECT OF CALCIUM ON THE STABILITY OF PURIFIED FACTOR VIII

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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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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A STUDY ON THE RECOVERY OF FACTOR VIII PROCOAGULANT ACTIVITY FROM RECALCIFIED, HEPARINISED, CITRATED PLASMA

10.1055/s-0038-1644156 ◽

1987 ◽

Author(s):

A M Cumming ◽

R T Wensley ◽

S E Cottrell ◽

I W Delamore

Keyword(s):

Factor Viii ◽

Membrane Filtration ◽

Specific Activity ◽

Protein S ◽

Procoagulant Activity ◽

Routine Manner ◽

Acid Citrate Dextrose ◽

The Stability ◽

Citrate Dextrose ◽

Plasma Heparin

This study has been carried out to investigate the potential for increasing the recovery of factor VIII procoagulant activity (factor VIII:C),in cryoprecipitates and concentrates, by the use of heparin anticoagulant. Jhctor VIII:C in citrated plasmahas been shown to be stabilised by recalcification and heparinisation of the plasma. Donations of substantially platelet-free and platelet-product-free plasma, anticoagulated by acid citrate dextrose formula A anticoagulant (ACD A), were collected using a combined membrane filtration/centrifugation plasmapheresis device (the HemaSciences "Autopheresis C"(R)). Samples of this plasma were heparinised (over a range from0.1 to 12.8 iu/ml) and physiological Ca2+ levels were restored, levels of fibrinopeptide A (FpA) of less than 5 ng/ml were measured in all samples(including following the incubation of plasmas at 21 °C for 24 hours). This indicated minimal thrombin generation at plasma heparin concentrations as low as 0.1 iu/ml. Fibrinogen degradation fragment BJ3 1542 levelsin these plasmas were comparable to those in ACD A control plasmas and there was no upward trend with increasing heparin concentration. This suggested that heparin-induced formation of plasmin would not adversely affect the stability of factor VIII:C in plasma treated in this way. Cryoprecipitates were prepared in a routine manner from ACD A plasmas collected, by plasmapheresis, into bags containing sufficient heparin and Cacl2 to achieve plasma heparin levels of 0.1 iu/ml and physiological Ca2+ concentrations. (As a result of thissecondary heparinisation, there was no infusion of heparin into the donors). Analysis of the cryoprecipitates revealed no resolubilisation problems, a significant (P<0.02) gain inthe yield of factor VIIIsC comparedwith ACD A control cryoprecipitates, and a mean factor VIIIsC specific activity of 0.11 iu/mg of total protein (S. D. 0.03, 6 experiments). Pools of plasma collected in the above manner are currently being fractionated to evaluate the resulting factor VIII concentrates.

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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 ◽

10.1182/blood-2020-142522 ◽

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.

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

Blood ◽

10.1182/blood.v110.11.2686.2686 ◽

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.

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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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647295 ◽

1990 ◽

Vol 64 (02) ◽

pp. 251-255 ◽

Cited By ~ 8

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.

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DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY

10.1055/s-0038-1643613 ◽

1987 ◽

Author(s):

M Ph Verbeet ◽

R F Evers ◽

A Leyte ◽

H L Lamain ◽

A J J Van Ooyen ◽

...

Keyword(s):

Factor Viii ◽

Recombinant Proteins ◽

Cleavage Site ◽

Specific Activity ◽

Expression System ◽

Full Length ◽

Procoagulant Activity ◽

Expression Vectors ◽

Recombinant Fviii

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.

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Immunologic Approach to the Study of Factor VIII

10.1055/s-0039-1680465 ◽

1977 ◽

Author(s):

Leon W. Hoyer

Keyword(s):

Factor Viii ◽

Carrier State ◽

Procoagulant Activity ◽

Related Protein ◽

Low Protein ◽

Biochemical Analyses ◽

And Function ◽

Deficiency Diseases

In contrast to direct biochemical analyses, immunologic studies of factor VIII are less limited by that factor’s lability and low protein concentration. Both human and heterologous antifactor VIII have been used in these studies. Human antibodies inactivate factor VIII procoagulant. activity but they do not fix complement or form immunoprecipitates. Their interaction appears to involve the formation of an immune complex with a univalent component distinct from that which has been designated factor VIII-related protein. This component is synthesized—but remains nonfunctional—in hemophilia, and at least two different defects have been distinguished. Very different reactions can be identified using heterologous antibodies. Although many of these antibodies do not react directly with the procoagulant site, they do fix complement, precipitate with factor VIII-related protein, and inactivate ristocetin cofactor activity. These antibodies are useful for classification of factor VIII-deficiency diseases according to the relative levels of factor VIII procoagulant activity and factor VIII-related antigen; identification of the hemophilic carrier state; characterization of heterogeneity in factor VIII structure; and purification of factor VIII procoagulant activity by immunoadsorbent chromatography. In general, the information obtained by immunologic studies has complemented that from direct biochemical analyses. Both approaches contribute to our evolving understanding of factor VIII structure and function.

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A New Functional Coagulation Assay for Measurement of Total Factor V Activity in Human Plasma and Its Application to Samples from Patients with Disseminated Intravascular Coagulation.

Blood ◽

10.1182/blood.v104.11.2983.2983 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2983-2983

Author(s):

John A. Samis ◽

Kenneth A. Stewart ◽

Cheng Hock Toh ◽

Colin Downey ◽

Michael E. Nesheim

Keyword(s):

Disseminated Intravascular Coagulation ◽

Specific Activity ◽

Factor V ◽

Plasma Samples ◽

Single Chain ◽

Intravascular Coagulation ◽

One Stage ◽

Normal Human ◽

Coagulation Assay ◽

The One

Abstract Factor V (F.V) circulates as an inactive single chain procofactor. Only upon cleavage and activation with thrombin to F.Va does it function as a cofactor in the F.Xa-mediated conversion of prothrombin to thrombin. The conventional F.V coagulation assay measures the one-stage clotting activity, which most likely reflects the trace of F.V that is activated in the sample or becomes activated during the assay. Since F.V is activated by thrombin to F.Va, a second (two-stage) assay is crucial to fully assess the total F.V potential activity in the plasma sample. For this assay, normal human pooled plasma (diluted 100- to 500-fold) was treated with thrombin (1–5 NIH Units/ml, final concentration) for various times at 37°C to fully activate the F.V. The added thrombin was inhibited with a 2-fold molar excess of Phe-Pro-Arg chloromethylketone (PPACK) and the sample re-assayed. This second assay measures the total F.V activity of a sample. Maximal 10-fold activation of plasma F.V activity occurred with 2Units/ml thrombin after 1 to 2min at 37°C and this activity was stable for 30min prior to PPACK addition. Control experiments indicated that the levels of PPACK used did not affect the one-stage F.V activity of untreated plasma samples but were required to inhibit the added thrombin from clotting fibrinogen in the assay. Assay of timed plasma samples (n=48) from seven patients admitted to the Intensive Care Unit and subsequently diagnosed with disseminated intravascular coagulation (DIC), indicated that the total F.V activity, the specific activity (total F.V activity/antigen), and the activation quotient were decreased, on average, by 45%, 49%, and 31%, respectively, from that observed with normal human pooled plasma. The results indicate that the total amount of activatable F.V as well as its degree of activation decreases from normal as part of the pathogenesis of DIC in humans. Further, the decreased total F.V specific activity observed in DIC patient plasma samples indicates that the F.V that is present is less active per molecule than normal, possibly because of proteolytic inactivation. This total F.V clotting assay should provide new information about the biochemistry of F.V and its contribution to the pathogenesis of coagulation disorders.

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Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽

10.1182/blood.v108.11.1692.1692 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1692-1692

Author(s):

Jennifer Newell ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Time Course ◽

Specific Activity ◽

Factor Xa ◽

Factor X ◽

Wild Type ◽

Thrombin Cleavage ◽

Factor Ixa ◽

Factor Viiia ◽

Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

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ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS

10.1055/s-0038-1644044 ◽

1987 ◽

Author(s):

Debra D Pittman ◽

Louise C Wasley ◽

Beth L Murray ◽

Jack H Wang ◽

Randal J Kaufman

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Protein C ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Factor X ◽

Proteolytic Activation ◽

Thrombin Cleavage ◽

Cofactor Activity

Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.

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