THE INTERACTION OF PURIFIED FACTOR VIII WITH PLATELETS

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

THE EFFECT OF CALCIUM ON THE STABILITY OF PURIFIED FACTOR VIII

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.

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.

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 Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies

1984 ◽  
Vol 218 (2) ◽  
pp. 601-608 ◽  
Author(s):  
T Lah ◽  
M Drobniĉ-Koŝorok ◽  
V Turk ◽  
R H Pain
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Single Chain ◽  
Guanidinium Chloride ◽  
Single Polypeptide Chain ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Kinetics Of ◽  
Chain Form ◽  
Covalent Complex

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

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

Blood ◽  
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.

Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1714-1714
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Kohei Tatsumi ◽  
Yuri Fujita ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Cleavage Rate ◽  
Physiological Concentration ◽  
C2 Domains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Km Value

Abstract Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

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

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.

scholarly journals The factor VIII protein and its function.

2016 ◽  
Vol 63 (1) ◽  
Author(s):  
Anna Mazurkiewicz-Pisarek ◽  
Grażyna Płucienniczak ◽  
Tomasz Ciach ◽  
Andrzej Płucienniczak
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Catalytic Efficiency ◽  
Fluid Phase ◽  
Coagulation System ◽  
Severe Bleeding ◽  
Factor X ◽  
Single Chain ◽  
Human Factor Viii ◽  
Coagulation Protein

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.

Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

1993 ◽  
Vol 70 (06) ◽  
pp. 0942-0945 ◽  
Author(s):  
Job Harenberg ◽  
Marietta Siegele ◽  
Carl-Erik Dempfle ◽  
Gerd Stehle ◽  
Dieter L Heene
Keyword(s):  
Tissue Factor ◽  
Binding Sites ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Factor X ◽  
Rebound Phenomenon ◽  
Tissue Factor Pathway ◽  
Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

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