Mechanisms of Plasmin-Catalyzed Inactivation of the Factor VIII.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1017-1017
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Fold Increase ◽  
Clotting Factors ◽  
Fviii Activity ◽  
A1 Domain ◽  
Von Willebrand

Abstract Factor VIII (FVIII) functions as a cofactor for factor IXa in the intrinsic tenase complex. This tenase activity is down-regulated by activated protein C (APC) or factor Xa (FXa). Plasmin, the most potent fibrinolytic protease, inactivates FVIII as well as other clotting factors. However, the mechanism of FVIII inactivation by plasmin is poorly understood. FVIII activity reached to the peak value of ~2-fold increase at 3 min after the addition of plasmin in a one-stage clotting assay. Then, the activity was decreased rapidly and was undetectable within 30 min. This time-dependent reaction was not affected in the presence of von Willebrand factor and phospholipid. The activation of FVIII by plasmin was an ~50% level of that by FXa. The rate constant (min-1) of inactivation of FVIIIa by plasmin possessed ~11.3- and ~2.5-folds greater than those by FXa and APC in the presence of protein S, respectively. SDS-PAGE analysis showed that plasmin cleaved the 90~210-kDa heavy chain of FVIII to 50, 48,45, 40, and 38-kDa fragments via 90-kDa fragment. Using western blot and N-terminal sequence analyses, these fragments derived from the heavy chain were identified as A11-372, A1337-372-A2, A11-336, A2, and A137-336, respectively, by cleavages at Arg372, Arg740, Lys36 and Arg336 in the A1 domain. On the other hand, the 80-kDa light chain was cleaved to 67-kDa fragment via 70-kDa fragment by cleavages at Arg1721 and Arg1689, respectively, consistent with the pattern of cleavage by FXa. However, the cleavage at Arg336 by plasmin was much quicker than that at Arg372, contrast with that by FXa. Furthermore, this cleavage was faster than that by APC, consistent with rapid inactivation of FVIII. In addition, the cleavage at Arg336 of FVIIIa by plasmin was faster than that of isolated A1 or A1/A3-C1–C2 dimer, different with that by FXa. These results demonstrate the importance of cleavage at Arg336 for the mechanism of plasmin-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A2 domain that may interact with plasmin.

scholarly journals Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

1977 ◽  
Author(s):  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gel Filtration ◽  
Protein S ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1036-1036
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Heavy Chain ◽  
Light Chain ◽  
Early Phase ◽  
Factor Viia ◽  
Clotting Factors ◽  
Initiation Phase ◽  
A1 Domain

Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1702-1702
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Tsukasa Suzuki ◽  
Kunihiro Hattori ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Neutralizing Antibodies ◽  
Active Form ◽  
Activated Protein C ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heat Denaturation ◽  
Factor X ◽  
Fviii Activity

Abstract Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.

scholarly journals Characterization of des-(741–1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa

1995 ◽  
Vol 312 (1) ◽  
pp. 49-55 ◽  
Author(s):  
M J S H Donath ◽  
R T M de Laaf ◽  
P T M Biessels ◽  
P J Lenting ◽  
J W van de Loo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Xa ◽  
Single Chain ◽  
Factor Ixa ◽  
Fusion Site ◽  
Von Willebrand ◽  
Willebrand Factor

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Cleavage Of Reduced Factor VIII/VON Willebrand Factor Subunits By Thrombin Or Factor Xa

1981 ◽  
Author(s):  
G A Vehar
Keyword(s):  
Factor Viii ◽  
Ammonium Sulfate ◽  
Von Willebrand Factor ◽  
Activated Protein C ◽  
Factor Xa ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Bovine Plasma ◽  
Von Willebrand ◽  
Willebrand Factor

Purified Factor VIII/von Willebrand Factor (FVIII/vWF) was obtained from bovine plasma using ammonium sulfate and glycine precipitations, tricalcium citrate adsorption, and Sepharose CL-4B gel chromatography. The protein was concentrated with ammonium sulfate and dialyzed against 0.05 M Tris-HCl, pH 7.4, containing 0.15 M NaCl. Partial reduction of these preparations was obtained using a 2.5 hr incubation at 20°C with 5 mM dithioerythritol, followed by dialysis to remove the reductant. The addition of a 19 to 1 molar ratio (110 to 1 wt. ratio) of FVIII/vWF to thrombin resulted in a time dependent cleavage of the reduced preparation, as detected by SDS gel electrophoresis. A 50 min. digest resulted in the cleavage of the 220,000 dalton subunit to products of 145,000 and 140,000 daltons. Factor Xa, in the presence of calcium and phospholipid, also cleaved the reduced protein when present at a 14 to 1 molar ratio (75 to 1 wt. ratio) of FVIII/vWF to factor Xa . A 50 min. digest resulted in fragments of 200,000, 175,000, and 55,000 daltons. No cleavage occurred under these conditions if the native, unreduced FVIII/vWF was used. Factors XIIa, XIa, IXa and activated protein C did not cleave the reduced or unreduced preparations. The observed cleavages of FVIII/vWF by thrombin or factor Xa do not, however, correlate with the alteration of any known function of this protein complex.

VON WILLEBRAND FACTOR PROTECTS FACTOR VIII FROM INACTIVATION BY ACTIVATED PROTEIN C AND PROTEIN S

1987 ◽  
Author(s):  
S Joost ◽  
A Koedam ◽  
Joost C M Meijers ◽  
Jan J Sixma ◽  
Bonno N Bouma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Activated protein C (APC) inactivated the cofactors factor V (FV) and factor VIII (FVIII). In the case of FV, this reaction and the respective roles of Ca2+ , phospholipids and protein S have been well documented. We investigated the role of protein S and von Willebrand factor (VWF) on the inactivation of FVIII.Purified human factor VIII (3 units/ml) was incubated with protein C (0.2 μg/ml) in the presence of 8 μg/ml phospholipid, 5 mM CaCl, and 1 unit/ml hirudin. Factor VIII coagulant activity decreased with a pseudo first-order rate constant of 0.09 min . The reaction rate increased linearly with the concentration of prot^ig S in the incubation mixture. 12I-FVIII was incubated under the same conditions. SDS-polyacrylamide gel electrophoresis showed cleavage products of Mr 43 and 22 kDa. High Mr bands (FVIII-heavy chain) ranging fromMr 108 to208 kDa disappeared while the Mr 80 kDa FVIII-lightchain remained unchanged. The degradationpattern was not changed by addition of protein S.The FVIII-VWF complex was reconstitutedby mixing the two components (±2 units VWF/units FVIII) and lowering the calcium concentration to 2 mM. The inactivation of the FVIII-VWF complex by APC proceeded at a 15- to 20-fold slower rate as compared to the isolated FVIII, indicating a protection of FVIII by VWF. Protein S exhibited no cofactor activity on the inactivation of FVIII-VWF by APC. The protective effect of VWF was lost completely after activation of the FVIII-VWF complexwith thrombin (0.05 units/ml).When FVIII (0.1 units/ml) was added toplasma of a patient with severe von Willebrand's disease, 96% of its activitywas lost in 20 min after the addition of APC. All of the FVIII activity was retained when haemophilic plasma was used. Mixing experiments showed that one unit ofVWF unit FVIII is needed to fully protec FVIII against APC. These results may explain the observed lability of FVIII in von Willebrand's disease patients.

INACTIVATION OF FACTOR VIII BY ACTIVATED PROTEIN C AND PROTEIN S

1987 ◽  
Author(s):  
P J Fay ◽  
S I Chavin ◽  
F J Walker
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Ix ◽  
Heavy Chains ◽  
Factor Va

Human factor VIII has been isolated from factor VIII concentrates. The isolated protein is composed of a heavy chain and light chain. The heavy chain was heterogenous with respect to molecular weight ranging from 110-170 kDa. The light chain appeared as a 81/84 kDa dimer, 'when factor VIII was treated with activated protein C in the presence of calcium and phospholipids factor VIII procoagulant activity was rapidly lost. Analysis of the activated protein C catalyzed cleavage products of factor VIII indicated that loss of activity was correlated with cleavage of the heavy chains. The heavy chains appeared to be converted into 93 kDa and 53 kDa peptides. A separate factor VIII preparation has been prepared that contained only a 93 kDa heavy chain as well as the 81/83 kDa light chain. When this preparation was inactivated with activated protein C, a pathway in which the 93 kDa peptide was degraded into a 68 kDa peptide which was subsequently degraded into 48 and 23 kDa polypeptides. This result suggested that the 53 kDa polypeptide was not derived from the 93 kDa domain of the heavy chain, but must have been derived from the variable molecular weight portion of the heavy chain. These results suggest that activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. Protein S has been observed to be a protein cofactor both the anticoagulant and proteolytic action of activated protein C with factor Va. It is thought that protein S forms a lipid bound complex with activated protein C which then can rapidly inactivate factor Va. When factor VIII was inactivated in the presence of both activated protein C and protein S the rate of activity loss was enhanced. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products including the 93, 68, and 53 kDa polypeptides. In an analogous reaction, the addition of factor Xa has been observed to inhibit the inactivation of factor Va by activated protein C. The addition of factor IX to the factor Vlll-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Finally, as both factor Va and factor VIII have structural similarities and are substrates for activated protein C the possibility that they might compete as substrates was tested. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.

THE INTERACTION BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Joost A Koedam ◽  
Rob J Hamer ◽  
Nel H Beeser-Visser ◽  
Etienne Jap Tjoen San ◽  
Kees Schippers ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Disulfide Bonds ◽  
Factor Xa ◽  
Exchange Chromatography ◽  
Solid Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.

APC Resistance and other Haemostatic Variables during Pregnancy and Puerperium

1999 ◽  
Vol 81 (04) ◽  
pp. 527-531 ◽  
Author(s):  
U. Kjellberg ◽  
N.-E. Andersson ◽  
S. Rosén ◽  
L. Tengborn ◽  
M. Hellgren
Keyword(s):  
Factor Viii ◽  
Plasminogen Activator ◽  
Protein C ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Protein S ◽  
Reference Level ◽  
Soluble Fibrin ◽  
Prothrombin Fragment ◽  
D Dimer

SummaryForty-eight healthy pregnant women were studied prospectively and longitudinally. Blood sampling was performed at 10-15, 23-25, 32-34 and 38-40 weeks of gestation, within one week and at eight weeks postpartum. Classic and modified activated protein C ratio decreased as pregnancy progressed. In the third trimester 92% of the ratios measured with the classic test were above the lower reference level whereas all modified test ratios were normal. Slight activation of blood coagulation was shown with increased levels of prothrombin fragment 1+2, soluble fibrin and D-dimer. Fibrinogen, factor VIII and plasminogen activator inhibitor type 1 and type 2 increased. Protein S and tissue plasminogen activator activity decreased. Protein C remained unchanged. No correlation was found between the decrease in classic APC ratio and changes in factor VIII, fibrinogen, protein S, prothrombin fragment 1+2 or soluble fibrin, nor between the increase in soluble fibrin and changes in prothrombin fragment 1+2, fibrinogen and D-dimer.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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