Exposure of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1951-1951
Author(s):  
Michael A. Bukys ◽  
Tivadar Orban ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Factor Viii ◽  
Anion Binding ◽  
Factor V ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Factor X ◽  
Clotting Time ◽  
Experimental Conditions ◽  
Membrane Bound ◽  
Cofactor Activity

Abstract The intrinsic tenase complex and the prothrombinase complex are composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. Incorporation of the protein cofactor in both complexes results in a substantial increase in the catalytic efficiency of both enzymes, factor IXa and factor Xa, for cleavage and activation of factor X and prothrombin respectively, resulting in normal hemostasis. The procofactors, factor V (FV) and factor VIII (fVIII), do not interact with the components of prothrombinase and intrinsic tenase respectively and must be activated. α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate binding necessary for efficient catalysis. α-Thrombin catalyzes the activation of fV and fVIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available) and a recombinant prothrombin molecule rMz-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a sulfated pentapeptide (DY(SO3−)DY(SO3−)Q, named D5Q1,2) as exosite-directed inhibitor of thrombin. D5Q1,2 was found to increase thrombin time in a dose dependent manner yielding an eight-fold increase in thrombin time at 250 μM in the presence of 10 nM α-thrombin. This clotting time was equivalent to the thrombin time obtained with 10 nM β-thrombin alone. The clotting time of rMZ-IIa was increased four-fold compared to the clotting time of α-thrombin under similar experimental conditions. α-Thrombin readily activated fV following cleavages at Arg709, Arg1018, and Arg1545 and fVIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors by α-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave fV at Arg1545 and fVIII at both Arg372 and Arg1689. The former is required for expression of factor Va (fVa) cofactor activity while the latter two cleavages are a prerequisite for expression of factor VIIIa (fVIIIa) cofactor activity. β-Thrombin was found to cleave fV at Arg709 and fVIII at Arg740, albeit less efficiently than α-thrombin. D5Q1,2 inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules with a rate similar to that observed for the activation of fV and fVIII by α-thrombin. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. These data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Our data also show that a sulfated pentapeptide inhibits several procoagulant ABE-I-related functions of α-thrombin and provide a target as well as the scaffold for the synthesis of an exosite-directed anticoagulant molecule that could inhibit and/or attenuate therapeutically thrombin function in individuals with thrombotic tendencies.

scholarly journals A Novel Variant of Factor VIII Yields Cofactor Activity without Proteolysis between the A1 and A2 Domains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3771-3771
Author(s):  
Manjunath Goolyam Basavaraj ◽  
Sriram Krishnaswamy
Keyword(s):  
Factor Viii ◽  
Research Funding ◽  
Kinetic Studies ◽  
Proteolytic Cleavage ◽  
Activate Factor ◽  
Factor V ◽  
Factor X ◽  
High Concentration ◽  
One Stage ◽  
Cofactor Activity

Abstract Factor VIII (FVIII) has a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2), and intracellular processing within the B domain results in its secretion as a heterodimeric procofactor consisting of a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2). Proteolytic cleavage by thrombin at R372, R740, and R1689 removes the B-domain, releases it from vWF by cleaving the a3-acidic region and activates FVIII to the heterotrimeric cofactor, FVIIIa (A1-a1/A2-a2/A3-C1-C2). The requirement for cleavage following a1 to generate active cofactor sets FVIII/FVIIIa apart from factor V, its structural and functional homolog. FVIII binds its cognate protease, factor IXa (FIXa) with high affinity in a membrane-dependent way. However, this complex does not efficiently activate factor X (FX) until R372 is cleaved, indicating that this cleavage reaction is important in facilitating FX recognition and its enhanced activation by intrinsic tenase complex. We speculated that separation of A1-a1 and A2-a2 domains through cleavage at R372 might be replicated by inserting a linker between A1-a1 and A2-a2 domains even when the 372 site was rendered uncleavable. We chose canine FVIII (cFVIII) to test our ideas due to its higher expression levels in cell culture compared to human FVIII. We made cDNA constructs encoding cFVIII variants with flexible (GGGGS, [GGGGS]3, GGGGGG) or rigid (EAAAK, [EAAAK]3, PAPAP) linkers between A1-a1 and A2-a2 domains on the R366Q backbone (corresponding to R372 in human FVIII). All constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Clotting times of cFVIII variants with flexible linkers were very similar to cFVIII R366Q without linker (~64 sec). In contrast, clotting times of cFVIII variants with rigid linkers were consistently lower (16 - 26 sec). cFVIII variants (R366Q, R366Q-EAAAK, R366Q-PAPAP) were purified from 15L of expression media each. In one stage aPTT assays purified cFVIII variants (10 nM) showed clotting times (R366Q-EAAAK, 33.3 s, R366Q-PAPAP, 26 s) that were considerably shorter than the R366Q variant (52.2 s) and more in line with wild type cFVIII (cFVIII-WT,19.3 s). To assess possible cleavage at the 366 site, purified cFVIII variants were analyzed by SDS-PAGE following treatment with a high concentration of thrombin. As expected, cFVIII-WT was quantitatively cleaved at R366, R734 and R1689 to produce bands corresponding to A1-a1, A2-a2 and A3-C1-C2. In contrast, both R366Q and R366Q-PAPAP variants were resistant to cleavage at 366, yielding bands corresponding to A1-a1-A2-a2 and A3-C1-C2. The cFVIII-R366Q-EAAAK variant was susceptible to some cleavage, likely at the lysine present within linker. We also performed steady state kinetic studies of FX activation using limiting concentrations of IXa, saturating concentrations of cFVIII variants pretreated with thrombin, membranes and increasing concentrations of FX. Vmax/[E] for FXa formation was 140 ± 7 min-1 with cFVIIIa-WT, 4 ± 0.3 min-1 with cFVIIIa-R366Q, and 42 ± 4 min-1 with cFVIIIa-R366Q-PAPAP. The Km for FX remained unaffected for all variants. The results indicate that the impaired cofactor activity of the FVIII variant that is not cleaved at the 366 site can be substantially rescued by insertion of the PAPAP linker after 366Q. Further optimization of the linker might completely correct defective FX activation associated with the lack of proteolytic cleavage between A1-a1 and A2-a2 domains. Our study indicates that cleavage at the 366 site is not an absolute determinant of FVIII activation and provides new insights into the function of FVIIIa as a cofactor within the intrinsic tenase complex. Disclosures Goolyam Basavaraj: Bayer: Research Funding; Grifols: Research Funding. Krishnaswamy:Portola: Research Funding; Baxalta: Consultancy; Janssen Research & Development: Research Funding.

Factor V Enhances the Cofactor Function of Protein S in the APC-Mediated Inactivation of Factor VIII: Influence of the Factor VR506Q Mutation

1996 ◽  
Vol 76 (02) ◽  
pp. 208-214 ◽  
Author(s):  
K Váradi ◽  
J Rosing ◽  
G Tans ◽  
I Pabinger ◽  
B Keil ◽  
...  
Keyword(s):  
Factor Viii ◽  
Reaction System ◽  
Protein S ◽  
Factor V ◽  
Dependent Manner ◽  
Plasma Samples ◽  
Factor X ◽  
Formation Factor ◽  
Free Protein ◽  
Plasma Factor

SummaryFactor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chro-mogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell’s viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.

Coagulation Findings in Rats with Experimental Hypersplenism and Their Changes after Splenectomy and after Administration of Adrenaline

1970 ◽  
Vol 23 (03) ◽  
pp. 593-600
Author(s):  
P Pudlák ◽  
I Farská ◽  
V Brabec ◽  
V Pospíšilová
Keyword(s):  
Factor Viii ◽  
Normal Control ◽  
Normal Values ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Factor V ◽  
Thrombin Time ◽  
Factor Ii ◽  
Recalcification Time

Summary1. The following coagulation changes were found in rats with experimental hypersplenism: a mild prolongation of the recalcification time, shortened times in Quick’s test, a lowered activity in plasma thrombin time and shortened times in the partial thromboplastin test. Concentrations of factor II, V, VII (+X), VIII and X did not differ from those of normal control rats.2. The administration of adrenaline to hypersplenic rats induced the correction of the partial thromboplastin test, Quick’s test and plasma thrombin time to normal values. Concentrations of coagulation factors were not significantly changed. An increase was found in factor V.3. Splenectomy performed in hypersplenic rats was followed by a shortened recalcification time, a prolongation of the partial thromboplastin test and of the test with partial thromboplastin and kaolin. A prolongation was also observed in Quick’s test. Complete correction of plasma thrombin time was not observed. The concentration of factor VII increased.4. The administration of adrenaline to splenectomized rats with experimental hypersplenism did not induce any significant changes with the exception of a corrected plasma thrombin time and a decreased concentration of factor VIII.5. A different reaction of factor VIII to adrenaline in normal and hypersplenic rats is pointed out.

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.

scholarly journals Evaluation of anticoagulant activity of aqueous extract of Cestrum nocturnum

2017 ◽  
Vol 8 (4) ◽  
pp. 525
Author(s):  
Chandra Kishore Tyagi ◽  
Deenanath Jhade ◽  
Sunil Kumar Shah
Keyword(s):  
Aqueous Extract ◽  
Anticoagulant Activity ◽  
Ethanolic Extract ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Clotting Time ◽  
Standard Procedures ◽  
Prolonged Aptt ◽  
Pharmacologically Active

<p>The study evaluated anticoagulant properties of the aqueous extract of <em>Cestrum nocturnum</em> using aPTT-Activated Partial Thromboplastin Time, PT- Prothrombin Time &amp; TT-Thrombin Time as standard procedures.</p><p>For <em>in vitro</em> coagulation assays, aqueous extract of plant prolonged APTT, TT, and PT clotting times in a dose-dependent manner (Table 7). It prolonged APTT clotting time from 45 ± 2 (2mg/mL) to 82.2 ± 2.63s (10mg/mL), PT clotting time from 20.4 ± 1.49 (2mg/mL) to 31.4 ± 2.15s (10mg/mL), and TT clotting time from 9.2 ± 1.16 (2mg/mL) to 17.4 ± 1.01s (10mg/mL) at the concentration of 2 to 10mg/mL. Heparin prolonged APTT and PT clotting times more than 111.8s and 40.8s, respectively, at a concentration of 1 IU/mL. Heparin prolonged TT clotting times more than 20.6s at a concentration of 1 IU/mL.</p><p>The phytochemical screening of the plant confirm the presence of saponin in the water and ethanolic extract, Alkaloid in all the extract except hexane extract, tannin in water, ethanol and methanol extract, amino acid in water and ethanolic extract, carbohydrate in water and methanolic extract and triterpenoids and glycoside were absent in all the extracts. The results demonstrated that the aqueous extract of <em>Cestrum nocturnum</em> possesses pharmacologically active anticoagulant principles that could be isolated and evaluated for clinical or physiological purposes.</p>

HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII

1987 ◽  
Author(s):  
F A Ofosu ◽  
G J Modi ◽  
M R Buchanan ◽  
J Hirsh ◽  
M A Blajchman
Keyword(s):  
Factor Viii ◽  
Antithrombin Iii ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Inhibitory Effects ◽  
Efficient Inhibitor ◽  
System 1 ◽  
Prothrombin Activation

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.

scholarly journals Feiba Immuno: A Preparation with Factor Eight Inhibitor Bypassing Activity

1977 ◽  
Author(s):  
F. Elsinger
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Active Principle ◽  
Factor Viii Inhibitor ◽  
Factor V ◽  
In Vitro Experiments ◽  
Factor X ◽  
Viii Inhibitor

FEIBA IMMUNO is a preparation in which a new activity is generated capable of bypassing factor VIII. The preparation which is used to treat patients with inhibitors (especially inhibitors to factor VIII) is standardized in FEIBA units, i.e. in terms of its in vitro capacity to shorten the activated PTT of a factor VIII inhibitor plasma.It could be concluded from different in vitro experiments that none of the classic’ activated coagulation factors is responsible for the factor VIII bypassing reaction; FEIB-activity seems to be correlated to a new complex of coagulation factors.To get an answer to the question which coagulation factors are essential for FEIB-activity, we tried to generate this activity from different deficient plasmas; from these experiments the following conclusions could be drawn:, the presence of at least factors VII, IX, and X is essential for the generation of the molecular species responsible for factor VIII as well as factor X bypassing activity, but factor V is not bypassed. This activity is not factor Xa itself. Factors VIII and V are not necessary for the generation of this active principle, but factor V is finally needed for its bypassing action.

Singlet Oxygen Inactivates Fibrinogen, Factor V, Factor VIII, Factor X, and Platelet Aggregation of Human Blood

2000 ◽  
Vol 97 (6) ◽  
pp. 473-480 ◽  
Author(s):  
Thomas W Stief ◽  
Jürgen Kurz ◽  
Manfred O Doss ◽  
Jawed Fareed
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Singlet Oxygen ◽  
Human Blood ◽  
Factor V ◽  
Factor X

Factor V as Anticoagulant Cofactor for Activated Protein C in Factor Va Inactivation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3075-3075
Author(s):  
Thomas J Cramer ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Protein C ◽  
Time Course ◽  
Activated Protein C ◽  
Protein S ◽  
Anticoagulant Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Anticoagulant Function ◽  
Apc Protein

Abstract Factor V (FV) is a cofactor that promotes inactivation of activated factor VIII (FVIIIa) by the activated protein C and protein S complex (APC/protein S). Cleavage in FV at Arg506 is required for proteolytic inactivation of FVa, but also for the anticoagulant function of FV as cofactor for APC in the inactivation of FVIIIa. This is demonstrated by the well known FVLeiden mutant with Arg506 mutated to glutamine (Q506), causing APC resistance due to both impaired sensitivity of Q506FVa to APC and reduced cofactor activity of Q506FV for APC inactivation of FVIIIa. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. Thus, we question whether the APC-mediated inactivation of FVIIIa is relevant to the FVLeiden thrombophilic phenotype. Rather, we hypothesized that FV can function as an anticoagulant cofactor for the APC/protein S complex in the inactivation of activated FV (FVa). To test this hypothesis, we designed a coagulation assay initiated by tissue factor that was sensitive to FV but was insensitive to FVIII. FV was titrated into FV deficient plasma and clotting times were measured in absence and presence of APC to determine an APC sensitivity ratio (APCsr). An increase in the APCsr was observed as the level of FV was increased, suggesting an anticoagulant function of FV. Similar titrations were done with Q506FV, showing no increase in clotting time when APC was present and an APCsr of 1.0 in the presence of Q506 FV. Control experiments confirmed that this clotting assay was insensitive to the presence or absence of FVIII; thus, these assays were reflecting FVa inactivation. The potential anticoagulant effect of FV as cofactor for APC in FVa inactivation was further investigated by monitoring proteolysis of purified FVa by APC over time using SDS PAGE. Recombinant purified FVa was labeled with a fluorescent dye, and then subjected to proteolysis by APC/protein S in the absence or presence of FV in a time course. The resulting FVa fragments seen on SDS gels reflected the known cleavages at Arg306 and Arg506, and the FVa cleavage products were quantified by digital fluorescent scanning of the gel. FV stimulated a small but statistically insignificant increase in the rate of FVa cleavage by APC/protein S. Thus, in our experimental conditions, we found a significant anticoagulant effect of FV in clotting assays that were sensitive to FV but not sensitive to FVIII whereas in purified reaction mixtures there was not a significant enhancement by FV of APC proteolysis of FVa. These data contrasting FV’s apparent APC-cofactor activities between plasma and purified reaction mixtures lead us to speculate that other factors or mechanisms present in plasma also contribute to the anticoagulant function of APC in a FV dependent manner.

Blood Coagulation and Fibrinolysis in Haemorrhagic Stroke

1978 ◽  
Vol 39 (01) ◽  
pp. 084-088 ◽  
Author(s):  
Rama Kanta Dube ◽  
P V B Rao ◽  
P K Saha ◽  
B C Katiyar ◽  
B Dube
Keyword(s):  
Platelet Count ◽  
Prothrombin Time ◽  
Fibrinolytic Activity ◽  
Haemorrhagic Stroke ◽  
Factor V ◽  
Thrombin Time ◽  
Clotting Time ◽  
Plasma Recalcification Time ◽  
Recalcification Time ◽  
Coagulation And Fibrinolysis

SummaryStudies of 11 patients with haemorrhagic stroke revealed no significant change in kaolin cephalin clotting time, prothrombin time, thrombin time, PF 3 availability, platelet count and factor V and VIII during the first week. Plasma fibrinogen was significantly increased while factors VII+X were decreased (borderline significance). Prolongation of plasma recalcification time and decrease in heparin tolerance reached borderline significance. There was moderate, but significant, increase in serum antithrombin activity and plasma (euglobulin fraction) fibrinolytic activity.

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