The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation

1979 ◽  
Author(s):  
J. Rosing ◽  
G. Tans ◽  
J.W.P. Govers-Riemslag ◽  
R.F.A. Zwaal ◽  
H.C. Hemker
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor V ◽  
Factor X ◽  
Factor Va ◽  
Prothrombin Activation ◽  
Thrombin Formation

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

scholarly journals Characterization of an inducible endothelial cell prothrombin activator

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2989-2994 ◽  
Author(s):  
L Liu ◽  
GM Rodgers
Keyword(s):  
Thrombin Generation ◽  
Interleukin 1 ◽  
Factor Xa ◽  
Apparent Molecular Weight ◽  
Factor V ◽  
Factor X ◽  
Exogenous Factor ◽  
Km Value ◽  
Prothrombin Activation

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

Kinetic Analysis Of Prothrombin Activation By Des (l-44) Factor Xa

1981 ◽  
Author(s):  
W F Skogen ◽  
C T Esmon ◽  
A C Cox
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Phospholipid Analysis ◽  
Factor Va ◽  
Gla Domain ◽  
Dissociation Constant Kd ◽  
Prothrombin Activation

The region of factor X containing the gla residues was released by mild chymotryptic digestion and the resulting Des (1-44) factor X was converted to its activated form with the Russell’s viper venom factor X activator as described by others. The modified enzyme, factor Xa(-gd), retains its ability to activate prothrombin, the activation was still accelerated by factor Va, but the activation was no longer accelerated by phospholipid. The interaction between factor Va and either factor Xa or factor Xa(-gd) was studied kinetically. The rate of prothrombin activation was measured as a function of increased factor Va concentration in reaction mixtures where the factor Xa or factor Xa(-gd), prothrombin, and Ca2+ concentrations were constant. In the absence of phospholipid, analysis of the above data indicated an apparent dissociation constant (Kd’) of factor Xa for factor Va of 3.6 x 10-8 M. The removal of the gla domain lowered the apparent affinity of factor Xa for factor Va to 2.3 x 10-6 M indicating a role of the gla domain in the factor Xa-factor Va interaction even in the absence of phospholipid. Although the affinity was lowered, the V max of the reaction was identical for factdr Xa and factor Xa(-gd) in the presence of saturating concentrations of factor Va. With the addition of phospholipid, the Kd’ of factor Xa for factor Va was 4.7 x 10-10 M and V max increased some 297 fold. Phospholipid had no effect on the Kd’ of the factor Xa(-gd)-factor Va complex (Kd’ s 2.3 x 10-6 M) and V max was unaltered. These results demonstrate that phospholipid has little or no effect on factor Va function when factor Xa has lost its gla mediated Ca2+ binding sites.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

The Human Prothrombin-Activating Enzyme

1969 ◽  
Vol 22 (01) ◽  
pp. 045-067 ◽  
Author(s):  
K Deggeller ◽  
J Vreeken
Keyword(s):  
Linear Relationship ◽  
Factor Xa ◽  
Enzyme Concentration ◽  
Factor V ◽  
Factor X ◽  
Active Centers ◽  
Factor Va

SummaryThe formation and action of human prothrombin-activating enzyme is described. The study of the formation of the enzyme leads to the following conclusions :1. The enzyme is formed from factor V, factor X and phospholipid in the presence of calcium. If one of the reagents is omitted no activity develops.2. Factor V and factor X need activation by thrombin and for instance Russell Viper Venom, respectively.3. A linear relationship exists between the inverse of factor Va concentration and the inverse of enzyme concentration.4. A linear relationship exists between the inverse of factor Xa concentration and the inverse of enzyme concentration.5. A linear relationship exists between the inverse of phospholipid concentration and the inverse of enzyme concentration at small phospholipid concentration.6. A linear relationship exists between the phospholipid concentration and the inverse of enzyme concentration at high phospholipid concentration.The study of the action of the enzyme leads to the conclusion that human prothrombin is substrate and an inhibitor if present in excess.The observed phenomena are best explained by the hypothesis that factor Va and factor Xa adsorb onto the phospholipid surface. When both factors are adsorbed close together they are active as an enzyme. This activity depends on two active centers, probably one derived from factor Va and one from factor Xa.

Prothrombin Activation Is Increased among Asymptomatic Carriers of the Prothrombin G20210A and Factor V Arg506Gln Mutations

2000 ◽  
Vol 84 (09) ◽  
pp. 396-400 ◽  
Author(s):  
Steve Humphries ◽  
Belinda Smillie ◽  
Lily Li ◽  
Jacqueline Cooper ◽  
Samad Barzegar ◽  
...  
Keyword(s):  
Factor Viia ◽  
Conclusive Evidence ◽  
Factor Ix ◽  
Factor Vii ◽  
Prothrombin G20210a ◽  
Factor V ◽  
Factor X ◽  
Coagulation Proteins ◽  
Prothrombin Activation

SummaryThe risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50–61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G20210A and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment F1+2 [mean ± SD, 0.88 ± 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 ± 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 ± 0.24 among non-carriers for either mutation]. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.

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.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1695-1695
Author(s):  
Michael A. Bukys ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amino Acid Region ◽  
Factor Va ◽  
Initial Cleavage ◽  
Membrane Bound ◽  
Prothrombin Activation ◽  
Thrombin Formation

Abstract Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown that a pentapeptide encompassing amino acid sequence 695–699 from the COOH-terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits prothrombin activation by prothrombinase. The peptide was found to be a competitive inhibitor of prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds prothrombin in competition with the binding of the substrate to the enzyme, and inhibits prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. To understand the mechanism of inhibition of thrombin formation by DYDYQ we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by fully assembled prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for prothrombin activation, depending on the incorporation of factor Va in prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in prothrombin most likely has two interactive sites for the components of prothrombinase, a factor Va interactive site and a factor Xa binding site.

scholarly journals Experimental Studies on Factor VIII Inhibitor Bypassing Activity

1977 ◽  
Author(s):  
H. Vinazzer
Keyword(s):  
Factor Viii ◽  
Calcium Chloride ◽  
Experimental Studies ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Factor V ◽  
Factor X ◽  
Tissue Thromboplastin ◽  
Viii Inhibitor ◽  
Thrombin Formation

The exact action of factor VIII inhibitor bypassing activity (FEIBA) is still unclear. For this reason, a series of experimental studies was carried out. Procoagulant activities were examined by standard one-stage methods while factor Xa and thrombin were measured by chromogenic substrates. Activities of factors II, VII, IX, and X were similar to PPSB fractions. In addition, low factor V activity and a phospholipid were detected. No activated factor X was present in FEIBA but there was a trace amount of 2.1 NIH units of thrombin per 100 FEIBA units. On addition of calcium chloride slow thrombin formation could be observed which however, reached 1100 NIH units of thrombin per 100 FEIBA units within an incubation time of 10 min. The velocity of thrombin formation was greatly enhanced by addition of a PTT reagent and of thromboplastin respectively. Factor Xa on the other hand, was neither formed after addition of calcium chloride nor by a PTT reagent. Tissue thromboplastin however, activated Xa from FEIBA in the same manner as a PTT reagent plus barium sulfate plasma. From these results, the conclusion could be drawn that thrombin could readily be made available from FEIBA while activation of Xa either needed the complete endogenous pathway or the presence of tissue thromboplastin. The procoagulant activity of FEIBA therefore, could be attributed to direct thrombin formation. By this process, an activation of the clotting mechanism in plasmas deficient in endogenous coagulation factors, and a complete independence from the presence or absence of a specific antibody could be explained.

scholarly journals Studies of the prothrombin activation pathway utilizing radioimmunoassays for the F2/F1 + 2 fragment and thrombin--antithrombin complex

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 1086-1097 ◽  
Author(s):  
JM Teitel ◽  
KA Bauer ◽  
HK Lau ◽  
RD Rosenberg
Keyword(s):  
Disseminated Intravascular Coagulation ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor V ◽  
Human System ◽  
Normal Individuals ◽  
Kinetics Of ◽  
Prothrombin Activation

Abstract We have evaluated the efficacy of utilizing radioimmunoassays (RIAs) for prothrombin activation fragments (F2/F1 + 2) and for thrombin-- antithrombin complex (TAT) in purified systems and in whole blood. During venipuncture, appropriate anticoagulants were employed in order to prevent the generation of thrombin and factor Xa. The RIAs were shown to be specific for F2/F1 + 2 as well as TAT and did not interact with other plasma components. Initially, thrombin generation was studied in a purified human system of prothrombin, antithrombin, factor Xa, and factor V as well as phospholipid and Ca++. Under these conditions, the kinetics of F2/F1 + 2 and TAT generation were virtually superimposable. However, when factor V was omitted from the reaction mixture, a significantly greater amount of F2/F1 + 2 as compared to TAT was observable. Subsequently, prothrombin activation was monitored during the spontaneous coagulation of freshly drawn blood. Throughout the entire course of thrombin generation, the observable rate of formation of F2/F1 + 2 was considerably greater than that of TAT. We have examined the levels of F2/F1 + 2 and TAT in normal individuals. Our studies indicate that the concentrations of F1 + 2 and TAT average 1.97 nM and 2.32 nM, respectively. We have also quantitated the concentrations of F2/F1 + 2 and TAT in patients with disseminated intravascular coagulation. In these individuals, the levels of both components are elevated. However, the ratio of F1 + 2 to TAT ranges from 2.37 to 5.55. Thus, we conclude that under in vivo conditions, prothrombin activation is characterized by the accumulation of a stable precursor, such as prethrombin-2, and that this phenomenon may be related to an alteration of factor V function.

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