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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-01-0290.
REVIEW ARTICLE
From the Department of Biochemistry, University
of Vermont, College of Medicine, Burlington; the Department of
Chemistry, Cleveland State University, Cleveland, OH; and the
Department of Molecular Cardiology, The Lerner Research Institute, The
Cleveland Clinic Foundation, Cleveland, OH.
The desirable process of hemostasis and the
undesirable phenomena of thromboses are unequivocally consequences of
the generation of thrombin by the prothrombinase complex. This
stoichiometric complex of factor Xa and factor Va assembled on a
platelet membrane converts membrane-bound prothrombin into its active
products by 2 sequential peptide-bond cleavages. The functionality and
durability of the prothrombinase complex is subject to multiple
regulatory processes that may enhance or eliminate its function. Factor
Va, an essential component of prothrombinase, is both produced and destroyed by proteolytic events. The absence or dysfunction of factor
Va leads to hemorrhagic disease, whereas excessive longevity of the
active species is associated with thrombosis. Factor V is thus required
for a good outcome (Dr Jekyll) but also is a potential source of
disaster (Mr Hyde). In this review, we summarize the current state of
knowledge with respect to the good and the bad aspects of factor V
functionality and durability.
The factor Va molecule is generated by cleavage of its precursor
factor V. It contributes to the blood clotting reaction by binding with
factor Xa on a membrane surface to form the prothrombinase complex.
This complex is the essential activator of prothrombin to thrombin
during the blood clotting process and effectively increases the
activity of factor Xa by 5 orders of magnitude.
Factor V was discovered by Paul Owren in 19431 when, using
relatively primitive technology, he was able to deduce the existence of
a fifth component required for fibrin formation that he named "factor
V," thus beginning the era of Roman numerology for coagulation factors. This work, published in Lancet after War World
II,1 also identified the pro-cofactor nature of factor V
and the requirement for its activation by thrombin as well as its
participation in the generation of thrombin. Dr Owren's work defined
factor V as the activity in normal plasma that corrected the
prothrombin time (PT) of the plasma of a patient with factor V
deficiency.1,2 Following a long period of controversy,
factor V was isolated in 1979.3-6 The isolation
established the "biochemistry" period of blood clotting complexes
and launched the paradigm of membrane-bound complexes contributing to
the blood clotting process.
Human plasma factor V circulates at a concentration of 20 nM, as a
large single-chain pro-cofactor with an Mr of
330 000.4-6 In addition, approximately 20% of
the total human factor V found in whole blood is contained in the
platelet The binding protein multimerin is found in platelets and endothelial
cells and appears to be a carrier for platelet factor V. It contains an
RGDS motif as well as an epidermal growth factor domain.19,20 The multimerin monomers are linked via
interchain disulfide bonds to produce one of the largest proteins found
in platelets with a mass exceeding approximately 1 000 000 Da.
Following platelet activation, both factor V and multimerin are
released and dissociate from one another.19,20
The physiologic significance of factor Va for clot formation is clearly
demonstrated in mice in which complete deficiency of factor V results
in massive hemorrhage and death.21 However, humans who are
deficient in plasma factor V (< 2% factor V clotting activity)
although rare (prevalence of 1 in 1 million) show variable bleeding
tendencies.11,12,22 The physiologic relevance of factor Va
in the pathology of thrombosis is assured because of case reports of
familial thrombophilia associated with factor V mutations and defects
in the protein C pathway.23,24 In this review, we describe
the structure, activation, function, and inactivation of factor V and
discuss pathologic states associated with genetic mutations of the
factor V molecule.
The 80-kb factor V gene is located on chromosome 1 at
q21-2525 and contains 24 introns. Following transcription
it gives rise to a 6.8-kb mRNA.25-28 Synthesis of factor V
has been demonstrated by bovine aortic endothelial
cells,29 by a human hepatocarcinoma cell line
HepG2,30 and by human and guinea pig
megakaryocytes.31 The liver appears to be the principal
source of factor V in plasma and platelets.7
The human factor V molecule (Figure 1A)
is secreted after pre-propeptide processing as a 2196 amino acid
protein. The molecule is composed of triplicated A domains, a B domain,
and duplicated C domains.26-28 The A domains are
homologous to those found in plasma ceruloplasmin and factor VIII, each
of which are members of a copper-binding family.32 The C
domains are homologous to the slime mold protein
discoidin.26 The B domain is poorly conserved among the
various species of factor V that have been studied. The B domain of
human factor V that is ultimately released in the form of 2 heavily
glycosylated fragments of Mr 150 000 and
71 000 contain 44 semiconserved repeats of the form DLSQTT/NLSP. This
highly glycosylated region also contains 2 conserved repeats of 17 amino acids each.26
The factor V molecule undergoes multiple posttranslational alterations,
including sulfation, phosphorylation, and glycosylation (Figure 1A;
Table 1).33-39 Inhibition of
tyrosine sulfation by sodium chlorate results in a cofactor molecule
with one fifth the activity of the native molecule. Thus, sulfation
appears important for factor V/Va function.34,35 Factor Va
is also phosphorylated by a membrane-associated platelet casein kinase
II (CKII) enzyme on the heavy chain at Ser692 and by a
platelet-derived protein kinase C isoform on 2 sites of the light chain
(Figure 1A).36-38 Phosphorylation of the heavy chain of
factor Va at Ser692 increases the rate of inactivation of the cofactor
by activated protein C.38 The platelet CKII responsible
for factor Va phosphorylation is composed of Factor V has 19 cysteine residues (Table 1 and Figure 1A show
locations).41,42 Five of the cysteines are present as free Models of the A domains of the molecule based on the coordinates of ceruloplasmin have been described43-45; however, these hypothetical models of factor Va domains should be only provisionally interpreted until the true crystal structure of factor Va becomes available. The crystal structures of the recombinant C2 domain of the molecule have been reported.46 Bovine factor Va has been crystallized,47 and a preliminary account of the crystal structure of bovine factor Va has been reported.48
Factor V is the inactive precursor of factor Va that contributes
factor Xa receptor and catalytic effector functions to
prothrombinase. The complex formation between factor Xa and
factor Va membrane to form prothrombinase increases the rate at which
prothrombin is converted to Single-chain factor V does not bind factor Xa. Thus, the activation of
factor V to factor Va is essential to its biologic function. During
tissue factor-initiated blood coagulation, thrombin generation can be
divided into 2 phases; an initiation phase during which very small
amounts of thrombin are produced (~ 1%, 5 nM) and clotting is
observed; during a subsequent propagation phase 700- to 900-nM thrombin
is produced.50,55-57 The identity of the initial activator
of factor V has been a subject of controversy. Although factor
Xa-phospholipid, thrombin, and plasmin are all capable of activating
factor V to factor Va, the kinetic evaluation of tissue
factor-initiated blood clotting reactions shows that the initial
activator of factor V is Substrates for thrombin include platelets, fibrinogen, factor XI,
factor V, factor VIII, factor VII, and factor XIII. During the process
of prothrombin activation by prothrombinase, 2 forms of thrombin are
produced, meizothrombin and
Cofactor for factor Xa in prothrombinase The membrane-binding site of the factor V molecule, which is crucial to its function, is contributed by elements of the C2 and A3 domains contained within the light chain of the molecule.67-71 The binding of the light chain to a membrane surface involves both electrostatic and hydrophobic interactions with anionic/neutral membranes and penetration of the molecule into the membrane bilayer.67-72 The hydrophobic binding site of factor Va located on the middle portion of the A3 domain is suggested to interact with phospholipid and penetrate into the bilayer.67-69,72 The binding site of the cofactor located on the C2 domain of factor Va and reported to interact with anionic phospholipid was found to be exposed on the surface of the molecule as expected for a hydrophilic amino acid sequence.70,71,73,74 Two studies using recombinant factor V and the factor V C2 domain have demonstrated that most of factor V autoantibody inhibitors are directed to the C1-C2 domain of factor V and interfere with its binding to phosphatidyl serine-containing membranes.73,74 Kim et al73 using alanine-scanning mutagenesis demonstrated that several amino acid substitutions at the COOH-terminus of the light chain resulted in molecules with impaired binding to phosphatidyl serine.73 Because proper interaction of the cofactor with the membrane surface is required for the expression of factor Va cofactor activity, it must be concluded that these residues located at the carboxyl-terminal portion of factor V are critical for the interaction of the activated molecule with the cell surface. Overall, the data suggest that initial rapid factor Va-membrane interaction is most likely first mediated through the anionic lipid-binding domain on the C2 domain. Following this diffusion rate-limited interaction,75 a hydrophobic interaction, potentially involving the A3 domain of the molecule,67-69 tightly anchors the molecule to the membrane core.72 Binding and penetration of the molecule into the membranes result in a stable, high affinity (Kd ~ 2.5 nM) factor Va-phospholipid interaction.67 The physiologically relevant membranes for blood coagulation are provided by platelets and vascular cells.76,77 The membranes of these cells are lipid-protein complexes composed principally of phosphatidyl serine, phosphatidyl choline, and phosphatidyl ethanolamine.78-80 The selective qualities associated with cell binding sites for prothrombinase remain poorly understood.A fundamental contribution of factor Va to prothrombinase function is
the retention of factor Xa on the membrane surface. Stopped-flow
reaction kinetic data show that both factor Va and factor Xa interact
with phosphatidyl-choline phosphatidyl-serine (PCPS) vesicles at
diffusionally controlled rates (107-108
M The membrane-bound factor Va-factor Xa complex cleaves membrane-bound
prothrombin to produce Cofactor for the APC/protein S complex in the inactivation of factor VIII Shen and Dahlbäck90 reported that the intact pro-cofactor, single chain factor V, can act as a cofactor, contributing to the acceleration of inactivation of factor VIIIa by the activated protein C (APC)/protein S complex. Our laboratory demonstrated that factor V can accelerate the rate of factor VIII inactivation by APC, only when protein S was present.91 Under the conditions used, factor V was completely cleaved by APC early during the inactivation reaction and before 10% to 20% factor VIII activity was lost.91 APC-treated, membrane-bound factor V as well as -thrombin-activated factor Va (inclusive of the B
domain) produced an increase in the rate of inactivation of factor VIII
by the APC/protein S complex by 2-fold. In contrast, purified factor Va
(lacking the B domain) even at high concentrations does not show any
cofactor effect on the rate of the inactivation of factor VIII (± protein S).91 These observations suggest that the B region
fragments of factor V may be responsible for the cofactor effect of
factor V during the inactivation of factor VIII by the APC/protein
S complex.
Activated protein C (APC) down-regulates Protein C is activated to APC by During a one-stage plasma-clotting assay, the end point clot is produced when only approximately 10% of the total factor V present in plasma has been activated, approximately 7 pM of factor Xa is available, and less than 5% of the prothrombin activation process has occurred.53,56 When measured in a clotting assay, most of the activity of factor Va is perceived to be lost by cleavage at Arg506, because cleavage at Arg506 reduces the apparent affinity for factor Xa, and the binding of factor VaR506 to limiting amounts of factor Xa available becomes a major determinant of the activity of the factor Va molecule.105 However, when the reaction is measured at saturating concentrations of factor Xa, the cleavage at Arg506 of factor Va leads to the loss of only 25% to 40% of the factor Va cofactor activity. In contrast, cleavage at Arg306 leads to the ultimate inactivation of factor Va.96,105 Physical studies using ultracentrifugation and light scattering show that the products of cleavage of factor Va by APC at Arg306 and Arg506 dissociate to produce fragments, which correspond to the A1 domain of the molecule, noncovalently associated with the A3C1C2 (light chain), and fragments A2N and A2C that correspond to the amino and carboxyl termini of the A2 domain (Figure 1C, upper part).104 The inactivation rate of factor Va when explored as a function of APC concentration becomes independent of enzyme concentration, a clear signal that the final inactivation step involves not only cleavage but also dissociation of the A2N and A2C fragments from the rest of the molecule.101,104 The human pro-cofactor, factor V, is also inactivated by APC but only when bound to a membrane-surface. Human factor V is inactivated by cleavages at Arg306, Arg506, Arg679, and Lys994. In this instance, cleavage at Arg306 that occurs first appears to be the sole requirement for inactivation.96 In studies of the ability of endothelial cells to complement the
activation of protein C and the inactivation of factor Va, an
APC-independent cleavage was observed that resulted in an inactive product.105,106 In the presence of endothelial cells,
Hemorrhagic pathology: parahemophilia Factor V deficiency is extremely uncommon, occurring either because of a homozygous inheritance or because of a combination of defective alleles. However, humans with factor V deficiency are alive,8,11,12 whereas in mice the total lack of factor V is not consistent with survival.21 In empirical models conducted with purified reaction components and computer models of the blood coagulation system,-thrombin cannot be generated in the
absence of factor V when all inhibitors are present in the reaction
system.101,108,109 Conversely, humans with severe
truncations in the factor V molecule that do not allow any synthesis of
factor V by known conventional mechanisms have been observed to have
either manageable or no pathology associated with bleeding. These
observations suggest that there may be compensatory mechanisms present
in human blood, which either bypass or reduce the need for factor V in
generating levels of -thrombin required for survival.
Point mutations in the factor Va gene resulting in abnormal
factor V function are associated with amino acid substitutions located
on the entire coding region of factor V (Table
2). Deletions of entire portions of the
molecule, nonsense mutations, and mutations of the splice sites for the
introns have also been reported. However, multiple mutations resulting
in conserved substitutions (polymorphisms) also exist and have no
apparent consequence on factor V function. A compendium database on
factor V mutations, most of which result in parahemophilia, was
constructed by Dr Hans L. Vos (Hemostasis and Thrombosis Research
Center, Leiden University Medical Center, Leiden, The Netherlands;
e-mail: h.l.vos{at}lumc.nl) and is available on request.
Several representative mutations are shown in Table 2. Mutations in the
factor V gene result either in low circulating levels of the protein or
in the complete deficiency of factor V if the mutation results in a
stop codon in the middle of the reading frame (Table 2).
Defects in the inactivation of factor V/Va associated with thrombosis APC deficiency. Mature protein C is composed of several conserved domains, each of which is characteristic for the vitamin K-dependent proteins. Human protein C is encoded by an autosomal gene of approximately 10 kb located on chromosome 2q13-14.110,111 Absence of the protein or a defective circulating protein in plasma, which is the result of more that 160 different mutations, has severe consequences for the individuals bearing the mutations.112 Individuals who are heterozygous for a protein C mutation, resulting in low levels of circulating protein C because one of the 2 alleles does not produce the normal protein, variably suffer from venous thrombosis.113 However, homozygous protein C deficiency, characterized by the complete absence of circulating protein C, is a very serious condition with life-threatening thrombotic symptoms. This severe condition usually presents immediately after birth, in the form of purpura fulminans with the only remedy being the infusion of protein C.114-116 Congenital APC resistance. Factor VLEIDEN. In 1993, Dahlbäck et al23 observed that plasma from some individuals with venous thrombosis had a reduced response to APC.23 When APC is introduced into normal plasma, there is a prolongation of clotting time. In plasma from some patients, higher concentrations of APC were required to obtain similar prolongations of clotting time. This condition was called APC resistance. This observation coupled with data that identified the Arg505/Arg506 APC cleavage site in the bovine and human factor Va molecules95,96,117 led to the identification of factor VLEIDEN.118 This polymorphism occurs as a consequence of G1691A in the factor V gene, leading to the substitution of an Arg506Gln of the human factor V molecule.118 Arg506 is the initial cleavage site for APC on the heavy chain of human factor Va and facilitates the subsequent lipid-dependent inactivation cleavage at Arg306.96,101,105 Because this abnormal molecule does not possess the APC-cleavage site at Arg506, factor VaLEIDEN is inactivated by APC at approximately one tenth the rate of normal factor Va.119 However, inactivation still proceeds through cleavage at Arg306.97,119-124 Because elimination of the latent activity of membrane-bound factor V occurs because of an initial cleavage at Arg306, factor VLEIDEN is inactivated by APC at the same rate as normal factor V.119 Platelet-factor Va and factor VaLEIDEN inactivation by APC proceed at similar rates and are delayed when compared with their plasma equivalents.125,126 Thus, on the surface of platelets there is little difference in the inactivation rates of the 2 molecules, both of which are protected from inactivation. Two groups have reported patients with thrombotic disorders that were heterozygous for an amino acid substitution at the Arg306 cleavage site.127,128 As a consequence of the factor VLEIDEN mutation, the partial loss of activity of the normal factor Va molecule that occurs following cleavage at Arg506, resulting in diminished interaction between factor Va and factor Xa, is not observed. The pathology of factor VLEIDEN is associated in vivo with thrombotic manifestations, which are observed in both homozygous and heterozygous individuals.129-133 Venous thrombosis is more common among those individuals homozygous for the Arg506Gln substitution. Statistical analyses suggest that the relative risk of a thrombotic episode in individuals heterozygous for factor VLEIDEN is 7-fold higher that the risk for healthy individuals.134 In contrast, homozygous factor VLEIDEN individuals have an 80-fold higher risk of thrombosis than individuals with the normal factor V gene.133,134 No correlation has been observed between arterial thrombosis and the existence of factor VLEIDEN.135-137 The frequency of factor VLEIDEN in patients with arterial thrombi is approximately 5%, a value similar to the frequency of the mutation present in the normal population. An explanation for this phenomenon is potentially found in the composition of the clot. The composition of a thrombus is dependent on the surface-promoting coagulation and the hemodynamic factors regulating blood circulation. Although venous thrombi are most commonly formed in areas of stasis and are composed of fibrin with few platelets, arterial thrombi are formed in areas of high blood flow and are composed mainly of platelets with less fibrin. It is possible that plasma-derived factor Va and factor VaLEIDEN are the predominant cofactors for prothrombinase assembly on the venous side. In contrast, on the arterial side, the predominant cofactor pool participating in prothrombinase at the site of injury may be platelet factor Va (or platelet factor VaLEIDEN). Because both platelet factor VaLEIDEN and normal factor Va cofactors are equally resistant to inactivation by mutation, APC resistance would not be selectively associated with arterial thrombosis. Many studies have shown that the thrombotic risk in homozygous patients carrying the factor VLEIDEN mutation is influenced by other acquired and congenital risk factors but less dramatic than the risk of thrombosis for patients homozygous for protein C and protein S deficiencies.138-142HR2 haplotype. The factor V allele His1299Arg (also known as R2 haplotype) is marked by A4070G in the factor V gene.143 This mutation (in the B region) is a marker that cosegregates with several other polymorphisms encoding several amino acid changes in the factor V molecule.143-146 It has been reported that individuals carrying the factor V His1299Arg mutation have mild APC resistance and an increase in the risk of venous thrombosis. Thus, this ensemble of polymorphisms represents per se a potential thrombotic risk factor.146,147 We have described a thrombotic family with the R2 haplotype and several symptomatic members.147 One of these individuals was found to be doubly heterozygous for the factor V HR2 haplotype and for the Tyr1702Cys mutation. The latter mutation was shown to be at the origin of factor V deficiency, resulting in the absence of circulating normal factor V.147,148 Thus, because the factor V allele predicting the Tyr1702Cys substitution and encoding for a non-R2 haplotype factor V is not expressed at the protein level, the plasma of this patient contains only molecules encoded by the R2 haplotype.148 This individual had also mild APC resistance and was classified as a pseudohomozygous R2 haplotype. We have found in this individual that the factor V molecule that is characterized by the mutations included in the R2 haplotype is resistant to APC inactivation because of impaired cleavage by APC at both Arg506 and Arg306.148 These findings suggest the possibility that one or more of the amino acid substitutions, which are predicted by the R2 haplotype, impair factor Va cleavage by APC at Arg506/Arg306. The patient studied was also a carrier of the Asp2194Gly substitution that is tightly associated with the R2 haplotype and is the most likely candidate for the observed APC resistance of factor Va. Studies by Kim et al73 using alanine-scanning mutagenesis suggested that several amino acid residues within the COOH-terminal portion of the C2 domain of factor V are crucial for the interaction of the cofactor with phosphatidyl serine.73 Among those, the Asp2194Ala substitution had impaired binding to phosphatidyl serine and defective cofactor activity. However, the factor V molecule from carriers of the R2 haplotype possesses normal procoagulant activity. The R2 haplotype demonstrates resistance to APC because of impaired cleavage at both Arg506 and/or Arg306.148 Thus, it is possible that the factor Va mutations surrounding Asp2194 impair its inactivation by APC. Acquired APC resistance. Activated protein C resistance may also be observed with elevated levels of homocysteine.149,150 Factor V contains 5 unpaired cysteines, 2 in the heavy chain, 2 in the light chain, and 1 in the B region41,42 (Table 1). These cysteines can incorporate homocysteine at physiologically relevant concentrations associated with hyperhomocystinemia and, as a consequence, display a form of acquired APC resistance.151 Influence of factor Va on fibrinolysis.
Regulation of factor V activity also contributes to the function
of the fibrinolytic cascade.152-154 A fibrinolysis
inhibitor named thrombin-activatable fibrinolysis inhibitor (TAFI) is
activated by the -thrombin formation, also results in enhanced activation of TAFI and
impaired removal of the fibrin clot. These data also suggest that the
enhanced activation of prothrombin associated with factor
VLEIDEN may increase the resistance of the fibrin clot to
lysis156,157 in individuals with hemophilia A, producing a
milder hemostatic defect.
TFPI and APC The dynamic process of factor V inactivation by APC is synergistic with the tissue factor pathway inhibitor (TFPI) inhibition during the tissue factor-initiated blood coagulation process.158-162 This inhibition occurs because the-thrombin activation of factor V is a sequential phenomenon with
cleavage at Arg709 occurring first to produce the heavy chain. However,
factor Va activity is associated with cleavage at Arg1545 and formation
of the light chain, which occurs more slowly. Once cleaved at Arg709,
the partially activated membrane-bound factor V molecule can be rapidly
cleaved by APC at Arg306 and Arg506, leading to inactivation. If the
inactivation process is sufficiently vigorous, it can occur prior to
the formation of the light chain of the molecule; hence, factor Va
activity is nullified.162 These regulatory processes
display synergy between the dynamic APC system and the stoichiometric
TFPI inactivation system.162 In vitro experimental data
suggested that a combination of factor VLEIDEN and
low normal levels of TFPI could produce unregulated generation of
thrombin because of the absence of the appropriate synergy between 2 dynamic regulatory processes.162,163 Transgenic mice obtained by crossing mice with reduced levels of TFPI with mice homozygous for factor VLEIDEN resulted in mice with a
lethal phenotype with fibrin deposition in multiple
organs.164 The observation of disseminated thrombosis in
mice homozygous for the factor VLEIDEN mutation and low
levels of TFPI is in keeping with the hypothesis suggested by in vitro
data.163 Thus, reduced TFPI levels may be a contributor to
thrombosis in association with factor VLEIDEN.
Hemophilia and factor VLEIDEN The importance of the timely regulation of factor Va cofactor activity is illustrated by a potentially beneficial effect of factor VLEIDEN for individuals with severe hemophilia A. The net effect of factor VIII deficiency is reduced-thrombin
formation rate; conversely, individuals with the factor
VLEIDEN mutation have sustained -thrombin
formation. Significant differences in pathology between
unrelated patients carrying the same factor VIII missense mutations
have been reported.165 Although these factor VIII
mutations most commonly result in a phenotype characterized by severe
bleeding in individuals carrying the normal factor V gene, 2 patients
with hemophilia heterozygous for the factor VLEIDEN
mutation have been reported who presented with mild/moderate bleeding episodes. More recently, an individual with severe hemophilia B (< 1% factor IX activity) but heterozygous for the factor
VLEIDEN mutation presented with only a mild bleeding
diathesis.166 Because factor VaLEIDEN is
inactivated by APC more slowly, a hemophiliac patient with the factor
VLEIDEN gene may have a milder bleeding syndrome because of
the increased durability of prothrombin activation.167
These data suggest that the variability in the expression of single
defects associated with hemophilia A and B may in part be because of
the high frequency of the factor VLEIDEN mutation in the
normal population and that hemophiliac patients should also be
regularly screened for the Arg506Gln mutation.
Parahemophilia and factor VLEIDEN A pseudohomozygous APC resistance, predisposing to thrombosis, may occur as a consequence of the combination of 2 defects in factor V that are associated with a quantitative reduction in factor V and heterozygous inheritance of the factor VLEIDEN mutation. The main laboratory findings are the presence of a low APC sensitivity ratio (APC-SR) and low plasma values of factor V antigen and activity.168-171 In this case the normal factor V allele is silent, and only the factor VLEIDEN allele is expressed. As a consequence, the patient who is genetically heterozygous for factor VLEIDEN is phenotypically homozygous for the mutation.171
Overall, factor V can be thought of as a Dr Jekyll and Mr Hyde. Dr
Jekyll, the good doctor, provides a useful support for individuals
(hemostasis) and Mr Hyde, the rapacious monster, causes destruction
(thrombosis). Four stages of the participation of the factor V molecule
in the hemostatic process are illustrated in Figure
2 in cartoon form. The factor V molecule
is presented with the activation peptide "B domain" illustrated as
a loop structure which connects the heavy and light chain
(A2-A3) domains of the factor Va molecule. The
single chain factor V interacts with membranes through its carboxyl
terminal region. Factor V is activated to Factor Va by thrombin (IIa)
which excises the B domain leaving the noncovalently-associated light
and heavy chains of the factor Va molecule. The membrane bound factor
Va molecule binds factor Xa through regions of both the light and heavy
chains. These interactions together with factor Xa membrane binding
provide the tightly associated enzymatic complex (prothrombinase) which
converts prothrombin (II) to thrombin (IIa). APC binds competitively
with factor Xa to factor Va, primarily through light chain
interactions. APC cleaves at 3 sites of the heavy chain of the factor
Va molecule resulting in the dissociation of the A2 domain
as 2 fragments, A2N and A2C. The resulting
product (factor Vai) composed of the A1 domain
noncovalently associated with the membrane-bound light chain binds APC
but will no longer function efficiently in generating thrombin. We need
factor V to have a normal blood clotting physiology; however, the
unmanaged evolution of factor V activation can cause severe pathology.
However, sometimes Mr Hyde can abrogate the damage caused by another
monster (hemophilia A or B), providing overall a beneficial support to
the individual.
Submitted February 14, 2002; accepted July 29, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-01-0290.
Supported by Merit Award R37 HL34575 from the National Institutes of Health (K.G.M.) and by Established Investigator Award 0040100N from the American Heart Association (M.K.).
Reprints: Kenneth G. Mann, Department of Biochemistry, Given Building, Health Science Complex, University of Vermont, College of Medicine, Burlington, VT 05405; e-mail: kmann{at}zoo.uvm.edu.
1. Owren PA. Parahaemophila: haemorrhagic diathesis due to absence of a previously unknown clotting factor. Lancet. 1947;1993:446-448. 2. Owren PA, Cooper T. Parahemophilia. Arch Intern Med. 1955;95:194-201[Medline] [Order article via Infotrieve]. 3. Mann KG, Nesheim ME, Tracy PB. Molecular weight of undegraded plasma factor V. Biochemistry. 1981;20:28-33[CrossRef][Medline] [Order article via Infotrieve].
4.
Nesheim ME, Foster WB, Hewick R, Mann KG.
Characterization of factor V activation intermediates.
J Biol Chem.
1984;259:3187-3196
5.
Suzuki K, Dahlbäck B, Stenflo J.
Thrombin-catalyzed activation of human coagulation factor V.
J Biol Chem.
1982;257:6556-6564
6.
Kane WH, Majerus PW.
Purification and characterization of human coagulation factor V.
J Biol Chem.
1981;256:1002-1007
7.
Tracy PB, Eide LL, Bowie EJW, Mann KG.
Radioimmunoassay of factor V in human plasma and platelets.
Blood.
1982;60:59-63 8. Tracy PB, Giles AR, Mann KG, Eide LL, Hoogendoorn H, Rivard GE. Factor V (Quebec): a bleeding diathesis associated with a qualitative platelet factor V deficiency. J Clin Invest. 1984;74:1221-1228[Medline] [Order article via Infotrieve].
9.
Janeway CM, Rivard GE, Tracy PB, Mann KG.
Factor V Quebec revisited.
Blood.
1996;87:3571-3578
10.
Hayward CP, Cramer EM, Kane WH, et al.
Studies of a second family with the Quebec platelet disorder: evidence that the degradation of the alpha-granule membrane and its soluble contents are not secondary to a defect in targeting proteins to alpha-granules.
Blood.
1997;89:1243-1253 11. Montefuso MC, Duga S, Asselta R, et al. A novel two base pair deletion in the factor V gene associated with severe factor V deficiency. Br J Hematol. 2000;111:1240-1246[CrossRef][Medline] [Order article via Infotrieve]. 12. Guash JF, Cannegieter S, Reitsma PH, van't Veer-Korthof ET, Bertina RM. Severe coagulation factor V deficiency caused by a 4 bp deletion in the factor V gene. Br J Hematol. 1998;101:32-39[CrossRef][Medline] [Order article via Infotrieve]. 13. Nesheim ME, Nichols WL, Cole TL, et al. Isolation and study of an acquired inhibitor of human coagulation factor V. J Clin Invest. 1986;77:405-415[Medline] [Order article via Infotrieve].
14.
Camire RM, Pollak ES, Kaushansky K, Tracy PB.
Secretable human platelet-derived factor V originates from the plasma pool.
Blood.
1998;92:3035-3041
15.
Viskup RW, Tracy PB, Mann KG.
The isolation of human platelet factor V.
Blood.
1987;69:1188-1195
16.
Monkovic DD, Tracy PB.
Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin.
J Biol Chem.
1990;265:17132-17140
17.
Hayward CP, Warketin TE, Horsewood P, Kelton JG.
Multimerin: a series of large disulfide-linked multimeric proteins within platelets.
Blood.
1991;77:2556-2560 18. Hayward CP, Bainton DF, Smith JW, et al. Multimerin is found in the alpha-granules of resting platelets and is synthesized by a megakaryocytic cell line. J Clin Invest. 1993;91:2630-2639[Medline] [Order article via Infotrieve].
19.
Hayward CP, Hassel JA, Denomme GA, Rachubinski RA, Brown C, Kelton JG.
The cDNA sequence of human endothelial cell multimerin: a unique protein with RGDS, coiled-coil, and epidermal growth factor-like domains and a carboxyl terminus similar to the globular domain of complement C1q and collages type VIII and X.
J Biol Chem.
1995;270:18246-18251
20.
Hayward CP, Furmaniak-Kazmierczack E, Cieutat AM, et al.
Factor V is complexed with multimerin in resting platelet lysates and colocalizes with multimerin in platelet alpha-granules.
J Biol Chem.
1995;270:19217-19224 21. Cui J, O'Shea KS, Purkayastha A, Saunders TL, Ginsburg D. Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature. 1996;384:66-68[CrossRef][Medline] [Order article via Infotrieve].
22.
Murray JM, Rand MD, Egan JO, Murphy S, Kim HC, Mann KG.
Factor VNew Brunswick: Ala221-to-Val substitution results in reduced cofactor activity.
Blood.
1995;86:1820-1827
23.
Dahlbäck B, Carlsson M, Svensson PJ.
Familial thrombophilia due to a previously unrecognized mechanism by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.
Proc Natl Acad Sci U S A.
1993;90:1004-1008
24.
Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM.
The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects.
Blood.
1991;78:890-894 25. Cripe LD, Moore KD, Kane WH. Structure of the gene for human coagulation factor V. Biochemistry. 1992;31:3777-3785[CrossRef][Medline] [Order article via Infotrieve].
26.
Jenny RJ, Pittman DD, Toole JJ, et al.
Complete cDNA and derived amino acid sequence of human factor V.
Proc Natl Acad Sci U S A.
1987;84:4846-4850 27. Kane WH, Ichinose A, Hagen FS, Davie EW. Cloning of cDNAs coding for the heavy chain region and connecting region of human Factor V, a blood coagulation factor with four types of internal repeats. Biochemistry. 1987;26:6508-6514[CrossRef][Medline] [Order article via Infotrieve].
28.
Kane WH, Davie EW.
Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders.
Blood.
1988;71:539-555
29.
Cerveny TJ, Fass DN, Mann KG.
Synthesis of coagulation Factor V by cultured aortic endothelium.
Blood.
1984;63:1467-1474 30. Wilson DB, Salem HH, Mruk JS, Maruyama I, Majerus PW. Biosynthesis of coagulation factor V by a human hepatocellular carcinoma cell line. J Clin Invest. 1984;73:654-658[Medline] [Order article via Infotrieve]. 31. Chiu HC, Schick P, Colman R. Biosynthesis of factor V in isolated guinea pig megakaryocytes. J Clin Invest. 1985;75:339-346[Medline] [Order article via Infotrieve].
32.
Mann KG, Lawler CM, Vehar GA, Church WR.
Coagulation factor V contains copper ions.
J Biol Chem.
1984;259:12949-12951 33. Hortin GL. Sulfation of tyrosine residues in coagulation factor V. Blood. 1993;76:946-952. 34. Pittman DD, Tomkinson KN, Michnick D, Selighsohn U, Kaufman RJ. Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity. Biochemistry. 1994;33:6952-6959[CrossRef][Medline] [Order article via Infotrieve].
35.
Pittman DD, Tomkinson KN, Kaufman RJ.
Post-translational requirement for functional factor V and factor VIII secretion in mammalian cells.
J Biol Chem.
1994;269:17329-17337
36.
Kalafatis M, Rand MD, Jenny RJ, Ehrlich YH, Mann KG.
Phosphorylation of factor Va and factor VIIIa by activated platelets.
Blood.
1993;81:704-719
37.
Rand MD, Kalafatis M, Mann KG.
Platelet coagulation factor Va: the major secretory platelet phosphoprotein.
Blood.
1994;83:2180-2190
38.
Kalafatis M.
Identification and partial characterization of factor Va heavy chain-kinase from human platelets.
J Biol Chem.
1998;273:8459-8466 39. Kumar HPM, Besman MJ, Lundblad RL, Jenny NS, Mann KG. Carbohydrate analysis of plasma factor V and factor VIII. Thromb Haemost [abstract]. 1999;Suppl 82:102a.
40.
Singh LS, Kalafatis M.
Sequencing of full-length cDNA encoding the 41. Xue J, Kalafatis M, Mann KG. Determination of the disulfide bridges in factor Va light chain. Biochemistry. 1993;32:5917-5923[CrossRef][Medline] [Order article via Infotrieve]. 42. Xue J, Kalafatis M, Silveira JR, Kung C, Mann KG. Determination of the disulfide bridges in factor Va heavy chain. Biochemistry. 1994;33:13109-13116[CrossRef][Medline] [Order article via Infotrieve]. 43. Villoutreix BO, Dahlback B. Structural investigation of the A domains of human blood coagulation factor V by molecular modeling. Protein Sci. 1998;7:1317-1325[Medline] [Order article via Infotrieve]. 44. Pellequer JL, Gale AJ, Getzoff ED, Griffin JH. Three-dimensional model of coagulation factor Va bound to activated protein C. Thromb Haemost. 2000;84:849-857[Medline] [Order article via Infotrieve]. 45. Zaitseva I, Zaitsev V, Card G, et al. The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centers. J Biol Inorg Chem. 1996;1:15-23[CrossRef]. 46. Macedo-Ribeiro S, Bode W, Huber R, et al. Crystal structures of the membrane-binding C2 domain of human coagulation factor V. Nature. 1999;402:434-439[CrossRef][Medline] [Order article via Infotrieve]. 47. Hockin MF. Regulation of the cofactor activity of factor Va: effects of proteolytic cleavage on intramolecular association, intermolecular complex assembly and cofactor function [doctoral thesis]. Burlington, VT: University of Vermont, Department of Biochemistry; 2000. 48. Everse SJ, Adams TE. Crystal structure of bovine factor Va. Abstracts Junior Faculty Scholar 43rd Annual Meeting of the American Society of Hematology. Washington, DC: American Society of Hematology; 2001:66.
49.
Nesheim ME, Taswell JB, Mann KG.
The contribution of bovine factor V and factor Va to the activity of prothrombinase.
J Biol Chem.
1979;254:10952-10962
50.
Hockin MF, Jones KC, Everse SJ, Mann KG.
A model for the stoichiometric regulation of blood coagulation.
J Biol Chem.
2002;277:18322-18333
51.
Butenas S, Brummel KE, Branda RF, Paradis SG, Mann KG.
Mechanism of factor VIIa-dependent coagulation in hemophilia blood.
Blood.
2002;99:923-930
52.
Butenas S, van't Veer C, Mann KG.
Evaluation of the initiation phase of blood coagulation using ultra sensitive assays for serine proteases.
J Biol Chem.
1997;272:21527-21533
53.
Brummel KE, Paradis SG, Butenas S, Mann KG.
Thrombin functions during tissue factor-induced blood coagulation.
Blood.
2002;100:148-152 54. Krishnaswamy S, Russel GD, Mann KG. The reassociation of factor Va from its isolated subunits. J Clin Invest. 1989;264:3160-3168.
55.
Lawson JH, Kalafatis M, Stram S, Mann KG.
A model for the tissue factor pathway to thrombin, I: an empirical study.
J Biol Chem.
1994;269:23357-23366
56.
Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG.
Blood clotting in minimally altered blood.
Blood.
1996;88:3432-3445
57.
van't Veer C, Mann KG.
Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II.
J Biol Chem.
1997;272:4367-4377
58.
Lee CD, Mann KG.
Activation/inactivation of human factor V by plasmin.
Blood.
1989;73:185-190
59.
Omar MN, Mann KG.
Inactivation of factor Va by plasmin.
J Biol Chem.
1987;262:9750-9755
60.
Kalafatis M, Mann KG.
The role of the membrane in the inactivation of factor Va by plasmin: amino acid region 307-348 of factor V plays a critical role for factor Va cofactor function.
J Biol Chem.
2001;276:18614-18623
61.
Zeibdawi AR, Pryzdial EL.
Mechanism of factor Va inactivation by plasmin: loss of A2 and A3 domains from a Ca2+-dependent complex of fragments bound to phospholipid.
J Biol Chem.
2001;276:19929-19936
62.
Krishnaswamy S, Church WR, Nesheim ME, Mann KG.
Activation of human prothrombin by human prothrombinase: influence of factor Va on the reaction mechanism.
J Biol Chem.
1987;262:3291-3299
63.
Nesheim ME, Mann KG.
The kinetics and cofactor dependence of the two cleavages involved in prothrombin activation.
J Biol Chem.
1983;258:5386-5391
64.
Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF, Hemker HC.
The role of phospholipids and factor Va in the prothrombinase complex.
J Biol Chem.
1980;255:274-283
65.
Rosing J, Zwaal RF, Tans G.
Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation.
J Biol Chem.
1986;261:4224-4228
66.
Doyle MF, Mann KG.
Multiple active forms of thrombin.
J Biol Chem.
1990;265:10693-10701
67.
Krishnaswamy S, Mann KG.
The binding of factor Va to phospholipid vesicles.
J Biol Chem.
1988;263:5714-5723
68.
Kalafatis M, Jenny RJ, Mann KG.
Identification and characterization of a phospholipid-binding site of bovine factor Va.
J Biol Chem.
1990;265:21580-21589 69. Kalafatis M, Rand MD, Mann KG. Factor Va-membrane interaction is mediated by two regions located on the light chain of the cofactor. Biochemistry. 1994;33:486-493[CrossRef][Medline] [Order article via Infotrieve].
70.
Ortel TL, Quin-Allen MA, Keller FG, Peterson JA, Larocca D, Kane WH.
Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras.
J Biol Chem.
1994;269:15898-15905 71. Ortel TL, Quinn-Allen MA, Charles LA, Devore-Carter D, Kane WH. Characterization of an acquired inhibitor to coagulation factor V: antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity. J Clin Invest. 1992;90:2340-2347[Medline] [Order article via Infotrieve].
72.
Lecompte MF, Bouix G, Mann KG.
Electrostatic and hydrophobic interactions are involved in factor Va binding to membranes containing acidic phospholipid.
J Biol Chem.
1994;269:1905-1910 73. Kim SW, Quin-Allen MA, Camp JT, et al. Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis. Biochemistry. 2000;39:1951-1958[CrossRef][Medline] [Order article via Infotrieve]. 74. Izumi T, Kim SW, Greist A, et al. Fine mapping of inhibitory anti-factor V antibodies using factor V C2 domain mutants: identification of two antigenic epitopes involved in phospholipid binding. Thromb Haemost. 2001;85:1048-1054[Medline] [Order article via Infotrieve].
75.
Krishnaswamy S, Jones KC, Mann KG.
Prothrombinase complex assembly: kinetic mechanism of enzyme assembly on phospholipid vesicles.
J Biol Chem.
1988;263:3823-3834 76. Bevers EM, Comfurius P, Zwaal RFA. Changes in membrane phospholipid distribution during platelet activation. Biochem Biophys Acta. 1983;736:57-66[Medline] [Order article via Infotrieve]. 77. Bevers EM, Comfurius P, VanRijn JLML, Hemker HC, Zwaal RFA. Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem. 1982;122:429-436[Medline] [Order article via Infotrieve].
78.
Toti F, Satta N, Fressinaud E, Meyer D, Freyssinet JM.
Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications, is an inherited disorder.
Blood.
1996;87:1409-1415
79.
Tracy PB, Mann KG.
Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va.
Proc Natl Acad Sci U S A.
1983;80:2380-2384 80. Smirnov MD, Ford DA, Esmon CT, Esmon NL. The effect of membrane composition on the hemostatic balance. Biochemistry. 1999;38:3591-3598[CrossRef][Medline] [Order article via Infotrieve].
81.
Krishnaswamy S, Mann KG, Nesheim ME.
The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.
J Biol Chem.
1986;261:8977-8984
82.
Krishnaswamy S.
Prothrombinase complex assembly: contributions of protein-protein and protein-membrane interactions toward complex formation.
J Biol Chem.
1990;265:3708-3718 83. Tucker MM, Nesheim ME, Mann KG. Differentiation of enzyme and substrate binding in the prothrombinase complex. Biochemistry. 1983;22:4540-4546[CrossRef][Medline] [Order article via Infotrieve].
84.
Kung C, Hayes E, Mann KG.
A membrane-mediated catalytic event in prothrombin activation.
J Biol Chem.
1994;269:25838-25848
85.
Pryzdial ELG, Mann KG.
The association of coagulation factor Xa and factor Va.
J Biol Chem.
1991;266:8969-8977 86. Kalafatis M, Xue J, Lawler CM, Mann KG. Contribution of the heavy and light chains of factor Va to the interaction with factor Xa. Biochemistry. 1994;33:6538-6545[CrossRef][Medline] [Order article via Infotrieve]. 87. Doyle MF, Haley PE. Meizothrombin: active intermediate formed during prothrombinase-catalyzed activation of prothrombin. Methods Enzymol. 1993;222:299-312[Medline] [Order article via Infotrieve].
88.
Bosckovic DS, Bajzar LS, Nesheim ME.
Channeling during prothrombin activation.
J Biol Chem.
2001;276:28686-28693 89. Wu JR, Zhouu C, Majumder R, Powers DD, Weinreb G, Lentz BR. Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor Xa in the absence of factor Va and in the absence and presence of membranes. Biochemistry. 2002;41:935-949[CrossRef][Medline] [Order article via Infotrieve].
90.
Shen L, Dahlbäck B.
Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa.
J Biol Chem.
1994;269:18735-18738
91.
Lu D, Kalafatis M, Mann KG, Long GL.
Comparison of activated protein C/protein S-mediated activation of human factor VIII and factor V.
Blood.
1996;87:4708-4717
92.
Esmon CT.
The regulation of natural anticoagulant pathways.
Science.
1987;235:1348-1352
93.
Lollar P, Parker C.
pH-dependent denaturation of thrombin-activated porcine factor VIII.
J Biol Chem.
1990;265:1688-1692
94.
Fay PJ, Haidaris PJ, Smudzin TM.
Human factor VIIIa subunit structure: reconstitution of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.
J Biol Chem.
1991;266:8957-8962
95.
Kalafatis M, Mann KG.
Role of the membrane in the inactivation of factor Va by activated protein C.
J Biol Chem.
1993;268:27246-27257
96.
Kalafatis M, Rand MD, Mann KG.
The mechanism of inactivation of human factor V and human factor Va by activated protein C.
J Biol Chem.
1994;269:31869-31880
97.
Nicolaes GAF, Tans G, Thomassen MCLGD, et al.
Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.
J Biol Chem.
1995;270:21158-21166
98.
Owen WG, Esmon CT.
Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.
J Biol Chem.
1981;256:5532-5535 99. Suzuki K, Kusumoto H, Deyashiki Y, et al. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J. 1987;6:1891-1897[Medline] [Order article via Infotrieve].
100.
Krishnaswamy S, Williams EB, Mann KG.
The binding of activated protein C to factor V and Va.
J Biol Chem.
1986;261:9684-9693 101. Hockin MF, Cawthern KM, Kalafatis M, Mann KG. A model describing the inactivation of factor Va by activated protein C (APC): bond cleavage, fragment dissociation and product inhibition. Biochemistry. 1999;38:6918-6934[CrossRef][Medline] [Order article via Infotrieve].
102.
Solymoss S, Tucker MM, Tracy PB.
Kinetics of inactivation of membrane-bound factor Va by activated protein C: protein S modulates factor Xa protection.
J Biol Chem.
1988;263:14884-14890
103.
Nesheim ME, Canfield WM, Kisiel W, Mann KG.
Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C.
J Biol Chem.
1982;257:1443-1447
104.
Mann KG, Hockin MF, Begin KJ, Kalafatis M.
Activated protein C cleavage of factor Va leads to dissociation of the A2 domain.
J Biol Chem.
1997;272:20678-20683
105.
Hockin MF, Kalafatis M, Shatos MA, Mann KG.
Protein C activation and factor Va inactivation on human umbilical vein endothelial cells.
Arterioscler Thromb Vasc Biol.
1997;17:2765-2775 106. Hockin MF, Kalafatis M, Cawthern KM, Simoni P, Mann KG. A novel cellular mechanism for factor Va inactivation. 40th Annual Meeting of the American Society of Hematology. Blood. 1998;92(suppl 1):739a.
107.
Undas A, Brummel K, Musial J, Mann KG, Szczeklik A.
PI(A2) polymorphism of beta(3) integrins is associated with enhanced thrombin generation and impaired antithrombotic action of aspirin at the site of microvascular injury.
Circulation.
2001;104:2666-2672 108. Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost. 1999;82:165-174[Medline] [Order article via Infotrieve].
109.
Butenas S, van't Veer C, Mann KG.
"Normal" thrombin generation.
Blood.
1999;94:2169-2178
110.
Foster DC, Yoshitake S, Davie EW.
The nucleotide sequence of the gene for human protein C.
Proc Natl Acad Sci U S A.
1985;82:4673-4677 111. Patracchini P, Aiello V, Palazzi P, Clzolari E, Bernardi F. Sublocalization of the human protein C gene on chromosome 2q13-q14. Hum Genet. 1989;81:191-192[CrossRef][Medline] [Order article via Infotrieve]. 112. Reitsma PH. Protein C deficiency: from gene defects to disease. Thromb Haemost. 1997;78:344-350[Medline] [Order article via Infotrieve]. 113. Simioni P, Kalafatis M, Tormene D, et al. Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (Protein CR-1L/propeptide). Thromb Haemost. 2001;86:1017-1022[Medline] [Order article via Infotrieve]. 114. Branson HE, Katz J, Marble R, Griffin JH. Inherited protein C deficiency and coumarin-responsive chronic relapsing purpura fulminans in a newborn infant. Lancet. 1983;19:1165-1168. 115. Estelles A, Garcia Plaza I, Dasi A, et al. Severe inherited "homozygous" protein C deficiency in a newborn infant. Thromb Haemost. 1984;52:53-56[Medline] [Order article via Infotrieve]. 116. Seligsohn U, Berger A, Abend M, et al. Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med. 1984;310:559-562[Abstract].
117.
Odegaard B, Mann KG.
Proteolysis of factor Va by factor Xa and activated protein C.
J Biol Chem.
1987;262:11233-11238 118. Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369:64-67[CrossRef][Medline] [Order article via Infotrieve].
119.
Kalafatis M, Bertina RM, Rand MD, Mann KG.
Characterization of the molecular defect in factor VR506Q.
J Biol Chem.
1995;270:4053-4057
120.
Kalafatis M, Haley PE, Lu D, Bertina RM, Long GL, Mann KG.
Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay.
Blood.
1996;87:4695-4707
121.
Rosing J, Hoekema L, Nicolaes GAF, et al.
Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C.
J Biol Chem.
1995;270:27852-27858
122.
Heeb MJ, Kojima Y, Greengard JS, Griffin JH.
Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V.
Blood.
1995;85:3405-3411 123. Aparicio C, Dahlbäck B. Molecular mechanisms of activated protein C resistance. Biochem J. 1996;313:467-472[Medline] [Order article via Infotrieve].
124.
Egan JO, Kalafatis M, Mann KG.
The effect of Arg306
125.
Camire RM, Kalafatis M, Cushman M, Tracy RP, Mann KG, Tracy PB.
The mechanism of inactivation of platelet-derived factor Va from normal and APC-resistant individuals by activated protein C.
J Biol Chem.
1995;270:20794-20800
126.
Camire RM, Kalafatis M, Simioni P, Girolami A, Tracy PB.
Platelet-derived factor Va/VaLeiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.
Blood.
1998;91:2818-2829
127.
Williamson D, Brown K, Luddington R, Baglin C, Baglin T.
Factor V Cambridge: a new mutation (Arg306
128.
Chan WP, Lee CK, Kwong YL, Lam CK, Liang R.
A novel mutation of Arg306 of factor V gene in Hong Kong Chinese.
Blood.
1998;91:1135-1139 129. Koster T, Rosendaal FR, de Ronde H, Brie TE, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study. Lancet. 1993;342:1503-1506[CrossRef][Medline] [Order article via Infotrieve].
130.
Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B.
Activated protein C resistance caused by Arg506 131. Voorberg J, Roelse J, Koopman R, et al. Association of idiopathic venous thromboembolism with single point mutation at Arg506 of factor V. Lancet. 1994;343:1535-1536[CrossRef][Medline] [Order article via Infotrieve].
132.
Sun X, Evatt B, Griffin JH.
Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia.
Blood.
1994;83:3120-3125 133. Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet. 1994;343:1536-1538[CrossRef][Medline] [Order article via Infotrieve].
134.
Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH.
High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance).
Blood.
1995;85:1504-1508
135.
Tripodi A, Mannucci PM.
Laboratory investigation of thrombophilia.
Clin Chem.
2001;47:1597-1606 136. Lillicrap D. The genetics of venous and arterial thromboembolism. Curr Atheroscler Rep. 2001;3:209-215[Medline] [Order article via Infotrieve]. 137. Segel GB, Francis CW. Anticoagulant proteins in childhood venous and arterial thrombosis: a review. Blood Cells Mol Dis. 2000;26:540-560[CrossRef][Medline] [Order article via Infotrieve]. 138. Gladson CL, Scharrer I, Hach V, Beck KH, Griffin JH. The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis. Thromb Haemost. 1988;59:18-22[Medline] [Order article via Infotrieve].
139.
Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM.
The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects.
Blood.
1991;78:890-894 140. Allaart CF, Poort SR, Rosendaal FR, Reitsma PH, Bertina RM, Briet E. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet. 1993;341:134-138[CrossRef][Medline] [Order article via Infotrieve].
141.
Bertina RM.
Protein C deficiency and venous thrombosis 142. Ozbek N, Tokel NK, Kayiran SM. Inherited combined deficiency of protein C and S. Eur J Haematol. 1999;63:138-139[Medline] [Order article via Infotrieve].
143.
Bernardi F, Faioni EM, Castoldi E, et al.
A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.
Blood.
1997;90:1552-1557 144. Lunghi B, Iacoviello L, Gemmati D, et al. Detection of new polymorphic markers in the factor V gene: association with factor V levels in plasma. Thromb Haemost. 1996;75:45-48[Medline] [Order article via Infotrieve]. 145. Castoldi E, Rosing J, Girelli D, et al. Mutations in the R2FV gene affect the ratio between the two isoforms in plasma. Thromb Haemost. 2000;83:362-365[Medline] [Order article via Infotrieve]. 146. Alhenc-Gelas M, Nicaud V, Gandrille S, et al. The factor V gene A4070G mutation and the risk of venous thrombosis. Thromb Haemost. 1999;81:193-197[Medline] [Order article via Infotrieve].
147.
Castoldi E, Simioni P, Kalafatis M, et al.
Combinations of four mutations (FVR506Q, FV H1299R, FVY1702C, PT20210G/A) affecting the prothrombinase complex in a thrombophilic family.
Blood.
2000;96:1443-1448
148.
Kalafatis M, Simioni P, Bernardi F.
Phenotype and genotype expression in pseudohomozygous R2 factor V.
Blood.
2001;98:1988-1989 149. Gemmati D, Serino ML, Moratelli S, Mari R, Ballerini G, Scapoli GL. Coexistence of antithrombin deficiency, factor V Leiden and hyperhomocystinemia in a thrombotic family. Blood Coagul Fibrinolysis. 1998;9:173-176[Medline] [Order article via Infotrieve].
150.
Kluijtmans LA, Boers GH, Verbruggen B, Trijbels FJ, Novakova IR, Blomm HJ.
Homozygous cystathionine beta-synthase deficiency, combined with factor V Leiden or thermolabile methylenetetrahydrofolate reductase in the risk of venous thrombosis.
Blood.
1998;91:2015-2018
151.
Undas A, Williams EB, Butenas S, Orfeo T, Mann KG.
Homocysteine inhibits inactivation of factor Va by activated protein C.
J Biol Chem.
2001;276:4389-4397 152. Nesheim ME, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Hemost. 1997;78:386-391[Medline] [Order article via Infotrieve].
153.
Bajzar L, Nesheim ME.
The effect of activated protein C on fibrinolysis in cell-free plasma can be attributed specifically to attenuation of prothrombin activation.
J Biol Chem.
1993;268:8608-8616
154.
Bajzar L, Manuel R, Nesheim ME.
Purification and characterization of TAFI, a thrombin-activatable fibrinolysis inhibitor.
J Biol Chem.
1995;270:14477-14484
155.
Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME.
A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activatable fibrinolysis inhibitor.
J Biol Chem.
1998;273:27176-27181
156.
Bajzar L, Kalafatis M, Simioni P, Tracy PB.
An antifibrinolytic mechanism describing the prothrombotic effect associated with factor VLEIDEN.
J Biol Chem.
1996;271:22949-22952
157.
Broze GJ Jr, Higuchi D.
Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma.
Blood.
1996;88:3815-3823 158. Broze GJ Jr, Lange GW, Duffin KL, MacPhail L. Heterogeneity of plasma tissue factor pathway inhibitor. Blood Coagul Fibrinolysis. 1994;5:551-559[Medline] [Order article via Infotrieve]. 159. Broze GJ Jr. Tissue factor pathway inhibitor and the revised theory of coagulation. Ann Rev Med. 1995;46:103-112[CrossRef][Medline] [Order article via Infotrieve].
160.
Huang Z, Wun T, Broze GJ Jr.
Kinetics of factor Xa inhibition by tissue factor pathway inhibitor.
J Biol Chem.
1993;268:26950-26955
161.
Novotny WF, Girard TJ, Miletich JP, Broze GJ Jr.
Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma.
J Biol Chem.
1989;264:18832-18837
162.
van't Veer C, Golden NJ, Kalafatis M, Mann KG.
Inhibitory mechanism of the protein C pathway on tissue factor induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor.
J Biol Chem.
1997;272:7983-7994
163.
van't Veer C, Kalafatis M, Bertina RM, Simioni P, Mann KG.
Increased tissue factor-initiated prothrombin activation as a result of the Arg506
164.
Eitzman DT, Westrick RJ, Xiaming Bi, et al.
Lethal perinatal thrombosis in mice resulting from the interaction of tissue factor pathway inhibitor deficiency and factor V Leiden.
Circulation.
2002;105:2139-2142
165.
Nichols WC, Amano K, Cacheris PM, et al.
Moderation of hemophilia A phenotype by the factor VR506Q mutation.
Blood.
1996;88:1183-1187
166.
Vianello F, Belvini D, Dal Bello F, et al.
Mild bleeding diathesis in a boy with combined severe haemophilia B (C10400
167.
van't Veer C, Golden NJ, Kalafatis M, Simioni P, Bertina RM, Mann KG.
An in vitro analysis of the combination of factor VLeiden and hemophilia A.
Blood.
1997;90:3067-3072 168. Simioni P, Scudeller A, Radossi P, et al. "Pseudo homozygous" activated protein C resistance due to double heterozygous factor V defects (factor V Leiden mutation and type I quantitative factor V defect) associated with thrombosis: report of two cases belonging to two unrelated kindreds. Thromb Haemost. 1996;75:422-426[Medline] [Order article via Infotrieve]. 169. Zehnder JL, Jain M. Recurrent thrombosis due to compound heterozygosity for factor V Leiden and factor V deficiency. Blood Coagul Fibrinolysis. 1996;7:361-362[Medline] [Order article via Infotrieve]. 170. Guasch JF, Lensen RPM, Bertina RM. Molecular characterization of a type I quantitative factor V deficiency in a thrombosis patient that is pseudo homozygous for activated protein C resistance. Thromb Haemost. 1997;77:252-257[Medline] [Order article via Infotrieve].
171.
Kalafatis M, Bernardi F, Simioni P, Lunghi B, Girolami A, Mann KG.
Phenotype and genotype expression in pseudohomozygous factor VLEIDEN. The need for phenotype analysis.
Arterioscler Thromb Vasc Biol.
1999;19:336-342   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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Top
Introduction
Background
subunits. The
CKII
SH, whereas the remaining 14 are involved in disulfide bridges forming several loops: three 26 amino acid residue 
-loops that
are composed of 154 and 155 residues, that represent the C1 and C2
domains (Figure 
2.
Controversy has existed with respect to whether 
1 sec
Ala and Arg506
the search for the second genetic defect.
Thromb Haemost.
2000;83:360-361
