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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:620-627.)
© 1997 American Heart Association, Inc.

Articles

Factor V^Leiden and Thrombophilia

Michael Kalafatis; ; Kenneth G. Mann

From the Department of Biochemistry, University of Vermont College of Medicine, Burlington.

Correspondence to Kenneth G. Mann, PhD, Department of Biochemistry, Given Building, Health Science Complex, University of Vermont College of Medicine, Burlington, VT 05405-0068.

Key Words: activated protein C • venous thrombosis • activated protein C resistance • factor V^Leiden • thrombophilia

	Introduction

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
Blood coagulation is initiated after injury to the vasculature and exposure of TF. TF is an integral single-chain glycoprotein that, when exposed to the circulating blood, binds to circulating factor VIIa and initiates the process of blood coagulation. Although the majority of factor VII molecules circulate in plasma as a single-chain, inactive zymogen, 2% of them possess a Ser protease active site but have poor catalytic activity.^{1 2} After binding to TF, the catalytic efficiency of the factor VIIa/TF complex increases by four orders of magnitude, and the enzymatic complex initiates a series of enzymatic reactions that lead to the generation of -thrombin.^{2 3} The prothrombin-activating enzyme prothrombinase is composed of the Ser protease factor Xa and cofactor factor Va, associated on the cell membrane.⁴ Factor Va is required for prothrombinase activity because it serves the dual function as a factor Xa receptor and a factor Xa catalytic effector on the cell surface. Both enzyme and cofactor are derived from plasma precursors by regulatory proteolytic events that involve -thrombin. These events include activation of factor VIII to factor VIIIa and of factor V to factor Va. -Thrombin, the major procoagulant enzyme of the blood coagulation cascade, is also paradoxically a major anticoagulant. Once formed, -thrombin binds to the endothelial cell receptor thrombomodulin and initiates the protein C pathway that leads to the formation of APC. The protein C pathway is composed of the zymogen protein C, thrombomodulin, and the accessory protein, protein S.⁵ APC downregulates -thrombin generation by inactivating factors Va and VIIIa and eliminating their participation in the prothrombinase and tenase complexes, respectively. Under physiological conditions, inactivation of factor VIIIa occurs in the absence of APC by dissociation of the A2 domain.^{6 7 8} Hence, APC is not essential for factor VIIIa inactivation.^{8 9} In contrast, proteolytic cleavage of factor Va by APC is required for inactivation of the cofactor and the arrest of its contribution to the procoagulant process.^{10 11} APC inactivates factor Va by impairing both its receptor and effector functions with respect to factor Xa. Thus, irregularities in the mechanism of inactivation of factor Va by APC may be associated with thrombotic episodes due to sustained prothrombin activation. The severity of the thrombotic episodes would therefore be correlated with the importance of the molecular basis of the defect. The physiological significance of the protein C pathway is assured because of case reports of familial thrombophilia associated with protein C, protein S, and thrombomodulin deficiencies. Recently, a mutation in factor V, the most important physiological substrate for APC, has been associated with familial thrombophilia.¹² The mutation was initially identified in plasma from individuals with venous thrombosis by a bioassay that identified a defect associated with "resistance" to the action of APC.¹² Subsequently, APC resistance has been found to be associated with a mutation at Arg⁵⁰⁶ in the factor V gene.¹³

	Structure-Function Relationships of Factor V

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
The autosomal factor V gene (80 kb) has 25 exons and is located within a 300-kb region of chromosome 1 (1q21-25) that also contains the genes for the selectin family.¹⁴ Factor V mRNA is 6.8 kb. While a portion of the factor V pool is stored in platelets,¹⁵ the majority circulates in plasma at a concentration of 20 nmol/L as a large, single-chain molecule with an M_r of 330 000.^{4 16} The cDNA and deduced amino acid sequence show that the molecule consists of 2224 amino acid residues, including a 28–amino acid leader peptide (Fig 1).^{17 18} Factor V contains triplicate A domains that share a high degree of homology with ceruloplasmin, duplicate C domains that share homology with discoidin, and a connecting B region that functions as an activation peptide. Factor V is processed to factor Va, its active form, by -thrombin^{16 19 20} through cleavage at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵. Factor Xa can also activate factor V²¹ by cleavage at Arg⁷⁰⁹ and Arg¹⁰¹⁸. The factor Va molecule produced by -thrombin cleavage is composed of a heavy chain of M_r 105 000 and a light chain of M_r 74 000. The heavy chain corresponds to the NH₂ terminus of the procofactor (residues 1-709) and comprises two A domains (residues 1-303 and 317-656) connected by a segment containing mostly basic amino acids (residues 304-316).¹⁸ The COOH-terminal portion of the heavy chain (residues 657-709) is rich in acidic amino acids. The light chain of the cofactor, which corresponds to the COOH terminus of the factor V molecule (residues 1546-2196), comprises one A domain (residues 1546-1877) and two C domains (residues 1878-2036 and 2037-2196)¹⁸ (Fig 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Structural features of human factor V. Factor V is composed of 2196 amino acids, plus a 28–amino acid leader peptide.17 18 The activating cleavage sites of the procofactor are shown at the top. The APC-inactivating cleavage sites are depicted at the bottom.11 All regions of the molecule known to participate in the expression of factor Va cofactor function are illustrated.24 25 26 32 33 34 35 36 The positions of the free Cys and disulfide bridges are also shown.22 23 "P" identifies phosphorylation sites on the molecule (one on the acidic carboxy-terminal part of the heavy chain and two on the light chain). The star at amino acid 221 identifies a single point mutation that is correlated with a defective function of the molecule.88

Factor V contains 19 Cys, 18 of which are contained in the light- and heavy-chain regions of the factor Va molecule. Fourteen of these Cys are involved in disulfide bridges, while the remaining four are present as free sulfhydryl groups. The disulfide-bridged loops in factor Va are highly conserved.^{22 23} Three nearly identical 26–amino acid residue -loops are present, one in each A domain (Fig 1). The A1 and A2 domains contain larger ß-loops, each of which contains 82 amino acids. There are two nearly symmetrical -loops composed of 154 and 155 residues, which encompass most of the C1 and C2 domains of the light chain of the cofactor. The factor V/Va molecule contains multiple potential N-linked glycosylation sites in the B region and on the heavy and light chains.¹⁸ The molecule is also phosphorylated by platelet kinases: by a membrane-associated casein kinase II–like enzyme on the heavy chain (at Ser⁶⁹²) and by a protein kinase C isoform at two sites on the light chain.^{24 25} Tyr⁶⁶⁵, Tyr⁶⁹⁶, and Tyr⁶⁹⁸ of the heavy chain as well as Tyr¹⁴⁹⁴, Tyr¹⁵¹⁰, and Tyr¹⁵¹⁵ of the B domain and Tyr¹⁵⁶⁵ of the light chain are believed to be sulfated.²⁶

Factor Va functions by binding factor Xa and prothrombin to a membrane surface contributed by activated platelets, monocytes, or damaged cells. The molecule expresses binding interactions for factor Xa (K_d=0.8 µmol/L),²⁷ prothrombin (K_d=10 µmol/L),²⁸ and acidic phospholipid–containing membranes (K_d=2.7 nmol/L).²⁹ The global K_d of membrane-bound factor Xa for membrane-bound factor Va is 0.7 nmol/L.^{30 31} The factor Xa binding site to factor Va is localized on two portions of the cofactor: one on the A1 domain and the other on the NH₂-terminal portion of the A3 domain (Fig 1).³² The binding site of the cofactor for prothrombin is localized on the heavy chain.^{28 33} Two lipid-binding sites with different requirements for interaction have been identified on the factor Va light chain.^{34 35 36} One, in the middle of the A3 domain, interacts with membranes containing neutral phospholipid, whereas the other binding site (on the C2 domain) requires the presence of anionic phospholipid and shows partial ionic binding characteristics (Fig 1).³⁶

	Assay for Factor V and Va Activity

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
Historically, factor V has been recognized as that activity in normal plasma that "corrected" the PT of plasma from a patient with a factor V deficiency.³⁷ In a PT-based assay for factor V, the clotting end point occurs when only small amounts of -thrombin (1% of the plasma concentration of prothrombin, or 15 nmol/L) are generated by still lesser amounts of prothrombinase (<10 pmol/L).³⁸ As the assay progresses, factor V is activated to factor Va, and a diminishingly small amount of factor Xa (10 to 20 pmol/L) is generated. Because of the small amount of factor Xa available in the assay, clotting assays are extremely sensitive to small changes in the affinity of factor Va for factor Xa. In contrast, when factor Va cofactor activity is measured in a purified prothrombinase assay (in which the conversion of prothrombin to -thrombin is quantitated), the reaction mixture generally contains factor Xa in the range of 1 to 10 nmol/L.^{10 11} These assay conditions, which provide a saturating concentration of factor Xa, permit discrimination of the differential loss of factor Va binding and effector functions on a quantitative basis. Comparisons of factor Va inactivation studies using prothrombinase assays with those using clotting assays have produced significant confusion in the evaluation of the mechanism by which factor Va is inactivated by APC.

	The Mechanism of Inactivation of Factors V and Va

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
The membrane-dependent inactivation of human factor Va occurs due to limited proteolysis by APC of the heavy chain at Arg⁵⁰⁶, Arg³⁰⁶, and Arg⁶⁷⁹. Complete inactivation of the normal factor Va molecule is associated with all three cleavages; however, cleavage at Arg³⁰⁶, which is responsible for the majority of the loss in cofactor activity, occurs only on the membrane-bound cofactor.^{10 11} In the absence of phospholipid vesicles, the human cofactor is cleaved at Arg⁵⁰⁶ and Arg⁶⁷⁹ and retains 80% of its cofactor activity after incubation with APC, as assessed in a prothrombinase assay using saturating concentrations of factor Xa (>1 nmol/L). In contrast, when factor Va cofactor activity is assayed in a clotting assay (ie, at limiting factor Xa concentrations, or 10 pmol/L), these cleavages typically result in the loss of 60% cofactor activity.^{28 33 39} With the use of analytical ultracentrifugation techniques, it has been shown that in the absence of a membrane surface, APC-cleaved factor Va has reduced affinity for factor Xa and prothrombin.^{27 28 33} These data demonstrate that the cleavages at Arg⁵⁰⁶ and Arg⁶⁷⁹ impair the cofactor's ability to interact with both factor Xa and prothrombin. The effective K_d of membrane-bound factor Va cleaved at Arg⁵⁰⁶ and Arg⁶⁷⁹ (factor Va_506/679) for factor Xa is raised from 0.7 to 3.9 nmol/L.⁴⁰ As a consequence, when measured in a prothrombin activation assay, the cleavages at Arg⁵⁰⁶ and Arg⁶⁷⁹ reflect a moderate loss in the quantitative affinity for prothrombinase assembly rather than a total loss of factor Va cofactor activity. When an anionic phospholipid surface is added, factor Va_506/679 is rapidly cleaved by APC at Arg³⁰⁶ to produce a cofactor molecule, factor Va_306/506/679, which is no longer capable of binding factor Xa.¹¹ Cleavage at Arg³⁰⁶ is associated with the complete loss of cofactor activity regardless of the assay used and the amount of factor Xa employed to measure factor Va cofactor activity.

When factor Va is inactivated by physiologically relevant concentrations of APC in the presence of a membrane surface, inactivation proceeds in a sequential fashion.^{9 10 11} The first cleavage at Arg⁵⁰⁶ appears necessary for optimum exposure of the cleavage sites at Arg³⁰⁶ and Arg⁶⁷⁹. Phosphorylation of the cofactor by a platelet-membrane casein kinase II–like enzyme at the COOH-terminal portion of the heavy chain (at Ser⁶⁹²)⁴¹ increases the rate of cofactor inactivation by APC due to acceleration of cleavage at Arg⁵⁰⁶. Protein S may also play a role in regulating the rate of these cleavages.^{10 42}

In contrast to the active cofactor factor Va, the procofactor, factor V, is inactivated by APC by initial cleavage at Arg³⁰⁶ only in the presence of a membrane surface. Subsequently, cleavage occurs at Arg⁵⁰⁶, Arg⁶⁷⁹, and Lys⁹⁹⁴. The role of these three cleavages on the latent cofactor activity of the membrane-bound procofactor remains to be identified.¹¹

	APC Resistance

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
Dahlbäck et al¹² observed that plasma from some individuals with venous thrombosis had an abnormal response to APC. Usually when APC is introduced into normal plasma that has been preincubated with an APTT reagent, there is a prolongation of the clotting time proportional to the amount of APC used. In plasma from some patients, higher concentrations of APC are required to obtain a similar prolongation of the clotting time. This condition was called "APC resistance."¹² Initially, this abnormal response to APC was thought to be associated with a new cofactor for the APC anticoagulant pathway (APC cofactor 2). After purification, however, "APC cofactor 2" was identified as factor V.⁴³

The molecular defect in patients with APC resistance was subsequently defined at the gene level by Bertina et al,¹³ who showed that individuals with APC resistance have a mutation in the factor V gene (a G-to-A substitution at nucleotide 1691), which results in an Arg⁵⁰⁶Gln mutation in the factor V molecule. As a consequence of the mutation, this abnormal molecule, called factor V^Leiden,^{13 44 45 46 47 48} does not possess the APC cleavage site at Arg⁵⁰⁶. When isolated from the plasma of patients homozygous for the Arg⁵⁰⁶Gln mutation, factor Va^Leiden is inactivated by APC at a rate slower than that observed for normal factor Va.⁴⁹ However, inactivation still proceeds as a consequence of cleavage at Arg³⁰⁶ (Fig 2).^{49 50 51} Although the bulk of data relating to the mechanism of inactivation of factor Va by APC published during the past several years appears to demonstrate that cleavage and inactivation of factor Va^Leiden occurs at a rate slower than that for normal factor Va^{49 50 51} (because the rate of cleavage at Arg³⁰⁶ appears to be accelerated^{11 49} by prior cleavage at Arg⁵⁰⁶), one study suggested that cleavages at Arg³⁰⁶ and Arg⁵⁰⁶ occurred randomly at similar rate constants defining the inactivation process and were only facilitated (accelerated) by the presence of a membrane surface.⁴⁰ Thus, no difference in inactivation rates between normal factor V and factor V^Leiden was observed.⁴⁰ The latter conclusion was based on a study performed at a 20:1 enzyme-to-substrate ratio in the absence of lipid (19 nmol/L factor Va incubated with 390 nmol/L APC). However, since the second phase of the assay (to measure activity) was performed in the presence of phospholipid, the design of this experiment was flawed, since the phospholipid-dependent cleavage (at Arg³⁰⁶) would have occurred rapidly in the second stage of the assay at the extreme enzyme-substrate ratio used and resulted in the observation of no cofactor activity. It is not clear why the authors used such high concentrations of APC in their experiments. One possible explanation is that the APC preparations⁴⁰ were not fully active. It is well known that although all APC preparations have similar esterase activity toward small chromogenic substrates, the anticoagulant activity with respect to factor Va inactivation can vary considerably from one preparation to another.⁵² In addition, since APC and factor Xa compete for factor Va binding,^{53 54 55} the high concentrations of APC that are carried over in the second stage of the assay would displace factor Xa from prothrombinase. Thus, the second stage of the assay will show reduced cofactor activity because of factor Xa displacement rather than proteolytic cleavage and inactivation of the cofactor by APC. In any event, under physiological conditions (ie, in the presence of low concentrations of APC), cleavage and inactivation of factor Va^Leiden occur at a slower rate than that of normal factor Va.^{49 50 51}

View larger version (25K): [in this window] [in a new window]   Figure 2. Cleavage of the heavy chain of normal factor Va and factor VaLeiden by APC. The heavy chain of normal human factor Va is cleaved at Arg506, Arg306, and Arg679. Cleavage at Arg306 is required for efficient exposure of the inactivating cleavage sites at Arg306 and Arg679. In the absence of the cleavage site at Arg506 (factor VaLeiden), cleavage at Arg306 and Arg679 occurs at a slower rate.49 Hatched boxes depict regions that are recognized by monoclonal antibodies HFVaHC#68 49 52 78 82 and HFVaHC#179 39 68 during cleavage of either normal factor Va (left) or factor VaLeiden (right).

We have shown that the rate of cleavage at Arg³⁰⁶ is selectively accelerated by approximately twofold in the presence of protein S.¹⁰ Subsequently, one study suggested that in the presence of protein S, the difference in inactivation rates between normal factor Va and factor Va^Leiden was eliminated because protein S accelerated cleavage of factor Va^Leiden at Arg³⁰⁶ to a much greater extent than cleavage of normal factor Va at the same site.⁴² It is noteworthy that APC resistance was first discovered in human plasma that contained physiological concentrations of protein S.¹² If protein S had been able to restore factor Va^Leiden sensitivity to APC, then no APC resistance phenomenon would have been observed. Thus, it is highly unlikely that protein S plays a major role in correcting the APC resistance phenomenon.

Because the latent activity of factor V is eliminated due to initial cleavage at Arg³⁰⁶, factor V^Leiden is inactivated by APC at a rate similar to that of normal factor V.⁴⁹ The reduced inactivation rate of factor Va^Leiden by APC compared with equivalent inactivation rates of both procofactor molecules (factor V^Leiden and normal factor V) confirms the observation made with normal factor Va; ie, cleavage at Arg⁵⁰⁶ facilitates subsequent cleavage at Arg³⁰⁶. These data also verify the significance of cleavage at Arg³⁰⁶ for inactivation of factors V and Va.¹¹

It has been reported that the intact procofactor, single-chain factor V, can act as a "cofactor" molecule resulting in the acceleration of the inactivation of factor VIIIa by the APC/protein S complex.⁴³ Subsequently, two studies have shown a significant effect of factor V on APC-mediated inactivation of factor VIIIa.^{56 57} In contrast, factor V^Leiden was found to have a diminished "cofactor" effect on the inactivation of factor VIIIa by the APC/protein S complex.⁵⁷ Thus, it was proposed that the thrombotic tendency in individuals with the Arg⁵⁰⁶Gln mutation might also be due to loss of the "cofactor" activity of factor V, which would result in the delayed factor VIIIa inactivation and increased thrombin generation that has been observed in the plasma of APC-resistant individuals. However, the reports that support these conclusions did not provide any evidence for the absence of cleavage of factor V by APC during the course of the assay.^{56 57} Recently, we have demonstrated that high concentrations of factor V can accelerate (by approximately twofold) the rate of factor VIII inactivation by APC only in the presence of the intact form of protein S.⁸ Immunoblotting experiments, however, demonstrate that factor V is completely cleaved within 1 minute and before 10% to 20% factor VIII activity is lost.⁸ APC-treated, membrane-bound factor V, as well as -thrombin–activated factor Va (including the B domain), gave similar results (ie, an increase in the rate of inactivation of factor VIII by the APC/protein S complex by twofold). In contrast, purified factor Va (without the B domain) showed no cofactor effect on the rate of inactivation of factor VIII by the APC/protein S complex.⁸ Thus, regardless of the physiological significance of these results (all obtained in the absence of von Willebrand factor), these data suggest that a portion of the B region of the procofactor facilitates inactivation of factor VIII. As a consequence, these findings provide a potential role for the B domain of factor V in the regulation of blood coagulation.

	Clinical Implications for Individuals Bearing the Arg506Gln Mutation

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
Thrombotic manifestations are observed in both homozygous and heterozygous individuals with factor V^Leiden. However, venous thrombosis is more common among those homozygous for the Arg⁵⁰⁶Gln substitution.⁵⁸ It is clear from many studies^{59 60 61 62 63 64 65 66} that the thrombotic risk in persons homozygous for factor V^Leiden is less important than that of those homozygous for protein C or protein S deficiency. Additionally, the risk of thrombosis in individuals with factor V^Leiden is influenced by acquired risk factors. The presence of factor V^Leiden in addition to other risk factors associated with thrombosis (ie, a defect associated with one of the components of the protein C pathway,^{59 60 61 62 63 64 65 66} use of oral contraceptives,⁶⁷ low levels of TF pathway inhibitor⁶⁸ ) exacerbates the risk of thrombosis. Statistical analyses demonstrate that the relative risk of a thrombotic episode for heterozygous factor V^Leiden individuals is sevenfold higher than that of normal individuals. In contrast, homozygous factor V^Leiden individuals have an 80-fold higher risk of thrombosis than do individuals bearing the normal factor V gene.⁵⁸ Furthermore, the risk of thrombosis among female carriers of the factor V^Leiden mutation who use oral contraceptives is increased more than 30-fold compared with women who do not use oral contraceptives and possess the normal factor V gene.⁶⁷ Thus, oral contraceptives, pregnancy, and trauma are a few of the known predisposing acquired risk factors that act synergistically to increase the risk of thrombosis in the presence of factor V^Leiden.

Although there is an increased incidence of deep venous thrombosis in patients with APC resistance, no correlation has been observed between arterial thrombosis and the existence of factor V^Leiden.⁶⁹ The frequency of factor V^Leiden in patients with arterial thromboemboli is 5%, a value similar to that of the frequency of the mutation in the normal population. Discrepancies in the magnitude of APC resistance between various laboratories relative to the generation of arterial thrombosis in patients carrying the factor V^Leiden mutation most probably reflect differences in technique and reagents (particularly of APC and the quality of the membrane surface).⁵²

	Thrombosis Associated With Combined Congenital Mutations

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
A recently published study showed that 73% of family members heterozygous for both the factor V^Leiden mutation and a defect in the protein C gene had experienced or will experience at least one thrombotic episode in their lifetime.⁷⁰ These data suggest that the combined defects would result in an increased risk of a thrombotic event in these individuals.

Protein C contains nine -carboxyglutamic acid residues at position 6, 7, 14, 16, 19, 20, 25, 26, and 29 of the light chain.⁷¹ These residues are involved in the Ca²⁺- and membrane-binding properties of the protein, a characteristic required for proper anticoagulant function. It has been established that -carboxylation of residues 7, 16, 20, and 26 is required for expression of the anticoagulant effect of APC.^{72 73} A hereditary thrombotic diathesis, which is manifested by arterial and venous thrombosis, was found to be associated with two point mutations in the light-chain region of protein C, ie, Glu²⁰Ala and Val³⁴Met (protein C^VERMONT).^{74 75} Studies using recombinant molecules demonstrated that whereas the mutation at Val³⁴ has no effect on the anticoagulant properties of APC^V34M, the malfunction of protein C^VERMONT can be attributed solely to the Glu²⁰Ala substitution.⁷⁶ Using recombinant APC with an Ala for a Glu at position 20 (APC^20A), we have shown that this molecule has impaired capability in inactivating normal factor Va⁷⁷ due to defective membrane interaction and thus, impaired cleavage at Arg³⁰⁶. Both the procofactor, factor V^Leiden, as well as the active cofactor, factor Va^Leiden, are resistant to complete inactivation by APC^20A. Cleavage and inactivation of both factor V^Leiden and factor Va^Leiden by wild-type recombinant APC occur at similar rates.⁷⁸ These findings demonstrate that in the absence of the cleavage site at Arg⁵⁰⁶, factor V^Leiden and factor Va^Leiden are equivalent substrates for APC, as previously suggested.^{11 49} These data also indicate that the combination of the factor V^Leiden mutation and a diminished APC function, as a consequence of either reduced plasma protein C concentration or a normal plasma concentration of a qualitatively abnormal protein C molecule, will result in prolongation of the lifetime of circulating factor Va^Leiden in plasma and lead to disruption of the balance between the anticoagulant and procoagulant mechanisms in favor of the latter.

Clinical evidence accumulated during the last 2 years demonstrates that the combination of the factor V^Leiden mutation and protein S deficiency also enhances a person's thrombotic risk.^{63 64 65 66} A recent study showed that 72% of family members with both the factor V^Leiden mutation and a defect in the protein S gene had experienced a thrombotic episode.⁶⁴ These data demonstrate that thrombosis-prone families with protein S deficiency may also be affected by another genetic risk factor. However, the data reported thus far do not allow reliable comparisons between the risk of thrombosis due to single-gene defects compared with that of the double defect because the number of individuals with a defective protein S gene is lower than that of individuals who carry the factor V^Leiden mutation. Furthermore, the biological role of protein S in the anticoagulant pathway has yet to be conclusively established.

	Indirect Effect of Factor VLeiden on Fibrinolysis

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
After injury to the vasculature, the temporary structural integrity of the vessel is ensured by the fibrin clot, which is composed of insoluble fibrin polymers and activated platelets. Removal of the fibrin clot is accomplished by the fibrinolytic cascade, which results in plasminogen activation. Plasminogen is efficiently activated by tissue plasminogen activator only in the presence of fibrin polymers that possess a COOH-terminal Lys. Recent reports have demonstrated that APC also has a profibrinolytic effect.^{79 80 81} This effect of APC in plasma is attributed to the capability of the molecule to downregulate -thrombin formation by inactivating factor Va. High amounts of -thrombin are in turn required to activate TAFI, a procarboxypeptidase-like molecule.⁸¹ Activated TAFI expresses its antifibrinolytic potential by removing COOH-terminal Lys from the partially degraded fibrin. Thus, fibrinolysis is impaired as a result of impaired plasminogen activation.

We have recently reported the profibrinolytic potential of APC in normal plasma and in plasma from APC-resistant individuals.⁸² Our data demonstrate that for a given concentration of APC, clot lysis is considerably delayed in plasma from individuals homozygous for the Arg⁵⁰⁶Gln substitution when compared with normal plasma. Similar results were found in a reconstituted system using purified reagents and either normal factor V or factor V^Leiden. When TAFI was omitted from the system, no APC effect on clot lysis was observed, regardless of the form of factor V. These data suggest that the resistance of factor Va^Leiden to inactivation by APC will result in sustained -thrombin formation, which in turn will enhance activation of TAFI. As a consequence, removal of the fibrin clot will be impaired.⁸² Thus, one may hypothesize that not only will the blood in individuals with factor V^Leiden have a tendency to clot more than in those with normal factor V (because of continuous activation of prothrombin), but once formed, the clot will also be more resistant to fibrinolysis because of sustained activation of TAFI.

	A Paradoxical Beneficial Effect of Factor VLeiden

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
Factor V deficiency, or parahemophilia, is a rare but significant hemorrhagic disorder similar to that observed in individuals who lack factor VIII. Since factor Va^Leiden is inactivated by APC at a slower rate than normal factor Va, patients who posses both factor Va^Leiden and a mutation in the factor VIII molecule (which would classify them as hemophiliacs) may have a milder bleeding syndrome than hemophiliac patients with normal factor V and a similar defect in the factor VIII molecule. Recently, Nichols et al⁸³ reported significant differences in pathology between two sets of unrelated patients carrying the same factor VIII missense mutations. These mutations usually result in a phenotype characterized as severe hemophilia A. However, although two patients with the normal factor V gene were clinically classified as severe hemophiliacs, two others heterozygous for the Arg⁵⁰⁶Gln mutation were classified as mild-moderate hemophiliacs.⁸³ These results were based on the levels of measurable factor VIII activity in a one-stage clotting assay using factor VIII–deficient plasma. The effect of factor VIII deficiency is a reduced rate of -thrombin formation, whereas in the case of factor V^Leiden, the effect is an increase in -thrombin formation. It appears that in the case of these two combined deficiencies, the extended lifetime of the abnormal factor Va molecule (factor Va^Leiden) is able to partially compensate for the abnormal factor VIII molecule, resulting in sufficient -thrombin generation to provide hemostasis.

	Conclusions

Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
A great deal of interest and data have been generated during the past 2 years because of the discovery of factor V^Leiden and the association of this genotype with thrombosis. Studies of factor V^Leiden and APC resistance have contributed enormously to our understanding of the roles of factor V, factor Va, factor Va_506/679, factor Va_306/679, and factor Va_506/306/679 in thrombosis and hemostasis. Factor Va is the central player of prothrombinase, and inactivation of the cofactor is a complex mechanism involving other processes in addition to APC cleavage of the heavy chain at Arg⁵⁰⁶, Arg³⁰⁶, and Arg⁶⁷⁹. Since the cofactor interacts with the membrane surface, APC, factor Xa, and protein S during normal blood clotting, alterations in any of these functions could potentially produce thrombosis. As a consequence, it is likely that factor V^Leiden will not be the end of the story vis à vis APC resistance.

Factor V and factor VIII are homologous proteins, displaying 40% identity between their activated forms. In contrast to the frequency at which the factor V^Leiden mutation is found in the human population, factor Va procoagulant defects are only rarely identified. The absence of frequent identification of parahemophilia in the human population is no doubt a consequence of the fact that factor V, unlike its factor VIII homologue, is an autosomal product. Since each individual inherits two genes encoding the factor V sequence, the presence of either gene product in theory would account for 50% of the factor V circulating in the blood. The factor V^Leiden mutation prolongs the procoagulant effects of factor Va; hence, inheritance of a single mutation can be associated with pathological consequences. However, whereas factor V^Leiden is present in the majority of patients with venous thrombosis, many individuals with venous thrombosis do not have this mutation. Thus, although the Arg⁵⁰⁶Gln substitution in the factor V molecule is by far the most common single point mutation that can be related to thrombosis, it remains puzzling that correlation of the mutation (at the genetic level) with a clearly defined pathology (phenotype) has yet to be established.^{84 85} Thus, since the presence of the mutation is not always associated with a disease state, we may conclude that the abnormal allele may not always be equivalently expressed. The existence of other compensatory mutations in individuals with APC resistance that could influence APC sensitivity of factor V should not be excluded.

It should be noted that factor VIII deficiency is associated with 174 known single base-pair substitutions in the coding region of the factor VIII gene.⁸⁶ These mutations in turn are associated with varying degrees of severity of hemophilia.⁸⁶ Whereas the DNA encoding factor VIII is much larger than that encoding factor V, the mRNA encoding both proteins is approximately the same size. Hence, it is likely that the frequency of mutation in the factor V molecule will be similar to that of factor VIII. Furthermore, in mice, complete deficiency of factor V results in massive hemorrhage immediately after birth and consequent death.⁸⁷ Thus, we would expect that severe mutations in human propositi would be rare. However, it is likely that additional gene mutations will be found in the factor V molecule that will be associated with APC resistance and thrombophilia.

	Selected Abbreviations and Acronyms

 

APC = activated protein C APTT = activated partial thromboplastin time PT = prothrombin time TAFI = thrombin-activatable fibrinolysis inhibitor TF = tissue factor

	Acknowledgments

 
This study was supported by Grants-In-Aid 9606277S (Vermont Affiliate) and 94017990 (National Center) from the American Heart Association (to M.K.) and Merit Award R37 HL34575 from the National Institutes of Health, Bethesda, Md (to K.G.M.).

Received November 7, 1996; accepted February 4, 1997.

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Top Introduction Structure-Function Relationships... Assay for Factor V... The Mechanism of Inactivation... APC Resistance Clinical Implications for... Thrombosis Associated With... Indirect Effect of Factor... A Paradoxical Beneficial Effect... Conclusions References

 
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