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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2181-2187.)
© 1995 American Heart Association, Inc.

Articles

Biochemical Prototype for Familial Thrombosis

A Study Combining a Functional Protein C Mutation and Factor V Leiden

Michael Kalafatis; Deshun Lu; Rogier M. Bertina; George L. Long; Kenneth G. Mann

From the Department of Biochemistry, University of Vermont, College of Medicine, Burlington (M.K., D.L., G.L.L., K.G.M.), and Hemostasis and Thrombosis Research Center, University Hospital, Leiden, Netherlands (R.M.B.).

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

	Abstract

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Abstract Resistance to activated protein C (APC) is associated with a single amino acid substitution in factor V (Arg⁵⁰⁶Gln, factor V Leiden) that results in delayed inactivation of the molecule by APC. The mutation is present in 20% of patients with a first episode of deep venous thrombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein C_Vermont). In protein C_Vermont, the substitution Glu²⁰Ala alone (rPC^20A) is responsible for the defective anticoagulant properties of PC_Vermont. It was recently established that a thrombotic episode occurred in 73% of family members who are heterozygous for both a functional protein C gene mutation and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor V^R506Q/Va^R506Q and rAPC^20A using recombinant APC and natural purified factor V^R506Q from patients homozygous for the Arg⁵⁰⁶Gln substitution. While wild-type recombinant APC (rAPC) slowly cleaves and inactivates factor V^R506Q and factor Va^R506Q, minimal cleavage of membrane-bound factor V^R506Q and Va^R506Q by rAPC^20A at Arg³⁰⁶ and Arg⁶⁷⁹ occurs, and no loss in cofactor activity is observed. Our data demonstrate that rAPC^20A cannot inactivate either factor V^R506Q or factor Va^R506Q at biologically relevant rates because of impaired cleavage at Arg³⁰⁶ and Arg⁶⁷⁹. The result is a stable procofactor and stabilization of an active cofactor in patients possessing both mutations. Our data provide a prototype of familial thrombosis that most likely would be manifested in vivo by the occurrence of massive thrombosis.

Key Words: thrombosis • factor V Leiden • protein C_Vermont • blood coagulation • APC resistance

	Introduction

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Factor V circulates in plasma as a single-chain high-molecular-weight inactive procofactor (M_r=330 000) that is cleaved to produce the active cofactor, factor Va.¹ The association of factor Va with factor Xa on a membrane surface forms prothrombinase, the enzymatic complex that activates prothrombin, with an efficiency five orders of magnitude greater than factor Xa acting alone.² Human factor V is activated by -thrombin after cleavage at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵.^{3 4 5} The product, factor Va, is composed of a heavy chain (M_r=105 000) containing the NH₂-terminal part of the procofactor (residues 1 through 709, A1-A2 domains) and a light chain (M_r=74 000) containing the COOH-terminal part of the factor V molecule (residues 1546 through 2196, A3-C1-C2 domains) that are noncovalently associated in the presence of divalent metal ions.^{6 7}

Factor Va is proteolytically inactivated by APC by three cleavages of the heavy chain, at Arg⁵⁰⁶, Arg³⁰⁶, and Arg⁶⁷⁹.^{8 9} Cleavage at Arg⁵⁰⁶ is necessary for efficient exposure of the inactivating cleavage sites at Arg³⁰⁶ and Arg⁶⁷⁹. Cleavage at Arg³⁰⁶ occurs efficiently only on the membrane-bound cofactor and is responsible for the loss of 70% to 80% of factor Va cofactor activity, whereas cleavage at Arg⁶⁷⁹ appears to be membrane independent and is responsible for the loss of the remaining cofactor activity. Elimination of the latent activity of the procofactor, factor V, by APC requires the presence of a membrane surface and is associated with four cleavages, at Arg³⁰⁶, Arg⁵⁰⁶, Arg⁶⁷⁹, and Lys⁹⁹⁴. The kinetically favored cleavage, at Arg³⁰⁶, is the inactivating cleavage site.⁹

Recent data demonstrate that a poor anticoagulant response to APC (APC resistance) is associated with a GA substitution at nucleotide 1691 in the factor V gene, resulting in an Arg⁵⁰⁶Gln mutation in the factor V molecule (factor V^R506Q, factor V Leiden).^{10 11 12 13 14 15 16 17 18} We recently demonstrated that inactivation of purified factor Va^R506Q by APC is delayed compared with normal factor Va.¹⁹ APC resistance has been suggested to be the most common risk factor for developing deep venous thrombosis.^{14 15 16 17 18} The association of a thrombotic syndrome and protein C deficiency was first established by Griffin et al.²⁰ Extensive studies of family members from thrombosis-prone APC-deficient families demonstrated that 50% of these heterozygous individuals had a thrombotic episode before the age of 45 years.^{21 22 23 24} A hereditary thrombotic diathesis manifested by arterial and venous thrombosis is associated with a type II protein C deficiency characterized by two single-point mutations that correspond to two amino acid substitutions in the protein C molecule: Glu²⁰Ala and Val³⁴Met.²⁵ We previously showed that rAPC^20A that accounts for the functional defect in protein C_Vermont²⁶ displays impaired Ca²⁺ and membrane binding²⁶ and impaired ability to inactivate normal plasma factor Va.²⁷ The reduced ability of rAPC^20A to inactivate plasma factor Va is the result of impaired cleavage of the cofactor at the membrane-dependent Arg³⁰⁶ site.^{9 27}

A recent study concluded that 73% of family members heterozygous for both a defective protein C gene and the Arg⁵⁰⁶Gln mutation experienced a thrombotic episode,²⁸ suggesting that the combination of these two defects is directly associated with thrombosis. The present study was undertaken to evaluate the effect of APC and rAPC^20A on factor V^R506Q and factor Va^R506Q and deduce a biochemical mechanism for the observed pathology.

	Methods

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Experimental Procedures
Materials, Reagents, and Proteins
HEPES, Sepharose CL-4B, PS, and PC were purchased from Sigma Chemical Co. Phospholipid vesicles composed of 75% PC/25% PS were prepared as described.²⁹ The chemiluminescent substrate, Luminol, was from DuPont–NEN Research Products. Normal human factor V and factor V from two unrelated patients (patient 1 [factor V_I] and patient 2 [factor V_II]) homozygous for the 1691 GA substitution in the factor V gene (which results in an Arg⁵⁰⁶Gln replacement in the factor V molecule), human prothrombin, and human -thrombin were purified as described.^{19 30 31 32 33} The APC sensitivity ratio was 1.13 for patient 1 and 1.14 for patient 2.¹⁸ Wild-type rPC, rAPC, and rAPC^20A were obtained as described.²⁶ Monoclonal antibody HFVa_HC#6, which recognizes an epitope located between amino acid residues 307 and 506 of the human factor V molecule, was prepared as described.¹⁹ The fluorescent thrombin inhibitor DAPA,³⁴ human plasma APC, and human factor Xa were provided as a gift by Paul Haley (Haematologic Technologies Inc). The -thrombin inhibitor hirudin was from Genentech. All reactions were performed in a buffer composed of 20 mmol/L HEPES, 0.15 mol/L NaCl, and 5 mmol/L CaCl₂, pH=7.4 [HBS(Ca²⁺)].

>Inactivation of Factor V^R506Q/Factor Va^R506Q by rAPC
Human factor V^R506Q and -thrombin activated factor V^R506Q (factor Va^R506Q) in HBS(Ca²⁺) were incubated with either rAPC or rAPC^20A in the presence of PC/PS vesicles. The concentrations of all reagents are given in the figure legends. At selected time intervals, aliquots of the mixture were assayed for cofactor activity as described with a Perkin-Elmer Instrument MPF-44A fluorescence spectrophotometer with a _ex 280-nm (slit at 8 nm), _em 550-nm (slit at 16 nm), and a 500-nm long-pass filter in the emission beam.^{8 34} In a typical experiment, a mixture composed of prothrombin (1.4 µmol/L), PC/PS vesicles (20 µmol/L), and DAPA (3 µmol/L) was incubated in the dark for 20 minutes. At selected time intervals, an aliquot of the mixture (1800 µL) was added to a cuvette containing 10 nmol/L factor Xa, and the baseline fluorescence was monitored for 15 to 20 seconds at room temperature. The reaction was initiated by the addition of the membrane-bound factor Va sample that was preincubated with APC. The reaction conditions (20 µmol/L PC/PS, 10 nmol/L factor Xa, and 1 nmol/L factor V/Va) were chosen to make the reaction dependent on factor Va activity. Thus, the rate of thrombin formation is linearly related to the amount of active cofactor (factor Va). The results are expressed as percent of initial cofactor activity as a function of time when inactivation of factor Va is studied and as percent of initial activity as a function of time when inactivation of factor V is studied. The difference in the expression of the results is related to the cofactor activity of the two molecules. Factor Va possesses cofactor activity, whereas factor V does not. However, when factor V cleavage by APC is studied, during the course of the assay, factor V is activated by factor Xa and displays similar cofactor activity as if the procofactor were activated by -thrombin before the assay.^{9 35} Thus, it is possible to measure alterations in factor V potential cofactor activity.⁹

At the same time intervals at which the activity was measured, the factor V/factor Va samples were also analyzed by SDS-PAGE according to the method of Laemmli.³⁶ After electrophoresis, transfer to nitrocellulose was performed as described,³⁷ and factor V fragments were probed with the monoclonal antibody HFVa_HC#6 as recently detailed using the chemiluminescent substrate Luminol.³⁸ The monoclonal antibody recognizes factor V fragments, as shown in the Table. The potential for contamination of the APC preparations with -thrombin was controlled for by addition of hirudin.

View this table: [in this window] [in a new window]   Table 1. Identity of Immunoreactive Fragments (for FVaHC#6) Deriving From Membrane-Bound Factor VR506Q/VaR506Q After APC Cleavage

	Results

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Inactivation of Factor V^R506Q and Va^R506Q by rAPC
We previously established that cleavage of factor V by APC at Arg³⁰⁶ is membrane dependent and inactivates the procofactor.⁹ The mutant procofactor, factor V^R506Q, is inactivated by APC in the presence of a membrane surface at a rate similar to that observed for normal plasma factor V, with cleavages at Arg³⁰⁶ and Arg⁶⁷⁹ occurring at similar rates.¹⁹ In contrast, natural purified factor Va^R506Q is inactivated by APC at a slower rate than normal factor Va.¹⁹ We also demonstrated that rAPC^20A, which has impaired membrane- and Ca²⁺-binding capabilities,²⁶ has impaired ability to cleave normal factor Va at Arg³⁰⁶.²⁷

The effects of wild-type rAPC and rAPC^20A on factor V^R506Q and factor Va^R506Q were investigated by use of natural purified factor V^R506Q/Va^R506Q, wild-type rAPC, and rAPC^20A. After 5 minutes of incubation with rAPC, membrane-bound factor V^R506Q retains 50% of its initial activity (Fig 1A, solid circles), whereas under similar experimental conditions no inactivation of membrane-bound factor V^R506Q by rAPC^20A was observed (Fig 1A, open circles). Minimal activity (10% remaining activity) is observed after a 2-hour incubation period of membrane-bound factor V^R506Q with rAPC. In contrast, after 2 hours of incubation with rAPC^20A, factor V^R506Q retains nearly 100% of its potential cofactor activity (Fig 1A, open circles). Previous data have demonstrated that under similar experimental conditions, factor Va is inactivated by APC faster than factor V, since cleavage at Arg⁵⁰⁶ accelerates the rate of the inactivating cleavage at Arg³⁰⁶.⁹ For example, under experimental conditions similar to those described in Fig 1B, when normal plasma factor Va was used instead of factor Va^R506Q, complete inactivation of the normal cofactor by APC occurred within 5 minutes, whereas factor V potential cofactor activity was abolished completely after 30 minutes of incubation.^{9 19} The data presented in Fig 1 show that factor Va^R506Q and factor V^R506Q are inactivated by rAPC at similar rates (compare Fig 1A, solid circles, with Fig 1B, solid squares). These data demonstrate that in the absence of the cleavage site at Arg⁵⁰⁶, factor V and factor Va are equivalent substrates for APC.

View larger version (16K): [in this window] [in a new window]   Figure 1. Graphs showing inactivation of factor VR506Q and factor VaR506Q by rAPC. A, Factor VR506Q (factor VI,18 19 110 nmol/L) was incubated with rAPC (2.2 nmol/L, ) or rAPC20A (2.2 nmol/L, ) in the presence of PC/PS vesicles (200 µmol/L) and hirudin (20 nmol/L) at 37°C. At selected time intervals, aliquots were assayed for activity as described8 34 using 10 nmol/L factor Xa. B, Factor VR506Q was incubated with -thrombin (12 nmol/L) for 10 minutes at 37°C. After the addition of hirudin (20 nmol/L), rAPC (2.2 nmol/L, ) or rAPC20A (2.2 nmol/L, ) were added to separate mixtures, and factor Va cofactor activity was assessed as described8 34 using 1 nmol/L factor VaR506Q and 10 nmol/L factor Xa (final concentrations). The results are expressed as percent of initial activity (at the steady state) in A and as percent of initial cofactor activity in B as a function of time after addition of rAPC or rAPC20A. At the same time intervals, aliquots of the mixture were also analyzed by SDS-PAGE (shown in Fig 2).

Analyses of the proteolytic fragments deriving from membrane-bound factor V^R506Q after incubation with rAPC and rAPC^20A were performed by immunoblotting using monoclonal antibody FVa_HC#6, which recognizes an epitope located between amino acid residues 307 and 506 of the factor V molecule (Table).¹⁹ The results demonstrate that cleavages at Arg⁶⁷⁹ before cleavage at Arg³⁰⁶ results in the transient appearance of an M_r=99 000 fragment (Fig 2A, lanes 2 through 6). Loss in activity of factor V^R506Q correlates primarily with cleavage at Arg³⁰⁶ and the appearance of an M_r=54 000 fragment containing the region 307 through 679 of the procofactor (Fig 2A, lanes 3 through 9). Appearance of this fragment requires two cleavages of factor V^R506Q: at Arg³⁰⁶ and Arg⁶⁷⁹.

View larger version (63K): [in this window] [in a new window]   Figure 2. Analysis of factor VR506Q and factor VaR506Q inactivation by rAPC. The samples assayed for activity in Fig 1 were also analyzed by SDS-PAGE after reduction with 2% ß-mercaptoethanol (5% to 15% linear polyacrylamide gradient), transferred to nitrocellulose, and probed with the monoclonal antibody HFVaHC#619 (200 ng of protein was applied per lane). A, Samples incubated with rAPC; lane 1, membrane-bound factor VR506Q control before addition of rAPC; lanes 2 through 9, factor VR506Q at 1, 3, 5, 10, 15, 30, 60, and 120 minutes after the addition of rAPC; lane 10, -thrombin factor VaR506Q control, no rAPC; lanes 11 through 18, factor VaR506Q at same time intervals as in lanes 2 through 9 after the addition of rAPC. B, Samples incubated with rAPC20A, at same time intervals (±-thrombin) as depicted in A. Position of the molecular weight markers is indicated at left in A and B. The position of factor Va heavy chain (amino acid residues 1 through 709) as well as the positions of the proteolytic fragments deriving from factor VR506Q or factor VaR506Q after cleavage at Arg679 (Mr=99 000, amino acid residues 1 through 679), Arg306 on factor VaR506Q (Mr=60 000, amino acid residues 307 through 709), or Arg306 and Arg679 on factor VR506Q and factor VaR506Q (Mr=54 000, amino acid residues 307 through 679) are also indicated. The star with arrowhead indicates fragments deriving from factor VR506Q after cleavage at Arg679 (Mr=99 000; A, lanes 2 through 6, and B, lanes 8 and 9) or after cleavage at Arg679 and Arg306 (Mr=54 000; A, lanes 3 through 9, and B, lane 9). The open arrowhead at right indicates a fragment that derives from factor VR506Q on prolonged incubation with -thrombin. This fragment, which is observed after activation of either normal plasma factor V or factor VR506Q by -thrombin and can be detected only after reduction, has an NH2-terminal sequence identical to the NH2-terminal sequence of factor V.27 The effect of this cleavage on factor Va cofactor activity by -thrombin remains to be identified.

Factor V^R506Q, although cleaved (Fig 2B, lanes 1 through 9), is not significantly inactivated by rAPC^20A after a 2-hour incubation period (Fig 1A, open circles). Analyses of the proteolytic fragments demonstrated that rAPC^20A slowly cleaves factor V^R506Q at Arg⁶⁷⁹, resulting in the generation of an intermediate of an M_r=99 000 fragment (denoted by a star with arrowhead in Fig 2B, lanes 8 and 9) that is very slowly cleaved at Arg³⁰⁶ to generate a small amount of the M_r=54 000 fragment (Fig 2B, lane 9).

Factor Va^R506Q [initial cofactor activity of 1.3 µmol/L IIa·min^-1·(nmol/L)^-1] retains 5% cofactor activity after 2 hours of incubation of the cofactor with rAPC (Fig 1B, solid squares). In contrast, no significant inactivation of membrane-bound factor Va^R506Q is observed after prolonged incubation with rAPC^20A (Fig 1B, open squares). Some limited slow cleavage by rAPC^20A at Arg³⁰⁶ occurs on the membrane-bound factor Va^R506Q, resulting in the generation of an M_r=60 000 fragment (amino acid residues 307 through 709, Fig 2B, lanes 13 through 17), which is followed by cleavage at Arg⁶⁷⁹, resulting in the generation of an M_r=54 000 fragment (Fig 2B, lanes 17 and 18). Previous data have demonstrated that rAPC^20A cleaves normal plasma factor Va at Arg⁵⁰⁶ and Arg⁶⁷⁹, resulting in the loss of 40% cofactor activity.²⁷ Our present data demonstrate that rAPC^20A does not significantly inactivate membrane-bound factor Va^R506Q (Fig 1B). Further slow cleavage at Arg³⁰⁶ occurs, while little cleavage at Arg⁶⁷⁹ is observed (Fig 2B). Altogether, these data suggest that prior cleavage at Arg⁵⁰⁶ on normal human plasma factor Va or cleavage at Arg³⁰⁶ on factor Va^R506Q facilitates cleavage at Arg⁶⁷⁹.

To compare cleavage and inactivation of membrane-bound factor Va^R506Q by rAPC^20A with cleavage of factor Va^R506Q by normal plasma APC in the absence of a membrane surface, factor Va^R506Q was assayed for cofactor activity after incubation with APC in the absence of PC/PS vesicles (Fig 3). Minimal loss in cofactor activity (Fig 3A) and limited cleavage at Arg³⁰⁶ were observed after a 3-hour incubation period of the mutant cofactor with APC in the absence of a membrane surface (Fig 3B, lanes 1 through 7, appearance of an M_r=60 000 fragment). The starting factor Va^R506Q solution (Fig 3B, lane 1) had a cofactor activity of 1.48 µmol/L IIa·min^-1·(nmol/L)^-1. After 3 hours of incubation of factor Va^R506Q with APC in the absence of PC/PS, 83% of its initial cofactor activity [1.23 µmol/L IIa·min^-1·(nmol/L)^-1] remained (Fig 3B, lane 7). Addition of PC/PS vesicles to the factor Va^R506Q–APC mixture resulted in the rapid increase of the M_r=60 000 fragment (Fig 3B, lanes 8 through 16). Appearance of this fragment, which corresponds to cleavage of the heavy chain of the cofactor at Arg³⁰⁶, correlates with loss in cofactor activity (80% loss in cofactor activity after 30 minutes of incubation).^{9 19} The appearance of the M_r=54 000 fragment corresponds to cleavage of the M_r=60 000 fragment at Arg⁶⁷⁹ and loss of the remaining cofactor activity. Our data demonstrate that cleavage at Arg³⁰⁶, which is membrane dependent, is required for inactivation of the cofactor. Thus, impaired inactivation of factor V^R506Q and factor Va^R506Q by rAPC^20A results in accumulation of active cofactor. The consequence of accumulation of a stable factor Va molecule would be manifested in vivo by a thrombotic episode in individuals possessing both mutations.

View larger version (46K): [in this window] [in a new window]   Figure 3. Membrane-dependent inactivation of factor VaR506Q. Factor VR506Q (110 nmol/L, factor VII)18 19 was incubated for 5 minutes at 37°C with -thrombin (12 nmol/L). After the addition of hirudin (40 nmol/L), plasma APC was added (2.2 nmol/L). At selected time intervals, aliquots of the mixture were withdrawn and assayed for cofactor activity as described8 34 using factor VaR506Q and factor Xa at 1 and 10 nmol/L, respectively. After 3 hours of incubation (vertical arrow), PC/PS vesicles were added (200 µmol/L) and incubation was allowed to proceed for 120 additional minutes. The results are expressed as percent of initial cofactor activity as a function of time after the addition of APC in graph A. At the same time intervals, aliquots of the mixture were mixed with 2% ß-mercaptoethanol/2% SDS, heated for 5 minutes at 90°C, and analyzed on a 5% to 15% (linear gradient) gel. After electrophoresis, the proteins were transferred to nitrocellulose, and immunoreactive fragments were detected with the monoclonal antibody HFVaHC#6 (B). Lane 1, factor VaR506Q control, no APC; lanes 2 through 7, factor VaR506Q with APC at 10, 20, 30, 60, 120, and 180 minutes. After 3 hours of incubation, PC/PS vesicles were added (200 µmol/L, indicated by the vertical arrow). Lanes 8 through 16 depict aliquots withdrawn at 1, 3, 5, 7, 10, 15, 30, 60, and 120 minutes after the addition of PC/PS vesicles to the APC–factor VaR506Q mixture. Lane 17 shows factor VaR506Q incubated at 37°C for 5 hours in the absence of APC and PC/PS vesicles. The cofactor activity of this sample is depicted in A by the dashed line (). Lane 18 shows membrane-bound factor VaR506Q (factor VaII18 19 ) after 30 minutes of incubation with APC as described.19 Approximately 150 ng of total protein was applied per lane. Position of the molecular weight markers is indicated at left in B.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our data demonstrate that rAPC^20A has no biologically relevant ability to cleave and inactivate both membrane-bound factor V^R506Q and factor Va^R506Q. Thus, the combination of the two defects renders not only the cofactor but also its precursor virtually immune to APC inactivation. Hence, individuals who possess both mutations will have a stable factor Va molecule that is not efficiently cleaved at Arg³⁰⁶ and a higher probability of producing a clotting event compared with individuals with only one of these mutations. Our results can explain recent findings showing that a thrombotic event had occurred in 73% of family members heterozygous for both a protein C mutation and the Arg⁵⁰⁶Gln mutation.²⁸

Although the association of a thrombotic syndrome and protein C deficiency has long been suspected,^{20 21 22 23 24 25 26 27 28} the definition of protein C deficiency as a common risk factor for thrombosis has been challenged because heterozygous protein C deficiency is found in apparently healthy individuals (0.1% to 0.3% of the normal population).³⁹ Heterozygosity for the Arg⁵⁰⁶Gln substitution in the factor V molecule is also found in 2% to 5% of the normal population.^{12 14} Recent data demonstrated the presence of the Arg⁵⁰⁶Gln mutation in 47 of 50 Swedish families with inherited APC resistance.⁴⁰ However, of 308 individuals investigated who were selected for APC resistance, 6% were homozygous and 47% were heterozygous for the mutation. Further, although 44% of the homozygous and 30% of the heterozygous individuals had experienced a thrombotic event,⁴⁰ these events occurred in 73% of family members who are heterozygous for both a protein C mutation and the Arg⁵⁰⁶Gln mutation.²⁸ Recent observations also demonstrate variability of thrombosis among siblings homozygous for the Arg⁵⁰⁶Gln mutation.⁴¹ In one family with three sons homozygous for the mutation, all were subject to at least one severe thrombotic episode and developed deep vein thrombosis. In contrast, the two daughters also homozygous for the mutation did not present any thrombotic episode or develop deep vein thrombosis at the time the study was performed. All five individuals and their parents (heterozygous for the mutation) had elevated levels of prothrombin fragment 1·2, indicating enhanced activation of prothrombin.^{42 43} These data suggest continuous activation of the coagulation mechanism in patients with the Arg⁵⁰⁶Gln mutation. Further, in the Leiden thrombophilia study, in which the Arg⁵⁰⁶Gln mutation in the factor V molecule was first identified, 6 of 7 symptomatic homozygous individuals were women, and 4 of them were using oral contraceptives.^{14 18} The use of oral contraceptives is known to increase the risk of thrombosis in individuals with the Arg⁵⁰⁶Gln mutation.⁴⁴ From all these studies, it is clear that the penetrance of thrombosis is defined by the genetic status of each individual and by acquired risk factors and/or triggering events. The overall data indicate that given the existence of a trigger that will "activate" clotting in individuals predisposed for that abnormality (ie, possessing the Arg⁵⁰⁶Gln mutation in factor V), this predisposition would be exacerbated in the presence of both the factor V and the protein C mutations.

APC expresses its anticoagulant activity by inactivating factor Va and factor VIIIa. Thus, impaired inactivation of both cofactors will result in the generation of thrombosis. However, recent data demonstrate that factor Va is the preferred substrate for APC compared with factor VIIIa.⁴⁵ Further, rAPC^20A has impaired capabilities in cleaving and inactivating factor VIII/VIIIa.⁴⁵ It is also well established that the absence of factor V is incompatible with the formation of a blood clot.^{38 46} On the other hand, it is also well documented that individuals with factor VIII deficiency have severe bleeding disorders (reviewed in Reference 47). Factor VIII deficiency (hemophilia A) is an X chromosome–linked bleeding disorder, whereas factor V deficiency (parahemophilia) is an autosomal-linked bleeding disorder. The bulk of data concerning factor VIII deficiency are most likely related to the survival of the patients with factor VIII deficiency, since very few patients with factor V deficiency were studied. Conversely, it has been demonstrated that 20% of patients with deep venous thrombosis have the factor V Leiden mutation.¹¹ No abnormalities in the factor VIII molecule were observed in these patients.¹⁰ Thus, it is possible to speculate that patients who possess both abnormalities (ie, the Arg⁵⁰⁶Gln mutation in the factor V molecule and a mutation in the factor VIII molecule) would not have either an increase in thrombotic tendency or a bleeding syndrome. It is thus possible that the molecular defect associated with the Arg⁵⁰⁶Gln mutation may compensate for a defective factor VIII in some hemophiliacs. As a consequence, a hemophiliac with a hyperactive factor Va (factor Va^R506Q) will have a milder bleeding tendency than a hemophiliac possessing a normal factor V molecule. Hence, it will be of great interest to verify whether some hemophiliacs with an unexplained mild bleeding syndrome are homozygous or heterozygous for the Arg⁵⁰⁶Gln mutation in the factor V gene.

In conclusion, our data suggest that homozygosity for a functional mutation in the protein C gene combined with homozygosity for the common congenital abnormality (ie, factor V Leiden) will result in accumulation of active cofactor, factor Va^R506Q, as a consequence of impaired cleavage at Arg³⁰⁶. Thus, we can predict that individuals with a stable factor Va molecule would manifest an enhanced propensity toward a thrombotic pathology requiring prophylactic therapy for survival.

	Selected Abbreviations and Acronyms

 

APC = activated protein C DAPA = dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide PC = 1-palmitoyl-2-oleoyl-phosphatidyl choline PS = 1-palmitoyl-2-oleoyl-phosphatidyl serine rAPC = recombinant activated protein C rAP rAPC20A = rAPC with a Glu20Ala substitution rPC = recombinant protein C

	Acknowledgments

 
This work was supported by merit award R37-HL-34575, grants PO1-HL-46703 and CO6-HL-39475 from the National Institutes of Health, and American Heart Association Grant-in-Aid 92011860. We thank Paul Haley for providing purified human plasma APC and human factor Xa and Dr William Church for ascites fluid.

Received August 8, 1995; accepted October 11, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
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2. 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. [Abstract/Free Full Text]

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