©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Molecular Defect in Factor V(*)

(Received for publication, December 16, 1994)

Michael Kalafatis (1) Rogier M. Bertina (2) Matthew D. Rand (1) Kenneth G. Mann (1)(§)

From the  (1)Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068 and (2)Hemostasis and Thrombosis Research Center, University Hospital, 2300 RC Leiden, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg Gln). Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor V(I), and patient II, factor V), who are homozygous for this mutation. The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation. The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va. In normal factor Va, cleavage at Arg generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg and Arg. Proteolysis at these sites leads to the appearance of M(r) = 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg is membrane-dependent and is required for complete inactivation. Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor Va(I) and factor Va retain approximately 50% of their initial activity. After 1 h of incubation with APC, factor Va(I) retains 20% of its initial cofactor activity whereas factor Va has 10% remaining cofactor activity. The initial loss in cofactor activity (70%) of membrane-bound factor Va(I) and factor Va during the first 10 min of the inactivation reaction is correlated with cleavage at Arg and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet. Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor V(I) and factor V, were inactivated following cleavage at Arg and Arg, with APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that: 1) cleavage at Arg is required for optimum exposure of the cleavage sites at Arg and Arg and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg by APC on membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals. Thus cleavage at Arg and Arg and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg for optimum exposure.


INTRODUCTION

The generation of alpha-thrombin is a central event of the blood coagulation process. This enzyme is generated by the catalyst prothrombinase, which is composed of factor Xa and its cofactor factor Va associated on a membrane surface in the presence of Ca. Factor V is a single chain procofactor molecule of M(r) = 330,000, which is activated during blood clotting to factor Va, by factor Xa and/or alpha-thrombin(1) . The active cofactor, factor Va, purified from human plasma, is composed of a heavy chain (M(r) = 105,000) containing the NH(2)-terminal part of the procofactor (residues 1-709, A1-A2 domains) and a light chain (M(r) = 74,000) containing the COOH-terminal part of the factor V molecule (amino acids 1546-2196, A3-C1-C2 domains)(1, 2, 3) , which are non-covalently associated. Factor Va is inactivated by proteolysis of the heavy chain by activated protein C (APC)(^1)(4, 5, 6, 7) . The mechanism of inactivation is an ordered and sequential event with cleavage at Arg necessary for optimum exposure of the inactivating cleavage sites at Arg and Arg(6) . Cleavage at Arg occurs only on the membrane-bound cofactor(6, 7) .

Resistance to APC is defined as a poor anticoagulant response of plasma to APC in a clotting assay like-activated partial thromboplastin time(8) . It has been recently reported that APC resistance is the most common identifiable defect among patients with deep venous thrombosis (8, 9, 10) . The abnormal response to APC has been linked to a plasma protein, which was identified as factor V because when purified human plasma factor V at 4 times the normal plasma concentration (76 nM) was added to the plasma from a patient with severe APC resistance, the patient's plasma recovered its APC sensitivity(11) . It was thus concluded that factor V acts as a cofactor for the APC anticoagulant pathway(11) . The molecular defect in APC-resistant patients was recently identified in several laboratories as a single point mutation at nucleotide 1691 in the factor V gene (G A substitution) predicting a single amino acid substitution in the factor V molecule, Arg Gln(12, 13, 14, 15, 16) . Thus, the bulk of data demonstrate that APC resistance is due to an abnormal factor V molecule rather than to a deficiency of a cofactor for APC as initially proposed (11) . However, the possibility that the mutant factor V is defective with respect to the APC cofactor activity of the protein S/factor V mixture during factor VIIIa inactivation (17) cannot yet be excluded. The present study was undertaken in order to examine the APC-mediated inactivation of the factor V molecule that does not possess a cleavage site for APC at amino acid residue 506.


EXPERIMENTAL PROCEDURES

Materials, Reagents, and Proteins

Hepes, soybean trypsin inhibitor, Sepharose CL-4B, 1-palmitoyl-2-oleoyl-phosphatidylserine (PS), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (PC) were purchased from Sigma. The chemiluminescent substrate, Luminol, was from DuPont NEN. Normal human factor V, human prothrombin, and human alpha-thrombin were purified as described(18, 19, 20, 21) . Human factor Va heavy chain that was used as an immunogen for the generation of the monoclonal antibody used in the present study was prepared as described(19) . Monoclonal antibody alphaHFVa#6 was prepared as described(22) . The antibody recognizes an epitope between amino acid residues 307-506 of the factor V molecule. The fluorescent thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide(23) , human plasma APC(24) , and human factor Xa (21) were provided by Paul Haley (Haematologic Technologies Inc., Essex Junction, VT). The alpha-thrombin inhibitor hirudin was from Genentech (South San Francisco, CA). Phospholipid vesicles composed of 75% PC and 25% PS were prepared as previously reported(25) . All reactions were performed in a buffer composed of 20 mM Hepes, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4 (HBS(Ca)).

Isolation of Patient Factor V

In the Leiden thrombophilia study (9, 13) nine patients with deep vein thrombosis were identified that are homozygous for the G A substitution in the factor V gene (resulting in one amino acid substitution, Arg Gln in the factor V molecule). Plasma was obtained in Leiden by manual plasmapheresis from two unrelated patients (patient I (90) and patient II (173)) after informed consent of the patients. Both patients were females, homozygous for the mutation (genotype 1691 AA), possessing normal levels of protein C and protein S. The APC-sensitivity ratio was 1.13 for patient 90 and 1.14 for patient 173. Plasma was collected on ACD (0.33% citric acid, 2.2% trisodium citrate, and 2.24% glucose). Directly after collection, the plasma was centrifuged for 20 min at 20,000 times g (at 4 °C) to render it platelet free. Following addition of soybean trypsin inhibitor (20 µg/ml) the plasma was frozen in 50-ml aliquots and stored at -80 °C until use. None of the patients was using oral anticoagulants at the time of plasmapheresis. Single chain factor V from the two unrelated patients was obtained following a previously described method with the omission of the phenyl-Sepharose column(18) , using an anti-factor Va light chain monoclonal antibody coupled to Sepharose CL-4B.

Factor V and Factor Va Proteolytic Inactivation by APC

Human factor V or alpha-thrombin-activated factor V (factor Va) in HBS(Ca) were incubated with APC in the presence of PCbulletPS vesicles. The concentrations of all reagents are given in the figure legends. At selected time intervals aliquots of the mixture were assayed for activity as described(7, 23) . At the same time intervals the factor V/factor Va samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When studying factor V inactivation the potential of contaminating alpha-thrombin in the APC preparations was controlled by addition of hirudin.

All gels were produced according to the method described by Laemmli (26) and stained with Coomassie Blue. In some experiments the proteins were transferred to nitrocellulose as described (27) and probed with the monoclonal antibody alphaHFVa#6 as recently detailed (28) using the chemiluminescent substrate Luminol. NH(2)-terminal sequencing of the patient's factor V and factor Va fragments from polyvinylidene difluoride membranes was performed as described (7) in the laboratory of Dr. Alex Kurosky (University of Texas, Medical Branch at Galveston, Galveston, TX).


RESULTS

Normal plasma factor V, factor V(I), and factor V in the presence of PCbulletPS vesicles demonstrate similar cofactor activity (750 nM IIa/min) following 15 min of incubation with alpha-thrombin. Following 5 min of incubation with APC, normal factor Va lost 95% of the heavy chain and virtually all of its cofactor activity ( Fig. 1and 2A). The pattern of cofactor activity loss corresponds to cleavage at Arg, followed by cleavage at Arg and Arg(6) . These cleavages result in the appearance of fragments of M = 45,000 and 30,000 and a doublet of M = 22,000/20,000 (Fig. 2A, where the M = 75,000 fragment intermediate of the inactivation process (lanes5 and 6) and the M = 30,000 fragment (lanes5-11) are detected). For the patient's factor Va, under similar experimental conditions, following 5 min of incubation of APC, the loss of approximately 90% of the heavy chain corresponds to a 50% loss in cofactor activity (Fig. 1, inset; Fig. 3A, lanes 4-6; and Fig. 2B, lanes 5-7). The complete disappearance of the heavy chain of the cofactor when using patient's factor Va corresponds to cleavage at Arg as determined by amino acid sequencing of the corresponding fragments. Cleavage at that position results in the generation of a M = 45,000 fragment and of a M = 62,000/60,000 doublet (Fig. 3A, lanes 4-6). Subsequently, complete loss in cofactor activity correlates with cleavage of the M = 62,000/60,000 doublet at Arg and generation of a M = 56,000/54,000 doublet (Fig. 3A, lanes 7-13). The position of the cleavage site at Arg is deduced from our previous findings (6) and from the fact that the fragments of M = 62,000 and 56,000 have the same NH-terminal sequence starting at amino acid residue 307, suggesting a precursor-product relationship. Thus, the difference between the two fragments resides at the COOH terminus. These results were confirmed using a monoclonal antibody directed against the heavy chain of the cofactor that recognizes an epitope located between residues 307-506 of the cofactor. In normal factor Va, during inactivation by APC, this antibody recognizes the M = 30,000 fragment (Fig. 2A), whereas when studying factor Va inactivation, the antibody recognizes the M = 62,000/60,000 and 56,000/54,000 doublets (which contain the region 307-709 and 307-679, respectively, of the factor Va heavy chain) (Fig. 2B). Thus, it must be concluded that cleavage only at Arg is responsible for the loss of approximately 30% in cofactor activity. Thus, cleavage at Arg is necessary for optimum exposure of the cleavage sites at Arg and Arg, which in turn are required for cofactor inactivation (Fig. 1, filled circles). In the absence of the cleavage site at Arg the rate of inactivation of factor Va is slower than that observed for normal factor Va (Fig. 1, filled triangles and filled squares).


Figure 1: Inactivation of membrane-bound factor Va by APC. Normal plasma factor V, factor V(I), and factor V (0.28 µM) were incubated with PCbulletPS vesicles (100 µM) for 5 min at 37 °C. alpha-Thrombin was added at 1 unit/ml (12 nM). Following 15 min of incubation at 37 °C hirudin was added (20 nM). The activity of factor Va was monitored as described (7, 23) in the presence of 1.4 µM prothrombin, 3 µM dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, and 20 µM PCbulletPS vesicles, using factor Va and factor Xa at 1 and 10 nM, respectively (final concentrations). APC was then added (5.4 nM). At selected time intervals aliquots were assayed for cofactor activity. The initial rates of thrombin formation were calculated and plotted as percent of initial cofactor activity as a function of time following addition of APC. Filled circles represent membrane-bound normal plasma factor Va treated with APC; the filled triangles depict membrane-bound factor Va treated with APC, whereas the filled squares show membrane-bound factor Va(I) inactivated by APC. The inset shows the analysis of the inactivation reaction during the first 10 min. At the same time intervals aliquots were withdrawn and analyzed by SDS-PAGE. The products resulting from the proteolytic inactivation of membrane-bound factor Va (filled triangles) are shown in Fig. 3A, whereas the comparison of the immunoreactivity of the fragments resulting from the inactivation of plasma-derived factor Va and factor Va(I) toward a monoclonal antibody that recognizes an epitope between amino acids 307-506 is displayed in Fig. 2, A and B.




Figure 2: Analysis of factor Va inactivation by APC. The samples assayed for cofactor activity in Fig. 1(normal plasma factor Va and factor Va(I)) were also analyzed on a 4-15% linear gradient SDS-PAGE. Following transfer to nitrocellulose fragments were visualized using monoclonal antibody alphaHFVa#6. Panel A, normal factor Va control; panel B, factor Va(I). Lane1, factor V in the presence of PCbulletPS vesicles, no alpha-thrombin, and no APC; lanes 2-4, factor V with alpha-thrombin at 2, 5, and 15 min; lanes 5-11, membrane-bound factor Va with APC at 1, 3, 5, 10, 15, 30, and 60 min. The position of the molecular weight markers is indicated at left of panelA.




Figure 3: Cleavage of membrane-bound factor Va and factor V by APC. Panel A, factor V was incubated with PCbulletPS vesicles and alpha-thrombin as described in the legend to Fig. 1. Following 15 min of incubation, hirudin and APC were added. At selected time intervals aliquots of the mixture, before and after addition of alpha-thrombin, were withdrawn and mixed with 2% SDS, 2% beta-mercaptoethanol, heated at 90 °C, and analyzed on a 4-15% linear gradient SDS-PAGE gel. The factor Va subunits and fragments were visualized by Coomassie Blue staining (the same aliquots were also analyzed for cofactor activity and are shown in Fig. 1, filled triangles): lane1, factor V, control; lanes2 and 3, factor V with alpha-thrombin at 5 and 10 min; lanes 4-13, factor Va and APC at 30 s and 2, 4, 7, 10, 15, 30, 45, 60, and 120 min. Lane14, factor V in the presence of PCbulletPS vesicles following 2 h of incubation at 37 °C. Panel B, factor V was incubated with PCbulletPS vesicles and APC as described in the legend to Fig. 4. At selected time intervals aliquots of the mixture were withdrawn and mixed with 2% SDS, 2% beta-mercaptoethanol, heated at 90 °C, and analyzed on a 4-15% linear gradient SDS-PAGE gel (at the same time intervals aliquots were also assayed for activity and are shown in Fig. 4, filled triangles). Lane1, factor V, control; lanes 2-10, factor V and APC at 1, 3, 5, 10, 15, 20, 30, 60, and 120 min. Position of the molecular weight markers (times 10) is indicated at the left of panelA.




Figure 4: Inactivation of membrane-bound factor V by APC. Normal plasma factor V, factor V(I), and factor V (0.22 µM) were incubated with PCbulletPS vesicles (200 µM) for 5 min at 37 °C. APC and hirudin were then added (4.3 and 20 nM, respectively). At various time intervals aliquots of the mixture were assayed for cofactor activity. The initial rates of thrombin formation (at the steady state) were calculated and plotted as percent of initial activity as a function of time following addition of APC. Filled circles represent membrane-bound normal plasma factor V treated with APC; the filled triangles depict membrane-bound factor V treated with APC, whereas the filled squares show membrane-bound factor V(I) inactivated by APC. The inset shows the analysis of the inactivation reaction during the first 30 min. At the same time intervals aliquots were withdrawn and analyzed by SDS-PAGE. The products resulting from the proteolytic inactivation of membrane-bound factor V (filled triangles) are shown in Fig. 3B, whereas the comparison of the immunoreactivity of the fragments resulting from the inactivation of plasma-derived normal factor V and factor V(I) toward a monoclonal antibody that recognizes an epitope located between amino acids residues 307-506 is displayed in Fig. 5, A and B. The dottedline at the bottom of the graph represents the progress of the reaction in the absence of added factor V (i.e. the generation of alpha-thrombin by factor Xa alone).




Figure 5: Analysis of factor V inactivation by APC. The samples assayed for activity in Fig. 4(normal plasma factor V and factor V(I)) were also analyzed on a 4-15% linear gradient SDS-PAGE. Following transfer to nitrocellulose, fragments were visualized using monoclonal antibody alphaHFVa#6. Panel A, normal factor V control; panel B, factor V(I). Lane1, factor V in the presence of PCbulletPS vesicles and no APC; lanes 2-10, membrane-bound factor V with APC at 1, 3, 5, 10, 15, 20, 30, 60, and 120 min. The position of the molecular weight markers is indicated at left of panelA.



In contrast to the results seen for the abnormal factor Va preparations, no difference in the inactivation rates for membrane-bound normal factor V and membrane-bound factor V(I) and factor V was observed (Fig. 4). These data confirm our previous findings that optimum cleavage at Arg on the membrane-bound procofactor does not require prior cleavage at Arg(6) . Analysis of the proteolytic fragments deriving from membrane-bound factor V following digestion by APC showed the appearance of a M(r) = 45,000 fragment, which is produced slightly faster than the M(r) = 54,000 fragment (Fig. 3B). NH(2)-terminal sequence of the two fragments demonstrated that the M(r) = 45,000 fragment has the same NH(2) terminus as factor V, whereas the M(r) = 54,000 fragment has an NH(2)-terminal sequence matching a portion of factor V starting at amino acid residue 307. These data were confirmed using the monoclonal antibody that recognizes the epitope located between amino acid residues 307-506 of the cofactor. Following cleavage by APC the antibody recognizes the M(r) = 30,000 fragment (Fig. 5A) from normal plasma factor V, whereas for factor V(I) inactivation, the antibody recognizes the M(r) = 54,000 fragment (Fig. 5B). Altogether these data lead to the conclusion that cleavage at Arg and Arg occurs almost simultaneously during inactivation of membrane-bound factor V(I) and factor V.


DISCUSSION

The inactivation of membrane-bound normal factor Va is an ordered and sequential event with the first cleavage occurring at Arg of the heavy chain (Fig. 6). This is followed by cleavage at Arg and Arg, which results in loss of cofactor activity. The M(r) = 75,000 fragment, which is the consequence of cleavage at Arg, is therefore an intermediate in the inactivation process (Fig. 6). Our data demonstrate that membrane-bound factor Va from patients with APC resistance lacking the cleavage site at Arg is still inactivated following cleavage at Arg and Arg (Fig. 6). However, the rate of inactivation is slower than in the normal molecule. Thus, cleavage at Arg appears to promote, but is not essential for, cleavage at Arg and Arg. In contrast, no differences in the inactivation rates are observed when studying inactivation of membrane-bound factor V from normal or APC-resistant individuals. Direct activity measurements, NH(2)-terminal sequencing, and immunoblotting experiments confirmed our previous findings (6) that, whereas rapid inactivation of membrane-bound factor Va requires cleavage at Arg prior to cleavage at Arg and Arg, membrane-bound human factor V does not require prior cleavage at Arg for inactivation. Since inactivation by APC of only the abnormal factor Va, not factor V, is retarded (hence factor Va lingers at the place of vascular injury) and since individuals with Arg Gln mutation have a poor anticoagulant response to APC, which in turn is associated with an increase in risk of developing deep vein thrombosis (7-fold increase for the heterozygous and 80-fold increase for the homozygous)(29) , it is likely that the primary physiological substrate for APC in vivo is factor Va, not factor V.


Figure 6: Schematic representation of the cleavage of human factor Va heavy chain during inactivation by APC. The heavy chain of the human cofactor (containing 709 amino acids) is composed of two A domains (A1-A2) associated through a connecting region (amino acids 304-316)(2) . Normal plasma factor Va is rapidly inactivated following three cleavages of the heavy chain; cleavage at Arg (k(1)), which is required for optimum exposure of the cleavage sites at Arg and Arg (k(2)), produces a M(r) = 75,000 intermediate. Thus, in the absence of a cleavage site at Arg, cleavage at Arg (k`(1)) and Arg (k`(2)), which are required for cofactor inactivation, occur much slower than on the precleaved (at Arg) heavy chain.



Recent data demonstrate that, in the absence of a membrane surface, while no cleavage at Arg occurs, factor Va is cleaved at Arg followed by cleavage at Arg resulting in loss of approximately 30% of cofactor activity(6) . We have also demonstrated that cleavage at Arg on the membrane-bound cofactor is impaired when using recombinant APC with the Glu Ala substitution (APC). Impaired cleavage at Arg results in a cofactor that is cleaved at Arg and Arg and still possesses 60% cofactor activity(30) . The present data, obtained using a factor V molecule that does not possess a cleavage site for APC at position 506, together with our previous findings (6, 30) suggest that: 1) cleavage at Arg on the membrane-bound cofactor is responsible for the loss in approximately 70% of cofactor activity; 2) cleavage at Arg, which is lipid-independent, is responsible for loss in the remaining (30%) cofactor activity. The overall data indicate that cleavage at Arg alone in normal factor Va has no consequence on cofactor activity; however, this cleavage is necessary for efficient exposure of the cleavage sites at Arg and Arg, which are associated with cofactor inactivation. In the absence of cleavage at Arg the inactivation process proceeds at a significantly reduced rate.

The study of the plasma from patients with APC resistance has been the subject recently of intense investigation worldwide. Surprisingly, the bulk of functional data generated has created much confusion among the scientific community rather than understanding(31, 32, 33, 34, 35, 36) . It has even been proposed that APC resistance may have disappeared with time in certain patients(35) . The possibility of an acquired and transient APC resistance has also been proposed(36) . Our data may explain some of these assay discrepancies.

It is preferable that individuals that suffered a thrombotic event should first be screened for the mutation as suggested (32) by DNA analysis and the results confirmed by the APC resistance assay. Finally, it is important to recognize that APC resistance (by clotting assay) is observed in healthy individuals (5% of the population) carrying the Arg Gln mutation, who do not present symptoms of deep vein thrombosis(9, 12, 13) . Additionally, it is well known that a single hit is in general insufficient to promote thrombosis. Thus, the existence of other (perhaps compensatory) mutations in the factor V gene that can influence APC sensitivity (i.e. alterations in the lipid binding sites of factor V or in the APC binding sites of the cofactor or a mutation in the phosphorylation site at Ser or a mutation that changes the three-dimensional structure of factor V) cannot and should not be excluded.


FOOTNOTES

*
This work was supported by Merit Award R37 HL34575 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Given Bldg., Health Science Complex, University of Vermont, College of Medicine, Burlington, VT 05405-0068.

(^1)
The abbreviations used are: APC, activated protein C; PS, 1-palmitoyl-2-oleoyl-phosphatidylserine; PC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We express our gratitude to Alex Kurosky and Steve Smith from the University of Texas, Medical Branch at Galveston, for NH(2)-terminal sequencing of factor V fragments from polyvinylidene difluoride membranes and to Paul Haley for providing purified plasma human factor Xa and human APC. We thank Scott Doscher for excellent technical assistance.


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