Mechanism of Factor Va Inactivation by Plasmin

LOSS OF A2 AND A3 DOMAINS FROM A Ca2+-DEPENDENT COMPLEX OF FRAGMENTS BOUND TO PHOSPHOLIPID*

Abed R. Zeibdawi and Edward L. G. PryzdialDagger

From the Research and Development Department, the Canadian Blood Services and the Department of Biochemistry, Microbiology, and Immunology, the University of Ottawa, Ottawa, Ontario K1G 4J5, Canada

Received for publication, May 31, 2000, and in revised form, February 1, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The coagulation cofactor Va (FVa) is a noncovalent heterodimer consisting of a heavy chain (FVaH) and a light chain (FVaL). Previously, the fibrinolytic effector plasmin (Pn) has been shown to inhibit FVa function. To understand this mechanism, the fragmentation profile of human FVa by Pn and the noncovalent association of the derived fragments were determined in the presence of Ca2+ using anionic phospholipid (aPL)-coated microtiter wells and large (1 µm) aPL micelles as affinity matrices. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed and their NH2 termini sequenced. These had apparent molecular weights and starting residues as follows (single letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828), 40(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from FVaL; and 65(A1), 50(A1), 45(A1), 34(S349), 30(L94), 30(M110), and 3 small <5(W457, W457, and K365) kDa from FVaH. Of these, 50(L1766), 48(1766), 43(Q1828), and 40(Q1828) spanning the C1/C2 domains, and 30(L94), but not the similar 30(M110), positioned within the A1 domain remained associated with aPL. These were detected antigenically during Pn- or tissue plasminogen activator-mediated lysis of fibrin clot formed in plasma. Chelation by EDTA dissociated the 30(L94)-kDa fragment, which was observed to associate with intact FVaL upon recalcification, indicating that the Leu-94 to Lys-109 region of the A1 domain plays a critical role in the FVaL and FVaH Ca2+-dependent association. By using domain-specific monoclonal antibodies and an assay for thrombin generation, loss of FVa prothrombinase function was coincident with proteolysis at sites in the A2 and A3 domains resulting in their dissociation. Inactivation of FV or FVa by Pn was independent of the thrombophilic R506Q mutation. These results identify the molecular composition of Pn-cleaved FVa that remains bound to membrane as largely A1-C1/C2 in the presence of Ca2+ and suggest that Pn inhibits FVa by a process involving A2 and A3 domain dissociation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Factor V (FV)1 is a central pro-cofactor in the blood coagulation cascade, which must be proteolytically activated to factor Va (FVa) for clot formation (1-4). Activated platelets, monocytes, and endothelial cells present at the site of vascular injury expose anionic phospholipid (aPL; e.g. phosphatidylserine (PS)) on their membrane surface providing a site of association for FVa and the serine protease factor Xa (FXa) in the presence of Ca2+. Once formed, this macromolecular complex, termed prothrombinase, enhances the FXa-mediated activation of prothrombin to thrombin by 5 orders of magnitude (5), which is necessary for physiological clot formation.

Human FV is secreted into plasma as a single chain glycoprotein (2196 amino acids) composed of three homologous A domains and two homologous C domains and a unique B domain arranged in the order NH2-A1-A2-B-A3-C1-C2-COOH (6, 7). FVa is a heterodimer composed of an NH2-terminally derived heavy chain (FVaH; A1/A2 domains; molecular mass = 104 kDa, amino acids 1-709) and a COOH-terminally derived light chain (FVaL; A3/C1/C2 domains; molecular mass = 74/71 kDa; amino acids 1546-2196). FVaH and FVaL are noncovalently associated in the presence of a Ca2+ (Kd = 6 × 10-9 M) (8), and both chains are essential for full biological activity (9, 10). Addition of a chelating agent such as EDTA disrupts this interaction (9-11) which can then be regained by recalcification (8, 10, 12, 13) resulting in the expression of a single Ca2+-binding site (Kd = 24 × 10-6 M) (13). Ca2+ binds to neither purified FVaH nor FVaL (13), suggesting the Ca2+-binding pocket is conformational or formed by both FVaH and FVaL. Localization of the Ca2+-binding site has been consequently elusive.

Proteolytic activation and inactivation of FV/FVa plays a central role in regulating coagulation. At the site of vascular injury, FV is activated through a series of cleavages predominantly by thrombin, at Arg-709, Arg-1018, and Arg-1545, which excises the B domain to yield FVa (7). Factor V is initially activated by FXa in an aPL and Ca2+-dependent manner (2). FVa is also inactivated by proteolysis. The well studied anticoagulant, activated protein C (APC), inhibits FVa by cleaving FVaH (14), leading to dissociation of the A2 domain (15). The known APC-dependent cleavage of FVaL has no known function. Both FV and FVa can also be inactivated by an additional protease, the fibrinolytic protease plasmin (Pn), although inactivation of FV is preceded by a brief phase of activation (16, 17). Pn further influences coagulation by inactivating factor VII (18), factor VIII (19), factor IX (20, 21), factor X/Xa (22, 23), and factor XII (24). Work from our laboratory has suggested a further communication mechanism between coagulation and fibrinolysis where inhibition of aPL-associated FVa coagulation activity by Pn results in a species that accelerates Pn generation (25). These combined observations strongly suggest that Pn participates in maintaining balance between the opposing hemostatic pathways, coagulation and fibrinolysis. To understand the anticoagulant function of Pn, the structural basis by which FVa coagulation cofactor activity is inhibited by Pn is reported in the current work. The unique fragmentation profile mediated by Pn has further enabled localization of the Ca2+-dependent contacts between FVaL and FVaH.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Chemicals and Reagents-- HEPES, EDTA, PS, phosphatidylcholine (PC), bovine serum albumin, polyethylene glycol (PEG, average molecular weight 8000) (Sigma), aprotinin (Calbiochem), H-D-Phe-pipecolyl-Arg-pNA.2HCl (S2238, Chromogenix), Tween 20 (Fisher), and a chemiluminescent detection system (ECL, Amersham Pharmacia Biotech) were obtained commercially. Small unilamellar vesicles (SUV; average diameter 50 nm) consisting of 75:25% mixture of PC/PS were prepared (25) and quantified as described (26). Large vesicles (LV; 300-600 nm) were made by extrusion using a Liposofast Basic apparatus (Avestin Inc.). LV consisting of 1-4 mg of 75:25% PC/PS was suspended in 1 ml of methanol in glass tubes and dried to a thin film under a steady stream of nitrogen at 4 °C. The phospholipid was resuspended in 0.5-1 ml of 20 mM HEPES, 300 mM sucrose, pH 7.4, vortexed for 1 min, and subjected to 10 freeze-thaw cycles. The suspension was then extruded through two 1-µm filters (Avestin, Inc.) and collected. The vesicles were then mixed 1:1 with 20 mM HEPES, 150 mM NaCl, pH 7.4 (HBS/Ca2+), and harvested by centrifugation at 13,000 × g at 22 °C for 5-10 min. The pellet was resuspended in HBS and quantified using an assay for total phosphorus (27).

Proteins-- Human FVa, FXa, prothrombin, plasminogen, Pn, monoclonal antibodies (mAb) specific for human FVaH (AHV-5146) and human FVaL (AHV-5112), and polyclonal sheep anti-human FV (PAHFV-S) were from Hematologic Technologies, Inc. mAb specific for FVa A3 domain (anti-FVaA3) was produced by Dr. R. Lemieux (Hema-Quebec) for which in-house recombinant FVaL produced using the baculovirus system and purified by electroelution was used as antigen. Polymerase chain reaction-screened R506Q homozygous plasma was a generous gift from Dr. M. C. Poon, University or Calgary, Canada. Rabbit antiserum to human plasminogen (Calbiochem), tissue plasminogen activator (tPA, Genentech), peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Inc.), peroxidase-conjugated goat anti-rabbit IgG (Promega), assayed reference plasma (Helena Laboratories), and thromboplastin (Sigma) were commercially obtained.

Factor Va Proteolysis by Pn-- FVa (0.1 µM) was incubated with Pn (0.1 µM) in the presence of SUV (250 µM) or LV (600 µM) and Ca2+ (2 mM) in HBS/Ca2+ at 22 °C. The digests were sampled over time and heated at 95 °C for 5 min in sample buffer (2% SDS, 2% 2-mercaptoethanol, 0.325 M Tris base, pH 6.5, 10% glycerol and 0.001% bromphenol blue). The digests were subjected to 10% SDS-PAGE (28), stained with Coomassie Blue, or electrotransferred to polyvinylidene fluoride (PVDF; Immobilon-P, Millipore) (29). The PVDF membrane was blocked at 22 °C for 1 h in milk (5%), and cleavage products of FVa were detected by Western analysis using chemiluminescence (ECL).

FVa was also cleaved by Pn when bound to aPL-coated microtiter wells prepared as described previously (25) except that the wells were blocked with gelatin (5 mg/ml) in HBS/Ca2+, and 0.001% Tween 20 was included in the wash buffer. After equilibration of the FVa with the aPL-coated well in HBS/Ca2+, the wells were washed and Pn (0.1 µM) in HBS/Ca2+ was added. At selected time points, four wells were quickly washed, and bound protein was eluted with 25 µl of SDS-PAGE sample buffer/well and pooled, heated to 95 °C for 10 min, and subjected to 10% SDS-PAGE. To visualize cleavage products, gels were either stained with Coomassie Blue or transferred to PVDF and detected with anti-FVaA3, anti-FVaL, anti-FVaH, or anti-human FV.

FVa Proteolysis by Pn in a Clot-- To determine whether Pn-cleaved FVa fragments observed in a purified system could also be identified in a physiological setting, FVa fragmentation in a lysing clot was studied. In these experiments, pooled plasma (2.5 µl of pooled plasma) was clotted by treatment with thromboplastin (80 µg/µl) after recalcification (10 mM CaCl2). Individual reactions were incubated for various times with Pn (50 nM) or tPA (0.5 nM) at 37 °C in a 7.5-µl final volume of HBS. The reactions were stopped by SDS, run on 10% SDS-PAGE, transferred to PVDF, and visualized by Western blotting with anti-FVaL and anti-FVaH antibodies. To resolve better the fragments of interest a concentration of acrylamide was used that in most cases does not allow the single chain FV to enter the gel (10%).

Amino Acid Sequencing-- NH2-terminal sequencing of Pn-mediated FVa fragments transferred to PVDF after reducing SDS-PAGE was conducted as described (30, 31). To sequence exclusively the FVa-derived fragments remaining bound to aPL in the presence of Ca2+ or EDTA, FVa digestions were conducted in the presence of LV, and bound fractions were separated by centrifugation prior to electrophoresis. Individual bands were excised for sequence analysis.

Effect of EDTA on Dissociation of Pn-cleaved FVa Fragments-- FVa (0.1 µM) was cleaved with Pn (0.1 µM) for selected time points on aPL-coated wells as described above except that an excess amount of aprotinin (50 kallikrein-inactivation units/ml; KIU/ml) was added to inhibit residual Pn activity. aPL-bound fragments were incubated at 22 °C for 3 h in HBS/Ca2+ or in HBS with EDTA (10 mM, HBS/EDTA) to allow for dissociation of Ca2+-dependent interactions. Protein samples were subjected to 12% SDS-PAGE, transferred to PVDF, and visualized with anti-FVaH and anti-FVaL. To determine whether Pn remains associated with the cleaved fragments, antiserum to human Pgn (30 ng/ml) was used on identical membranes.

FVa Subunit Dissociation Kinetics-- The 30-kDa fragment and FVaH dissociation with EDTA (10 mM) was monitored electrophoretically. FVa bound to aPL-coated wells was cleaved with Pn (0.1 µM) for 1 h, and Pn proteolytic activity was inhibited with aprotinin (50 KIU/ml). Wells were washed three times, and 200 µl of HBS/EDTA (10 mM) was added for selected time points. Dissociation of FVaH due to Ca2+ chelation was monitored after SDS-PAGE by Western blotting. In a parallel set of experiments, FVa cofactor activity for thrombin generation was assessed chromogenically (0.5 µM S2238). Following the last wash, factor Xa (0.06 µM) and prothrombin (1.0 µM) in 100 µl HBS/Ca2+ were added at 22 °C for 5 min, and the change in absorbance at 405 nm was monitored using a Vmax spectrophotometer (Molecular Devices).

Ca2+-dependent Association of the 30-kDa Fragment with FVaL-- To determine whether the 30-kDa fragment of FVaH can associate with FVaL, we had to initially obtain and concentrate sufficient amounts of the 30-kDa fragment. An aPL-coated plate was coated with FVa (300 ng/well) that was then cleaved with Pn (0.1 µM). Supernatants from all the wells (19 ml) were pooled, and the mixture was concentrated by repeated cycles of centrifugation on a Biomax-10 membrane (Millipore) to ~2 ml. To determine whether the 30-kDa fragment can associate with intact FVaL, FVaH was separated from aPL-immobilized FVaL with EDTA for 3 h at 22 °C. The 30-kDa fragment was then incubated in HBS/Ca2+ or HBS/EDTA with FVaL remaining bound to the aPL-coated well at 22 °C for 2 h and then at 37 °C for 30 min. The wells were washed, and remaining protein was eluted with sample buffer. Association of the 30-kDa fragment was evaluated with anti FVaH by Western blotting.

Inactivation of FV/FVR506Q and FVa/FVaR506Q by Pn-- Pooled normal plasma or R506Q homozygous plasma in HBS/EDTA was added to aPL-coated wells and was incubated for 30 min at 22 °C. The wells were washed three times with HBS/EDTA and then once with HBS/Ca2+. In one set of experiments, wells were incubated with 50 µl of HBS/PEG/Ca2+ for 5 min with gentle shaking. In another set of experiments, human FXa (5 nM) in HBS/PEG/Ca2+ was added to the wells and incubated for 5 min to preactivate the FV. The wells were then washed three times, and Pn (0.15 µM) was added in HBS/PEG/Ca2+ for various times. Following three more washes in HBS/Ca2+, FXa (1.0 nM) and prothrombin (1.0 µM) were added to the wells and incubated for 3 min. Thrombin activity was assessed chromogenically with S2238 (0.4 mM final).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

FVa Cleavage by Pn-- To identify fragments that are generated during inactivation of FVa by Pn in the presence of SUV and Ca2+, SDS-PAGE was conducted, which revealed a sequential cleavage pattern (Fig. 1). Both the 104-kDa FVaH and the 74/71-kDa doublet of FVaL were rapidly cleaved in a Pn-dependent manner within 1 h into products of ~50, 48, 45, 40, 34, 30, 12, and 7 kDa, along with several smaller fragments that migrated close to or at the dye front. To determine whether the digestion profile was aPL-dependent, experiments were performed in the absence of aPL vesicles but in the presence of Ca2+. It was found that the rate of digestion of the light chain was accelerated in the presence of aPL, whereas the rate of digestion of the heavy chain was not altered (not shown). Aprotinin completely inhibited the cleavage of FVa by Pn. No distinction in the size of fragments generated was observed when the digestion was performed in the absence or presence of aPL, although additional cleavages were observed when the interaction between FVaL and FVaH was inhibited by chelation (not shown).


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Fig. 1.   Time course of Pn cleavage of FVa bound to SUV. Purified human FVa (0.1 µM) was incubated with Pn (0.1 µM) in the presence of aPL-containing SUV (250 µM) in HBS/Ca2+ at 22 °C. At selected time intervals aliquots were withdrawn from the mixture and stopped immediately with SDS-containing sample buffer. Samples were separated on 10% SDS-PAGE and stained with Coomassie Blue. Incubation times (min) with Pn were as follows: 0 (lane 1), 1 (lane 2), 2 (lane 3), 4 (lane 4), 6 (lane 5), 8 (lane 6), 15 (lane 7), 30 (lane 8), 40 (lane 9), and 60 (lane 10).

Correlation between Pn-mediated FVa Fragments Remaining Bound to aPL and Loss of Cofactor Activity-- To determine which fragments remained noncovalently associated with aPL in the presence of Ca2+, FVa was equilibrated with aPL-coated microtiter wells, and fragments that remained bound to the plate after digestion with Pn were washed with buffer containing Ca2+. The remaining fragments were analyzed by Western blot using an FVaH- and two FVaL-specific mAb. These data showed that the fragments of 50, 48, 43, 40, and 30 kDa were light chain-derived (Fig. 2, A and B). Since the mAb in Fig. 2B recognizes the A3 domain, the completely different fragmentation pattern suggests the commercial mAb used in Fig. 2A has specificity for the COOH-terminal part of FVaL containing C1/C2. As shown, the 50- and 48-kDa FVaL fragments were converted to 43 and 40 kDa, and all remained bound to aPL. Since all aPL binding of FVa is mediated by FVaL, these fragments contain at least part of the Ca2+-independent membrane-binding site(s). Furthermore, we found that fragments of 50, 45, and 30 kDa were FVaH-derived and remained bound in the presence of Ca2+ (Fig. 2C). The 30-kDa FVaH-derived fragment also remained at least partly bound over the time course of the experiment. Comparable Ca2+-dependent complex of fragments was observed when beginning with bovine FVa (not shown). We did not observe a detectable difference in the rate of cleavage or the cleavage pattern, between experiments performed on aPL-coated plates, LV, or SUV (not shown). Furthermore, no detectable difference in the cleavage pattern was observed when the samples were electrophoresed under nonreducing conditions, indicating no disulfide-linked fragments were produced (not shown). Whereas all the aPL-bound bands, visualized by Coomassie Blue and Silver and polyclonal staining, were detected with the mAbs (not shown), an additional unbound 34-kDa fragment, released into solution upon cleavage, was observed with Coomassie Blue (Fig. 1).


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Fig. 2.   Time course of Pn cleavage of FVa subunits bound to aPL-coated wells. 96-Well microtiter wells were coated with aPL (0.3 µg) and then blocked with gelatin (5 mg/ml). The wells were preincubated with FVa (0.1 µM) for 30 min and then with Pn (0.1 µM) in HBS/Ca2+. At the selected time points, aPL-bound protein was removed from the wells with sample buffer and separated on 10% SDS-PAGE. The protein was transferred to PVDF and detected with anti-FVaL (A), anti-FVaA3 (B), or anti-FVaH (C). D, as in A, except that following incubation with Pn, FXa (0.06 µM), and prothrombin (1.0 µM) were added. Thrombin generation was monitored with S2238. The average of four experiments with standard deviation is shown. Incubation times (min) with Pn were as follows: 0 (lane 1), 1 (lane 2), 2 (lane 3), 4 (lane 4), 6 (lane 5), 8 (lane 6), 15 (lane 7), and 30 (lane 8), 40 (lane 9), and 60 (lane 10).

In order to correlate the appearance of degradation products with observed changes in FVa cofactor activity, fragments remaining bound to aPL were assayed for FVa function. Inactivation of FVa by Pn was rapid over the time course of the experiment (Fig. 2D). Following 8 min of incubation with Pn, aPL-bound FVa was completely inactive. The rapid loss of cofactor activity coincided with appearance of the 30-kDa A3 and the 48- and minor 50-kDa C1/C2-derived fragments and with the 45- and minor 50-kDa heavy chain-derived fragments.

Cleavage of FV and FVa by Pn in a Fibrin Clot-- By having established the fragment composition of purified Pn-inactivated FVa, we determined whether these fragments were also generated under more physiological conditions. Thromboplastin was added to plasma to generate a fibrin clot, and the FV/FVa fragmentation pattern was examined following either Pn- or tPA-induced clot dissolution. Western blots (Fig. 3) showed that the FVaH- and FVaL-derived fragmentation pattern was identical to those in our purified system regardless of whether Pn or tPA was used to induce clot fibrinolysis. These data support the physiological relevance of the purified system.


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Fig. 3.   Pn-cleaved FVa fragments in a lysing clot. Microcentrifuge tubes containing recalcified plasma were incubated with thromboplastin (80 µg/µl) in HBS at 37 °C for 3 min. Following clot formation, Pn (50 nM) or tPA (0.5 nM) was added to individual reactions for the indicated time points. The reactions were stopped with sample buffer, and the protein was separated on 10% SDS-PAGE, transferred to a PVDF membrane, and detected with anti-FVaL (top panels) or anti-FVaH (bottom panels). Pooled plasma (lane 1) incubation times (min) with Pn or tPA were as follows: 2 (lane 2), 15 (lane 3), 30 (lane 4), 60 (lane 5), 120 (lane 6), and 240 (lane 7).

Identification of Pn Cleavage Sites in FVa-- To identify the specific sites of Pn-mediated cleavage, amino acid sequence analyses were conducted on all Coomassie Blue-stained fragments derived from purified FVa. Fig. 4A shows the banding pattern of the fragments subjected to analysis, and the sequences are summarized in Fig. 4B. As expected, because of recognition by a single mAb, the 65-, 50-, 45-, and 30-kDa FVaH fragments overlap, and each contains at least a large portion of the A1 domain (starting at Leu-94). The 30-kDa FVaH band was found to be two co-migrating species, which differed by 16 amino acids starting at residues Leu-94 or Met-110 and hence were referred to as 30(L94) and 30(M110). The 34-kDa fragment not detected by the mAbs corresponded to the COOH-terminal end of FVaH and, unexpectedly, contained a conserved polymorphism at residue 352 (Leu to Thr) (Fig. 4B). This is the first report to our knowledge of this substitution, which is consistent with FVa being purified from pooled plasma. Once generated, the 34-kDa fragment was quickly proteolyzed into fragments of less than 7 kDa, of which several were sequenced. FVaL is cleaved by Pn to give 50-, 48-, 43-, and 40-kDa fragments that overlap as predicted by antigenicity and begin toward the COOH-terminal end of A3. By this method, a 30-kDa FVaL fragment corresponding to the NH2-terminal end of the light chain was distinguishable. This fragment was short lived and was processed into several smaller fragments of which two were sequenced.


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Fig. 4.   Identification of proteolytic cleavages in FVa by Pn. FVa (1.5 µM) was preincubated with aPL vesicles (300 µM) in HBS/Ca2+ for 15 min prior to cleavage by Pn (0.2 µM) at 22 °C for 1 h. The reaction was then stopped with SDS-containing sample buffer, heated at 95 °C for 5 min, and separated on large (12 × 14 cm) 8% SDS-PAGE. A, protein was transferred to PVDF membrane and stained with Coomassie Blue R-250 stain. B, NH2-terminal sequence of visible FVa fragments shown in A.

Ca2+-dependent Association of Pn-mediated FVa Fragments-- Since FVaH does not contain the aPL-binding site and is anchored to aPL through divalent metal ion-dependent interactions with FVaL, we investigated the effect of EDTA on the FVa fragments remaining bound to aPL. Digestion end point fragments that remained bound to aPL-coated wells after cleavage of FVa with Pn were subjected to an additional wash containing 10 mM EDTA. Interestingly, the 30-kDa component of FVaH and the 50- and 40-kDa FVaL-derived fragments dissociated from the aPL in the presence of EDTA (Fig. 5). Conversely, the FVaL 48- and 43-kDa fragments remained associated with aPL following EDTA treatment. Collectively, these data suggest that regions within the 48/43-kDa fragments of FVaL and the 30-kDa fragment of FVaH contribute to the Ca2+-dependent intramolecular association between FVaL and FVaH. It is unknown at present why apparent Ca2+-dependent differences exist between the 48/43- and 50/40-kDa fragments.


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Fig. 5.   Effect of EDTA on aPL-associated FVa fragments. FVa was treated with Pn (as in Fig. 2, 60 min), except that after incubating with Pn, Pn activity was inhibited with aprotinin (50 KIU/ml) in HBS/Ca2+ for 15 min. Wells were then incubated in HBS/Ca2+ (lane 1) or HBS/EDTA (lane 3) at 22 °C for 2 h. In lane 2, Pn was not added to the well prior to the addition of HBS/EDTA. Protein samples were separated on SDS-PAGE and transferred to PVDF. Western blot analysis was performed with a mixture of anti-FVaL and anti-FVaH.

To determine if the rate of EDTA-mediated dissociation of the FVaH-derived 30-kDa species from FVaL fragments is representative of dissociation of intact FVaH from intact FVaL, a time course was conducted (Fig. 6). Release of the 30-kDa species (Fig. 6A) or FVaH (Fig. 6B) from aPL-coated microtiter wells by EDTA was followed by Western blot. Although the general trend was comparable over the duration of the experiment, intact FVaH has an apparently faster initial phase of dissociation than the 30-kDa fragment. This may be indicative of a role for A2 in FVaL binding. As an additional means of following FVaL and FVaH dissociation, the effect of EDTA on FVa function was followed by measuring the loss of thrombin generation in a chromogenic assay (Fig. 6C). As expected, EDTA-mediated inhibition of FVa activity correlated with the dissociation of the 30-kDa fragment or intact FVaH from FVaL and are in agreement with the slow dissociation rates reported previously (8, 12). These results demonstrate that the Ca2+-dependent interactions observed for FVaPn are representative of intact FVa.


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Fig. 6.   EDTA-induced dissociation of FVaPn and FVa subunits and association of the 30-kDa species with FVaL. A, FVa was treated with Pn (as in Fig. 2, 60 min). After a brief wash with HBS/Ca2+, the wells were incubated with HBS/EDTA for the indicated times, and the 30-kDa FVaH fragment remaining bound to the aPL-coated well was detected by Western blotting. B, as in A except no Pn was included. C, as in B, except that following incubation with EDTA, factor Xa (0.06 µM), prothrombin (1.0 µM), and SUV (20 µM) were added. Thrombin generation was monitored with S2238. The average of triplicates with standard deviation is shown. Inset, as in B, except that following incubation with EDTA, Western blots were conducted to detect association of the 30-kDa fragment. Lane 1, no 30-kDa fragment added; lane 2, the 30-kDa fragment was added in the presence of HBS/Ca2+; lane 3, the 30-kDa fragment was added in HBS/EDTA.

The dependence of Ca2+ on the association of the 30-kDa FVaH fragment to intact FVaL was studied to investigate further whether the association we observed between Pn-mediated fragments of FVa reflect those within the intact molecule. aPL-coated microtiter wells were saturated with FVa and then treated with EDTA to dissociate FVaH, which was confirmed antigenically. The 30-kDa species of FVaH was then added in the presence of Ca2+, resulting in association specifically with intact FVaL (Fig. 6C, inset). Binding was found to be Ca2+-dependent since no detectable association was observed in the presence of EDTA. Significant Ca2+-dependent binding of the 30-kDa fragment to aPL-coated wells depended on the presence of FVaL (not shown). These data provide additional evidence that the 30-kDa fragment constitutes the FVaH-derived portion of a Ca2+-dependent contact region with FVaL.

Identification of aPL-bound Fragments following Pn Cleavage-- To identify by sequence analysis the species that remained bound to aPL in the presence or absence of Ca2+, LV were utilized as a preparative affinity matrix. In this experiment, aPL-bound FVa was pelleted by centrifugation after Pn treatment. As before, the 65- and 30-kDa FVaH-derived fragments and the 50-, 48-, 43-, and 40-kDa FVaL fragments were observed to remain bound to aPL in the presence of Ca2+. Of these only the 48- and 43-kDa fragments remained bound in EDTA (Fig. 7A). Sequence analyses (Fig. 7B) confirmed their identification. An unexpected observation was made concerning the co-migrating FVaH-derived 30-kDa fragments we identified in Fig. 4. After affinity fractionation, sequencing revealed that 30(L94) remained bound to aPL. In contrast, the similarly sized fragment starting at Met-110 was not observed to remain bound in the presence of Ca2+, suggesting that the 94-109-kDa region is important for the Ca2+-sensitive intrachain contact.


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Fig. 7.   Identification of the Pn-cleaved FVa fragments that dissociate in the absence of Ca2+. A, FVa (1.5 µM) was incubated with LV (600 µM) in HBS/Ca2+ and Pn (0.2 µM) as above. Proteolysis was stopped with aprotinin (50 KIU/ml). Following 3 h of incubation with EDTA, LV-bound fragments were separated by centrifugation. The pellet fraction (lane 1) and the supernatant fraction (lane 2) were then run on large (12 × 14 cm) 10% SDS-PAGE, transferred to PVDF, and stained with Coomassie Blue R-250 stain. B, NH2-terminal sequence of each fragment.

Loss of FVa and FVaR506Q Cofactor Activity by Pn-- To determine whether Pn-dependent inactivation of FV/FVa was affected by the common "Leiden" thrombophilic mutation, normal or R506Q FV was trapped from plasma on aPL-coated microtiter wells and cleaved by Pn. The activity profile of FV, as measured by thrombin generation in the presence of FXa, is shown in Fig. 8A. Pn treatment resulted in an initial activation and then inactivation. We observed that this change in FV activity by Pn was indistinguishable from that of FVR506Q. Incubation of FXa-preactivated FV with Pn resulted in an immediate loss of thrombin generating activity (Fig. 8B). An indistinguishable inactivation profile was detected when FVR506Q was used in these experiments. Collectively, these results clearly indicate that Pn-mediated activation/inactivation of FV and inactivation of FVa is independent of the R506Q substitution.


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Fig. 8.   Effect of Pn on plasma FV/FVa and FVR506Q/FVaR506Q prothrombinase activity. Pooled plasma or R506Q plasma was added to aPL-coated wells for 30 min. A, wells were incubated in HBS/PEG/Ca2+. B, human FXa (5 nM) was added for 5 min and washed. Pn (0.15 µM) was added at various times, inhibited with aprotinin, and then followed by FXa (1.0 nM) and prothrombin (1.0 µM). Thrombin activity was measured using S2238.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For over a decade, Pn has been known to induce a complicated cleavage pattern in FVa that causes complete inhibition of coagulation cofactor function. To understand how this functional effect is achieved, we have identified the location of the cleavage sites and present here a fragmentation map (Fig. 9A). This revealed that the A2 and A3 domains of FVa are proteolyzed at several sites resulting in their dissociation, which correlates to inactivation of the molecule. This process is analogous to the A2 dissociation correlated to the inactivation of FVa by APC (15) and for the spontaneous decay of FVIIIa activity (32). However, the loss of A3 from aPL-bound FVa as part of an inhibition mechanism is novel. Since FXa also cleaves FVa at Arg-348 and Arg-1765 without apparent loss of FVa coagulation cofactor activity (14), our results demonstrate that additional cleavages within these fragments and at Lys-1827 ultimately result in the observed complete loss of FVa activity. Cleavage of FVaH by Pn appears to occur by two cleavage pathways (Fig. 9B) that ultimately lead to the loss of the A2 domain and the generation of a 30-kDa doublet. In the major cleavage pathway, the 30-kDa species is generated from a prominent 45-kDa NH2-terminal fragment. In the minor cleavage pathway, the 30-kDa fragment is generated from a 50-kDa intermediate NH2-terminal fragment that is produced from a 65-kDa fragment. The COOH-terminal fragments from both pathways are quickly proteolyzed to smaller fragments that migrate close to or at the dye front (<5 kDa). In contrast, the 30-kDa fragment was observed to persist throughout the time of the experiment.


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Fig. 9.   Cleavage of FVa by Pn in the presence of Ca2+. A, Pn-mediated fragmentation map of FVa was constructed to scale based on NH2-terminal sequence and the apparent molecular weight of fragments which was relativized to the average molecular weight of an amino acid (110 Da). The estimated fragment number of amino acids in each is complicated by glycosylation not included in the estimate of fragment length. Disulfide linkages are indicated by thin lines. B, probable order of cleavage is shown. The predicted minor cleavage pathway is depicted by dotted arrows.

Cleavage of FVaL with Pn resulted in the rapid proteolysis of the 30-kDa (A3 domain) NH2-terminal fragment ultimately leading to dissociation of the A3 domain from the aPL-bound protein and hence the conversion of the 74/71-kDa FVaL into the 40- and 43-kDa COOH-terminal fragments. We suggest that the 50- and 48-kDa fragments are intermediates of the 40- and 43-kDa fragments, respectively, because of similar Ca2+-dependent aPL-binding properties of the 50/40- and 48/43-kDa species.

To our knowledge, this study is the first to describe the Pn cleavage sites within human FVa, although in a preliminary report Kalafatis and Mann (33) presented NH2-terminal amino acid sequence of Pn-cleaved fragments from bovine FVa. The identity of some of these species differed from those presented here for human FVa, with cleavages at Arg-348, Lys-1656, and Arg-1765 being in agreement.

To determine if the Pn-derived FVa fragments we observed in purified systems can occur in a physiological setting, we tested whether these fragments are generated following either Pn or tPA treatment of plasma-derived clot. The data showed that the 30-kDa fragment of FVaH and the four light chain-derived fragments are generated in clots lysed by either Pn or tPA. Together, these results support the hypothesis that at the site of clot formation, where the local concentration of the coagulation cofactor is elevated, Pn processing of FVa may have a physiological function. In further support of physiological relevance, Tracey et al. (34) previously reported the extent of FV proteolysis during thrombolytic therapy with tPA and found that cleavage of FV is primarily Pn-mediated. As an extension of this report, we found no detectable difference between fragments that remain bound to aPL following Pn treatment of FV or FVa (not shown). Independent observations of a FV fragmentation pattern in clots similar to ours have been made (35) where the authors speculated a Pn-dependent origin. We cannot, however, omit the possibility that cell-dependent processes may influence the cleavage profile, such as the requirement of cell-surface thrombomodulin for APC function or cathepsin G elastase from monocytes (36).

The contribution of aPL to the assembly of prothrombinase components, and to the localization of thrombin production to the site of injury, has been well documented (37-42). Furthermore, it has been shown that FVaL confers all of the aPL-binding function of FVa (11, 43-47) and that this interaction is Ca2+-independent (11, 46, 48). Several studies suggest that the C2 domain contains the aPL-binding region of FVaL (49, 50), whereas others have implicated the A3 (45) or both the A3 and C2 domains (47). By having identified the cleavages induced by Pn in FVa, we determined which fragments remained bound to aPL. Upon cleavage of FVa by Pn, four FVaL-derived fragments and one FVaH-derived fragment remained bound to aPL in the presence of Ca2+. Amino acid sequencing and antigenicity revealed that the four FVaL-derived species overlap, with the 50/48- and the 43/40-kDa fragments originating from a common cleavage site at Arg-1765 and Lys-1827, respectively. Each of these excludes a large portion of the A3 domain and includes the entire C2 domain, providing support for C2 function in aPL binding.

Of the four FVaL fragments that remained bound to aPL, only the 48- and 43-kDa fragments remained in the presence of EDTA. Since these are encompassed by the 50-kDa fragment and span the 40-kDa fragment, both of which dissociated from aPL due to chelation, an explanation is difficult to provide without further study. One possibility is related to the two forms of FVa that have been identified, designated FVa1 and FVa2, that differ in the COOH terminus of FVaL (51, 52). The difference has been suggested to be due to alternative glycosylation at Asp-2181 within the C2 domain (49, 53, 54) leading to a weaker association between FVa1 and aPL (51, 52, 55). Since we found that the 48-kDa fragment associated strongly with aPL, it is possible that the difference in the electrophoretic mobility between the 50- and 48-kDa species is due to derivation from FVa1 and FVa2, respectively. At this time we cannot exclude alternate processing at the COOH terminus by Pn resulting in the weaker aPL-binding 40-kDa fragment originating from the heavier 50-kDa species. An additional explanation for the different aPL-binding properties of overlapping fragments may be found in the two forms of the C2 domain recently observed by x-ray crystallography (56). These differ markedly in one of the three protruding hydrophobic loops implicated in aPL binding. Possibly, the Pn-mediated 50- and 40-kDa fragments originate from the FVaL type suggested by crystallography to have weaker aPL-binding. Notwithstanding, our observations provide further support for a role of the C2 domain in Ca2+-independent aPL binding.

It is well established that EDTA-dissociated FVaL and FVaH chains can reassociate in the presence of additional Ca2+ (Kd = 6 × 10-9 M) (8). Although Ca2+ does not bind to either of the individual FVa chains alone, it has been shown that a single high affinity binding site becomes available when the two chains are bound together (Kd = 24 × 10-6 M) (13). The first experimental evidence localizing the Ca2+-sensitive intrachain contact comes from our identification of two FVaH fragments with identical electrophoretic mobility. These differ at the NH2 terminus by only 16 amino acids. Of these, the fragment beginning at Leu-94 remained associated with FVaL-derived fragments in a Ca2+-dependent manner, whereas the similar species beginning at Met-110 did not interact with FVaL even in the presence of Ca2+. These data suggest that a Ca2+-sensitive site may be contained between Leu-94 and Lys-109. Consistent with this hypothesis, x-ray structure and molecular modeling, using homologous protein templates based on the ceruloplasmin and factor VIII A-type domains, have suggested a putative Ca2+ ion-binding site within the A1-A3 interface (57, 58). Interestingly, of the five negatively charged residues (Glu-96, Asp-102, Glu-108, Asp-111, and Asp-112) predicted to be involved in the Ca2+-binding pocket in the A1 domain (57), three residues are located between amino acids Leu-94 and Lys-109, and the remainder are neighboring. Although molecular modeling supports our finding that a Ca2+-sensitive region between Leu-94 and Lys-109 may exist, direct studies must be conducted to substantiate this likelihood.

To determine whether the observed Ca2+-dependent association between the fragments of Pn-cleaved FVa is representative of the intact FVaH/FVaL interaction, we demonstrated that the rates of EDTA-induced dissociation of the 30(L94) species from FVaPn and FVaH from intact FVa were comparable. Furthermore, the dissociation rate closely resembled the timing of FVa inactivation by EDTA. To demonstrate further the contribution of the region within FVaH corresponding to the 30-kDa fragment in the FVaL interaction, we followed the 30-kDa fragment association with aPL-bound intact FVaL. Our results demonstrate that the 30(L94) fragment can indeed associate with FVaL. Binding to the microtiter well was dependent on Ca2+, FVaL, and aPL. This observation is further evidence that the interaction between FVaH and FVaL is mediated at least in part through Leu-94 to Lys-345 within FVaH and Gln-1828 to Tyr-2196 (COOH-terminal half; i.e. C1/C2) in FVaL. Since EDTA is a nonselective chelator, we cannot exclude the role of other metal ions (10, 59) in this process in vivo; however, based on previous observations (10) and our data showing that EDTA-mediated dissociation of the 30(L94) fragment from FVaL is restored by recalcification, Ca2+ is strongly implicated.

In addition to the inhibitory function Pn has on FVa coagulant activity (16, 17), previous data from our laboratory has also suggested that Pn may convert FVa into a tPA accelerator (25). To determine whether these combined regulatory roles could be applied to a prevalent thrombotic risk factor, the effect of the R506Q "Leiden" mutation on Pn-mediated inactivation of FV/Va was studied. Our results showed that the rate of inactivation of FVR506Q/FVaR506Q by Pn is indistinguishable from normal FV/FVa, although the COOH termini of several small FVaH fragments we identified were predicted to be close to Arg-506. The R506Q mutation is known to inhibit the normal anticoagulant effects of APC on FV/Va (60). Therefore, the data presented here suggest that the essential physiological role of APC highlighted in thrombosis associated with FVaR506Q could be attenuated by administration of thrombolytics.

    ACKNOWLEDGEMENTS

We thank N. Lavigne for excellent technical assistance; T. Raynor and M. Derry for review of the manuscript; Dr. M. Issa (Canadian Blood Services, University of British Columbia) for sizing the LV; Dr. M. C. Poon (University of Calgary) for providing R506Q plasma; and S. Smith and Dr. A. Kurosky (Protein Chemistry Laboratory, University of Texas, Galveston Medical Branch) for expert amino acid sequence analysis. We thank Dr. J. Grundy for helpful suggestions regarding the manuscript and experimental design and for providing LV. We also thank Dr. R. Lemieux (Hema-Quebec) and Bogna Lasia for mAb production and purification, respectively.

    FOOTNOTES

* This work was supported by the Canadian Blood Services Research and Development Fund Grant CB50 928 and the Heart and Stroke Foundation of Canada Grant NA 4143.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger *To whom correspondence should be addressed: Canadian Blood Services, Research and Development Department, 1800 Alta Vista Dr., Ottawa, Ontario K1G 4J5, Canada. Tel.: 613-739-2201; Fax: 613-739-2426; E-mail: ed.pryzdial@bloodservices.ca.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M004711200

    ABBREVIATIONS

The abbreviations used are: FV, factor V; FVa, factor Va; Pn, plasmin; tPA, tissue plasminogen activator; APC, activated protein C; PC/PS, phosphatidylcholine/phosphatidylserine; FVaPn, Pn-cleaved FVa; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; LV, large vesicles; PVDF, polyvinylidene fluoride; mAb, monoclonal antibody; SUV, small unilamellar vesicles; KIU, kallikrein-inactivation units.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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