Phospholipid-Binding Domain of Factor VIII Is Involved in Endothelial Cell–Mediated Activation of Factor X by Factor IXa
Apparently quiescent, nonapoptotic endothelial cells mediate the activation of factor X by activated factor IX in the presence of its cofactor, activated factor VIII. In a previous study, we reported that during the activation of factor X, the interaction of the cofactor with the endothelial cell membrane clearly differs from the interaction of the cofactor with artificial lipid membranes. In the present study, we identified the peptide domain of factor VIII involved in the assembly of the enzyme-cofactor complex on the endothelial cell surface. With the use of monoclonal antibodies against different peptide sequences on the factor VIII light chain, it was observed that the lipid-binding region of the C2 domain on the factor VIII light chain mediates the assembly of the factor X–activating complex on the endothelial cell surface. In addition, a synthetic peptide that constitutes region Ala2318-Tyr2332 of the C2 domain and that is known for its ability to inhibit the binding of factor VIII to artificial lipid membranes also showed inhibition of the cofactor activity of factor VIII on endothelial cells. Thus, the carboxy-terminal part of the factor VIII light chain not only contains sites involved in lipid binding but also contains sites involved in complex assembly on the endothelial cell membrane.
The vascular endothelium plays a prominent role in maintaining the antithrombotic potential of the vessel wall (reviewed in Cines et al1). On the other hand, endothelial cells also possess the capacity to produce procoagulant and prothrombotic agents. For instance, when endothelial cells are exposed to certain perturbing agents, such as proinflammatory cytokines (eg, interleukin-1 and tumor necrosis factor-α) and bacterial lipopolysaccharides, they may express tissue factor on their surface, a powerful trigger of the extrinsic pathway of blood coagulation. Also, unperturbed endothelial cells are able to support the coagulation system. A number of studies (reviewed by Brinkman et al2) indicate that unperturbed endothelial cells are able to efficiently catalyze critical reactions of the intrinsic pathway of blood coagulation, including the activation of factor X by activated factor IX (factor IXa) in the presence of activated factor VIII (factor VIIIa).
Although it seems likely that negatively charged phospholipids, including phosphatidylserine (PS), play a prominent role in controlling the activation of factor X by the factor VIIIa-IXa complex,3 the molecular basis of the interaction of the factor X–activating complex with endothelial cells has remained poorly understood. It seems likely that the interaction of factor IXa and its substrate factor X with endothelial cells is mediated by their respective γ-carboxyglutamic acid domains.4,5⇓ In addition, a factor IXa–specific receptor could play a role in controlling the assembly of the factor X–activating complex.6 Little is known about the peptide regions of factor VIII required for its interaction with sites on the endothelial cell membrane involved in the procoagulant response. Previously, we and others have shown that factor VIII, by means of its light chain, readily binds to negatively charged phospholipid membranes.7–9⇓⇓ The carboxy-terminal C2 domain of factor VIII contains sites that are essential for its binding to negatively charged phospholipid surfaces.10–12⇓⇓ The present study was initiated to determine whether the C2 domain of factor VIII is also involved in the assembly of a functional factor X–activating complex on the endothelial cell surface. We have used monoclonal antibodies to the C2 domain and peptides that constitute phospholipid-binding sites of this domain to analyze the molecular recognition by endothelial cells. Furthermore, by using annexin V as a probe, we investigated the contribution of endogenous PS to the procoagulant property of endothelial cells.
Endothelial Cell Culture
Human umbilical vein endothelial cells were isolated and cultured on fibronectin-coated culture plates as described.13 To suppress apoptotic signaling, cells were cultured for 3 days in the presence of 100 μmol/L of the broad-spectrum caspase inhibitor z-Val-Ala-dl-Asp-fluoromethylketone (z-VAD, Alexis Biochemicals). After 2 days of culture, medium and z-VAD were refreshed. Apoptosis was induced by incubating the cells for 5 hours with 2 μmol/L staurosporine (Sigma Chemical Co).
Antibodies, Peptides, and Proteins
The anti–factor VIII monoclonal antibodies CLB-CAg 9, CLB-CAg 12, CLB-CAg 69, CLB-CAg 117, and CLB-CAg A have been described previously.14–17⇓⇓⇓ The antibodies ESH4 and ESH811,18⇓ were from American Diagnostica Inc. Purified immunoglobulins were dialyzed against 145 mmol/L NaCl and 25 mmol/L HEPES, pH 7.4. Peptides constituting the carboxy-terminal region of the factor VIII light chain, Val2293-Arg2307, Thr2303-Tyr2322, and Ala2318-Tyr2332, and a peptide constituting the heavy chain region Lys713-Ser727 were synthesized according to described methods.16 Peptide Thr2303-Tyr2322 was not soluble in aqueous solutions at neutral pH and, therefore, was not used in further experiments. The coagulation factors VIII, IXa, X, and Xa were prepared as described.19
Activation of Factor X on Endothelial Cells and Phospholipid Vesicles
Measurements of factor X activation on endothelial cells and on synthetic phospholipid vesicles composed of equimolar concentrations of L-α-PS and L-α-phosphatidylcholine were performed essentially as described.19 After the coagulation factors VIII (0.3 nmol/L) and IXa (0.3 nmol/L) were added to the incubation mixture, the reaction was started by the addition of the substrate factor X (200 nmol/L) and 1 nmol/L of factor Xa. The latter was added to ensure membrane-dependent activation of the cofactor.19
Annexin V Binding
Binding of annexin V to endothelial cells was assessed by using fluorescein isothiocyanate (FITC)-labeled annexin V (Boehringer-Mannheim Gmbh) as follows: Endothelial cells were harvested from 6-well culture plates by trypsinization and were washed once with culture medium and once with ice-cold buffer (25 mmol/L HEPES, 145 mmol/L NaCl, 5 mmol/L CaCl2, and 0.5% human serum albumin, pH 7.4). Subsequently, cells were resuspended in 200 μL ice-cold buffer containing 60 nmol/L annexin V–FITC. After incubation for 10 minutes on ice, 200 μL of 4% paraformaldehyde was added. Cells were washed, and bound annexin V was analyzed on a FACScan (Becton Dickinson).
Binding of factor VIII to immobilized L-α-PS (Sigma) was examined according to published protocols,10,20⇓ with some minor modifications. Wells of a 96-well microtiter plate were coated with 100 μL PS (3 μg/mL) in methanol. Plates were air-dried at room temperature and blocked with a 0.5% gelatin solution in buffer (50 mmol/L Tris, 150 mmol/L NaCl, and 5 mmol/L CaCl2, pH 7.4) for 1 hour at 37°C. Plates were washed with buffer containing 0.05% Tween 20 and incubated with 100 μL of factor VIII at a concentration of 0.3 nmol/L in buffer containing 0.5% albumin. After 2 hours at 37°C, plates were washed. Bound factor VIII was detected during a 1-hour incubation at 37°C with the peroxidase-labeled anti–factor VIII heavy chain monoclonal antibody CLB-CAg 9 in buffer containing 0.1% gelatin.
Significant differences were calculated by ANOVA, followed by the Tukey multiple comparison test.
Inhibitory Effects of Monoclonal Antibodies Against Factor VIII on Endothelial Cell–Mediated Factor X Activation
To identify the regions in factor VIII involved in its interaction with the endothelial cell membrane during the activation of factor X by factor VIIIa-IXa complexes, the effect of a variety of monoclonal antibodies to factor VIII on the cofactor activity of factor VIII on endothelial cells was investigated (Figure 1). Together with monoclonal antibody CLB-CAg A (directed against the factor IXa–binding site on the factor VIII-A3 domain), the antibodies CLB-CAg 117 and ESH4 (both directed against the C2 domain of the factor VIII light chain) were the most inhibitory. These data indicate the involvement of the C2 domain of factor VIII in the complex assembly on the endothelial cell membrane.
The inhibitory effect of CLB-CAg 117 was explored in more detail. This antibody inhibited the factor VIII-IXa–dependent activation of factor X not only on endothelial cells but also on synthetic phospholipid membranes. The extent of inhibition decreased with increasing lipid concentration (Figure 2A). This observation suggests competition between CLB-CAg 117 and phospholipids for binding to factor VIII. When studying the binding of factor VIII to immobilized PS, we observed a dose-dependent inhibition by CLB-CAg 117 (Figure 2B). Thus, inhibition of the cofactor activity of factor VIII on phospholipid membranes by CLB-CAg 117 apparently is due to inhibition of the interaction of factor VIII with PS. This mechanism might also be responsible for the inhibition of the cofactor activity of factor VIII on endothelial cells by this antibody.
In Table 1, the effect of antibodies on the cofactor activity of factor VIII on the endothelial cell surface is compared with their effect on the binding of factor VIII to immobilized PS. Of all antibodies tested, those interfering in the interaction of factor VIII with immobilized PS (CLB-CAg 117 and ESH4; see Figure 2B) also inhibited the cofactor activity of factor VIII on endothelial cells. Because these antibodies are directed against the factor VIII C2 domain, this observation suggests the involvement of lipid-binding regions of the factor VIII C2 domain in the interaction of factor VIII with the endothelial cell surface. This possibility was further explored by using synthetic peptides constituting a lipid-binding site on the C2 domain.
Inhibition of Factor VIII Cofactor Activity by Synthetic Peptides in Endothelial Cell–Mediated Activation of Factor X
The carboxy-terminal peptide sequence Thr2303-Tyr2332 on the factor VIII light chain has been implicated in lipid binding.10,21⇓ As shown in Figure 3A, a synthetic peptide that constitutes region Ala2318-Tyr2332 completely inhibited the factor VIII–dependent activation of factor X by factor IXa on endothelial cells at a concentration of 1 mmol/L. Half-maximal inhibition was achieved at 0.5 mmol/L peptide. Factor X activation in the presence of peptide Val2293-Arg2307 was similar to that in the presence of an irrelevant peptide (factor VIII heavy chain region Lys713-Ser727). Peptide Ala2318-Tyr2332 also inhibited the binding of factor VIII to immobilized PS (IC50 0.4 nmol/L, Figure 3B). These data suggest that the PS-binding carboxy-terminal part of the C2 domain is involved in the interaction of factor VIII with the endothelial cell surface and subsequent assembly of the factor X–activating complex.
Factor X Activation and PS Exposure
With the use of the PS-binding glycoprotein annexin V, exposure of PS on the surface of endothelial cells in the monolayer has been observed.22 We observed an inhibition of the cofactor activity of factor VIII on the endothelial surface by annexin V in a dose-dependent manner (Figure 4). This observation suggests that the PS constituent of the cell membrane mediates the assembly of the factor X–activating complex. Because PS exposure is associated with apoptosis (reviewed by Reutelingsperger and van Heerde23), we considered the possibility that the procoagulant response of endothelial cells is due to apoptosis-mediated PS exposure. However, z-VAD, a broad-spectrum inhibitor of caspases involved in the apoptotic cell death program,24 had no effect on the endothelial cell–mediated activation of factor X by factor VIII-IXa complexes (Table 2). Alternatively, we induced apoptosis in our cells by treating them with staurosporine. z-VAD clearly inhibited the enhanced activation of factor X on staurosporine-treated cells (Table 2). In addition, as shown in Figure 4 (inset), all cells bind annexin V in a Ca2+-dependent manner to a similar extent. z-VAD also did not reduce this binding, whereas the increase in binding of annexin V to staurosporine-treated cells was markedly inhibited (Table 2). These data indicate that procoagulant phospholipids are expressed on nonapoptotic endothelial cells and that the observed procoagulant effect is not due to apoptosis.
During the last 2 decades, it became clear that endothelial cells provide appropriate sites on their surface to facilitate the activation of factor X by factor IXa in the presence of its cofactor, factor VIII.1,2⇓ In the present study, we show that the lipid-binding region in the C2 domain of factor VIII is involved in factor VIII-IXa–dependent activation of factor X on endothelial cells. This was concluded from the following observations: First, the cofactor activity of factor VIII on endothelial cells was inhibited by the monoclonal antibodies CLB-CAg 117 and ESH4 (Table 1 and Figure 1). Both antibodies inhibit the interaction of factor VIII with phospholipid membranes (Figure 2B and Table 1) and are directed against the C2 domain of factor VIII.11,15⇓ The epitope of CLB-CAg 117 probably includes residues in region 2223–2332 and that of ESH4 most likely includes residues in the amino-terminus and carboxy-terminus of the C2 domain.25 Second, the cofactor activity of factor VIII on the endothelial cell surface was completely blocked by a synthetic peptide constituting residue Ala2318-Tyr2332 of the C2-domain of factor VIII (Figure 3A). This peptide constitutes part of the lipid-binding region Thr2303-Tyr2332 of factor VIII10,21⇓ and is known to inhibit the interaction of factor VIII with PS.10 The observation that all peptides and antibodies inhibiting the factor VIII–phospholipid interaction also inhibit the cofactor activity of factor VIII on endothelial cells strongly suggests that the cofactor activity is affected because of interference in membrane binding. Our observation that the inhibiting effect of CLB-CAg 117 decreases with increasing membrane concentration (Figure 2A) supports this hypothesis. Thus, it appears that phospholipids and endothelial cells share ≥1 binding site on the C2 domain of the factor VIII light chain.
Previously, it has been shown that the C2 domain of factor VIII appears to be entirely responsible for the binding of factor VIII to PS-containing membranes.12 Our results also point out to a major role of the C2 domain, including region Ala2318-Tyr2332, in the interaction of factor VIII with the plasma membrane of endothelial cells. However, homology modeling studies26 and analysis of the crystal structure of the C2 domain27 have revealed that residue Thr2303-Tyr2332 constitutes part of the β-sandwich core, exposing only Trp2313, Val2314, and Arg2320. In contrast, human factor VIII inhibitor antibodies to the C2 domain that inhibit the interaction of factor VIII with PS also recognize a synthetic peptide constituting the region Thr2303-Tyr2332,11 suggesting that this site is exposed. Furthermore, it appears that the C2 domain is susceptible to major conformational rearrangements that may be related to its binding functions.12,28,29⇓⇓ It is not clear whether these putative conformational rearrangements also are of importance in the regulation of the cofactor activity of factor VIII on endothelial cells.
One of the prominent features of the present study and also of previous studies13,22,30⇓⇓ is that apparently resting and unperturbed endothelial cells support essential steps in the coagulation cascade, including the factor VIII-IXa–dependent activation of factor X and the activation of prothrombin. This procoagulant state can be easily explained if one assumes that the endothelial cells examined undergo apoptosis, a process that readily leads to the exposure of negatively charged membrane lipids such as PS.23 Alternatively, it is possible that endothelial cells express PS constitutively. Pertinent to the latter view is our observation that the treatment of endothelial cells with z-VAD, an inhibitor of apoptosis,24 did not affect the procoagulant state of these cells (Table 2). In addition, as assessed by annexin V binding, unperturbed endothelial cells exhibit the exposure of PS (Figure 4, inset), which is not reduced on treatment with z-VAD (Table 2). Similarly, Bombeli et al30 reported binding of annexin V to endothelial cells that did not show any apoptosis-associated DNA fragmentation. The observed binding of annexin V to nonapoptotic endothelial cells is in concert with reports on the binding of annexin V to uninjured vessel walls.31,32⇓ Thus, in vitro and in vivo unperturbed endothelial cells express procoagulant phospholipids constitutively.
Our observation (Figure 4 and Table 2) and the observations of others22,30⇓ (ie, that annexin V binds to endothelial cells and, as such, inhibits the cofactor activity of factor VIII on endothelial cells) do not exclude the contribution of nonlipid membrane constituents. For example, evidence has been reported that binding of the zymogen factor IX (and probably also factor IXa) to endothelial cells is independent of phospholipids and is mediated by residues 3 to 11 of the lipid-binding γ-carboxy-glutamic acid domain of factor IX.4 On the other hand, studies on artificial lipid membranes have revealed that the phospholipid molecules themselves guide the specificity for binding coagulation factors. Specific binding of factor VIII to lipid membranes is mediated by the O-phospho-l-serine moiety of PS.33 During the activation of factor X on synthetic PS/phosphatidylcholine membranes, the light chain of factor VIIIa remains associated with the membrane.19 If one assumes that PS is the only binding site for factor VIII in the endothelial cell membrane, it is not clear why, as we reported previously,19 the activated light chain of factor VIII dissociates rapidly from the endothelial cell surface during the activation of factor X. It is possible that the amount of aminophospholipids exposed is sufficient to ascertain factor VIII–cofactor activity but is too low to facilitate a stable factor VIII–endothelial cell interaction. With respect to recent published models for the C2 domain, it also seems conceivable that on endothelial cells, nonlipid membrane constituents interfere with the penetration of hydrophobic side chains in the hydrophobic interior of the plasma membrane. Another possibility is that because of PS flip-flop34 in the cell membrane, there is a continuous binding and release of factor VIII.
Received October 29, 2001; revision accepted January 3, 2002.
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- ↵Gilbert GE, Arena AA. Activation of the factor VIIIa-IXa enzyme complex of blood coagulation by membranes containing phosphatidyl-l-serine. J Biol Chem. 1996; 271: 11120–11125.
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