Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells.

BACKGROUND: Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction. OBJECTIVES: To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS). METHODS: PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test. RESULTS: Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma. CONCLUSIONS: Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones.

Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation.

BACKGROUND: Histones are basic proteins that contribute to cell injury and tissue damage when released into the extracellular space. They have been attributed a prothrombotic activity, because their injection into mice induces diffuse microvascular thrombosis. The protein C-thrombomodulin (TM) system is a fundamental regulator of coagulation, particularly in the microvasculature, and its activity can be differentially influenced by interaction with several cationic proteins. OBJECTIVE: To evaluate the effect of histones on the protein C-TM system in a plasma thrombin generation assay and in purified systems. METHODS: The effect of histones on plasma thrombin generation in the presence or absence of TM was analyzed by calibrated automated thrombinography. Protein C activation in purified systems was evaluated by chromogenic substrate cleavage. The binding of TM and protein C to histones was evaluated by solid-phase binding assay. RESULTS: Histones dose-dependently increased plasma thrombin generation in the presence of TM, independently of its chondroitin sulfate moiety. This effect was not caused by inhibition of activated protein C activity, but by the impairment of TM-mediated protein C activation. Histones were able to bind to both protein C and TM, but the carboxyglutamic acid domain of protein C was required for their effect. Histones H4 and H3 displayed the highest activity. Importantly, unlike heparin, DNA did not inhibit the potentiating effect of histones on thrombin generation. CONCLUSIONS: Histones enhance plasma thrombin generation by reducing TM-dependent protein C activation. This mechanism might contribute to microvascular thrombosis induced by histones in vivo at sites of organ failure or severe inflammation.

Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia.

BACKGROUND: Activated protein C (APC) protects the host from severe sepsis. Endothelial protein C receptor (EPCR) is expressed on both hematopoietic leukocytes and non-hematopoietic endothelium, and plays a key role in protein C activation. OBJECTIVES: We explore the influence of EPCR deletion on the responses to lipopolysaccharide (LPS) and then determine whether the observed differences are due to loss of hematopoietic or non-hematopoietic EPCR. METHODS AND RESULTS: After LPS challenge, EPCR null (Procr(-/-)) mice exhibited more thrombin and cytokine generation, neutrophil sequestration in the lung and a higher mortality rate than Procr(+/-) mice. Procr(+/-) BM/Procr(-/-) (non-hematopoietic Procr(-/-)) and Procr(-/-) BM/Procr(+/-) (hematopoietic Procr(-/-)) chimeric mice were generated by bone marrow (BM) transplantation. Compared with control Procr(+/-) mice, non-hematopoietic Procr(-/-) mice exhibited reduced protein C activation by thrombin and exaggerated responses to LPS challenge, whereas Procr(+/-) mice and hematopoietic Procr(-/-) mice exhibited similar protein C activation by thrombin and similar responses to LPS challenge. CONCLUSIONS: EPCR deletion exaggerates the host responses to LPS primarily due to deficiency of EPCR on the non-hematopoietic cells.

Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor-alpha converting enzyme/ADAM17.

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor-alpha converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol-12-myristate 13-acetate (PMA)-stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA-stimulated EPCR shedding is completely blocked in fibroblasts from TACE-deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAG sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

The Ser219-->Gly dimorphism of the endothelial protein C receptor contributes to the higher soluble protein levels observed in individuals with the A3 haplotype.

The endothelial cell protein C receptor (EPCR) plays an important role in regulating blood coagulation and in activated protein C-mediated anti-inflammatory and antiapoptotic processes. Recent studies reported that there are polymorphisms in the human EPCR gene. One of the polymorphisms (haplotype A3) results in substitution of the Ser at residue 219 with Gly in the transmembrane domain. This haplotype is associated with increased plasma levels of soluble EPCR and is a candidate risk factor for thrombosis. We established stable cell lines expressing either the EPCR A1 (Ser at residue 219) or A3 (Gly at residue 219) haplotype. Both constitutive and PMA-stimulated shedding are five- to sevenfold higher in the A3 cell line than the A1 cell line. We also isolated human umbilical vein endothelial cells (HUVEC) from A1/A1 or A1/A3 origins. PMA-stimulated shedding is fourfold higher in HUVEC derived from A1/A3 origin than from A1/A1 origin. After PMA treatment, the rate of human protein C activation decreased 36% in HUVEC derived from A1/A3 origin, while it only decreased 18% in HUVEC derived from A1/A1 origin. These results indicate that the A3 haplotype does promote cellular shedding in either 293 or endothelial cells and therefore is likely directly contributory to the higher soluble EPCR levels seen in patients carrying this haplotype.

Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin.

Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.

Lipid oxidation enhances the function of activated protein C.

Although lipid oxidation products are usually associated with tissue injury, it is now recognized that they can also contribute to cell activation and elicit anti-inflammatory lipid mediators. In this study, we report that membrane phospholipid oxidation can modulate the hemostatic balance. Oxidation of natural phospholipids results in an increased ability of the membrane surface to support the function of the natural anticoagulant, activated protein C (APC), without significantly altering the ability to support thrombin generation. Lipid oxidation also potentiated the ability of protein S to enhance APC-mediated factor Va inactivation. Phosphatidylethanolamine, phosphatidylserine, and polyunsaturation of the fatty acids were all required for the oxidation-dependent enhancement of APC function. A subgroup of thrombotic patients with anti-phospholipid antibodies specifically blocked the oxidation-dependent enhancement of APC function. Since leukocytes are recruited and activated at the thrombus or sites of vessel injury, our findings suggest that after the initial thrombus formation, lipid oxidation can remodel the membrane surface resulting in increased anticoagulant function, thereby reducing the thrombogenicity of the thrombus or injured vessel surface. Anti-phospholipid antibodies that block this process would therefore be expected to contribute to thrombus growth and disease.

Antiphospholipid antibodies and the protein C pathway.

Among the mechanisms suggested for the prothrombotic activity of lupus anticoagulant and antiphospholipid antibodies is the direct inhibition of the anticoagulant activated protein C (APC) pathway. Although some pathological antibodies may be directed towards the proteins involved, we hypothesize that populations exist which selectively inhibit the APC complex as a result of differences in the phospholipid requirements of this complex as compared to those of the procoagulant complexes. The most prominent feature is the requirement for the presence of phosphatidylethanolamine in the membrane for APC anticoagulant function. This mimics the requirements for inhibitory activity of at least a subset of autoantibodies associated with thrombosis. The role of oxidation of the phospholipid in APC function and antibody reactivity is also discussed.

Metalloproteolytic release of endothelial cell protein C receptor.

Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increase in inflammatory diseases. In this study we examine the potential mechanisms involved in release of EPCR from cells. We find that EPCR is released from the surface of endothelium and transfected 293 cells by a metalloprotease in a constitutive fashion. The mass of soluble EPCR is 4 kDa less than intact EPCR. Release is blocked by either the hydroxamic acid based inhibitor, KD-IX-73-4 or by 1,10-phenanthroline, but not by matrix metalloprotease inhibitors. Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1beta, and hydrogen peroxide. Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein C activation to a limited extent. The influence of phorbol 12-myristate 13-acetate on both EPCR release and inhibition of protein C activation are enhanced by microtubule disruption with nocodazole. EPCR release is augmented by transfection of EPCR expressing 293 cells with caveolin, suggesting that release is caveolae dependent. These studies indicate that metalloproteolytic release of EPCR is a highly regulated process that is sensitive to both coagulation factors and inflammatory mediators.

Antibodies to thrombomodulin are found in patients with lupus anticoagulant and unexplained thrombosis.

OBJECTIVE: To test the hypothesis that thrombomodulin (TM) may be a target for lupus anticoagulant (LAC) antibodies. METHODS: A recombinant soluble form of TM was produced and used as an antigen for an ELISA to detect antibodies to TM (TMAB). Sixty-one samples from 58 patients identified by the coagulation laboratory as having a LAC and 200 patients with unexplained thrombosis were evaluated along with 201 healthy controls. RESULTS: Eighteen (30%) of the 58 patients with a LAC and 20 (10%) of 200 patients with unexplained thrombosis had antibodies to TM. Similar antibodies were found in only 4 (2%) of 201 normal controls. TMAB show selectivity for TM lacking chondroitin sulfate, but do not otherwise have an immunodominant region. The IgG from 6 patients with TMAB was purified, and it bound TM in our ELISA. Three of the 6 IgG fractions inhibited protein C activation 40% to 70% compared to no inhibition in 7 healthy controls. CONCLUSION: Some patients with LAC and unexplained thrombosis have antibodies to TM that may arise in response to TM that has been altered and lost its chondroitin sulfate attachment. Antibodies to TM may be an important risk factor for inflammation and thrombosis in these patients.

Inhibition of activated protein C anticoagulant activity by prothrombin.

In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of approximately 5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.

Lupus anticoagulants, thrombosis and the protein C system.

Although lupus anticoagulants (LAs) are immunoglobulins that inhibit procoagulant reactions in vitro, these molecules are associated with thrombosis in vivo. We and others have hypothesized that this may be due to selective targeting of the activated protein C (APC) anticoagulant pathway. Populations of antibodies that interact with protein C or protein S in ways that inhibit their activity are obvious candidates for such pathological molecules. However, it is less clear how populations that appear to bind to membrane surfaces might target the APC anticoagulant complex selectively. Studies now show that the membrane requirements of the APC anticoagulant complex are significantly different from those of the procoagulant reactions. The most dramatic difference is the requirement for the presence of phosphatidylethanolamine (PE) in the membrane for optimal APC function. The inhibitory activity of at least some LAs is enhanced by the presence of PE, but the anti-APC activity is enhanced even more, resulting in the plasma from these patients clotting faster than normal when APC is present. Structure-function studies have been undertaken to understand the PE dependence of this reaction better. Chimeric proteins in which all or part of the Gla domain of protein C has been replaced by the homologous region of prothrombin have been prepared. Unexpectedly, the PE dependence resides primarily in the C-terminal half of the Gla domain. Using liposomes of various composition, we found both the presence of the PE head group and unsaturation of the fatty acid chains are required for optimal inactivation of factor Va. It is hoped that a better understanding of the biochemistry of these reactions, combined with the use of the chimeric proteins described, will permit us to design better assays for the identification of pathologic LAs.

The effect of membrane composition on the hemostatic balance.

The phospholipid composition requirements for optimal prothrombin activation and factor Va inactivation by activated protein C (APC) anticoagulant were examined. Vesicles composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) supported factor Va inactivation relatively well. However, optimal factor Va inactivation still required relatively high concentrations of phosphatidylserine (PS). In addition, at a fixed concentration of phospholipid, PS, and APC, vesicles devoid of PE never attained a rate of factor Va inactivation achievable with vesicles containing PE. Polyunsaturation of any vesicle component also contributed significantly to APC inactivation of factor Va. Thus, PE makes an important contribution to factor Va inactivation that cannot be mimicked by PS. In the absence of polyunsaturation in the other membrane constituents, this contribution was dependent upon the presence of both the PE headgroup per se and unsaturation of the 1,2 fatty acids. Although PE did not affect prothrombin activation rates at optimal PS concentrations, PE reduced the requirement for PS approximately 10-fold. The Km(app) for prothrombin and the Kd(app) for factor Xa-factor Va decreased as a function of increasing PS concentration, reaching optimal values at 10-15% PS in the absence of PE but only 1% PS in the presence of PE. Fatty acid polyunsaturation had minimal effects. A lupus anticoagulant immunoglobulin was more inhibitory to both prothrombinase and factor Va inactivation in the presence of PE. The degree of inhibition of APC was significantly greater and much more dependent on the phospholipid composition than that of prothrombinase. Thus, subtle changes in the phospholipid composition of cells may control procoagulant and anticoagulant reactions differentially under both normal and pathological conditions.

Relocating the active site of activated protein C eliminates the need for its protein S cofactor. A fluorescence resonance energy transfer study.

The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa2 = 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 A, compared with 94 A for wild-type APC. The gamma-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 A observed for the APC. protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg306, but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC.protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg306. Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C.

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.

Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation.

Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (Km = 2.17 +/- 0.13 microM). With EPCR, two classes of sites are apparent (Km = 20 +/- 15 nM and Km = 3.2 +/- 1.7 microM). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM:EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Epitope-dependent selective targeting of thrombomodulin monoclonal antibodies to either surface or intracellular compartment of endothelial cells.

Internalization of antibodies to thrombomodulin (TM) may provide a mechanism for intraendothelial targeting of drugs or genes. This study characterized three monoclonal antibodies against human TM (mAb 1009,1029, and 1045) and examined their internalization by human umbilical vein endothelial cells (HUVEC). It assessed binding of antibodies to recombinant human TM containing chondroitin sulfate (complete, cTM) and TM lacking chondroitin sulfate (incomplete, iTM). Direct RIA, indirect RIA, and ELISA and competitive ELISA show that (1) mAb 1009 binds to both cTM and iTM independently of divalent cations; (2) binding of mAb 1029 to iTM requires divalent cations, while binding to cTM is cation-independent; (3) mAb 1045 binds selectively to cTM independently of divalent cations. Binding of all three antibodies to the surface TM in HUVEC at 4 degrees C was similar by indirect immunostaining. In permeabilized HUVEC, however, mAb 1009 and 1029 provide brighter intracellular staining than mAb 1045. Uptake of (125)I-mAb 1009 by HUVEC at 37 degrees C was significantly higher than that of (125)I-mAb 1045. Low temperature markedly suppresses binding of (125)I-mAb 1009 to HUVEC, but has no effect on (125)I-mAb 1045 binding. About 80% of radiolabeled mAb 1045 bound to HUVEC at 37 degrees C could be eluted by acidic buffer from the cell surface, but only 40% of mAb 1009 and 1029 was elutable at these conditions. About 70-80 % of (125)I in cell lysates was TCA-soluble after HUVEC incubation with either mAb 1009 and 1029, but only 10 and 2.5% of (125)I was TCA-soluble in cell lysates and medium after 90 min incubation with (125)I-mAb 1045 at 37 degrees C. Therefore, HUVEC internalize and degrade an mAb that reacts with iTM, yet do not internalize an mAb that reacts selectively with cTM (mAb 1045). This result implies that either HUVEC do not internalize cTM constitutively or mAb 1045 suppresses TM internalization. Therefore, antibodies recognizing different TM epitopes might provide targeting of drugs to different cellular compartments.