Platelet P2Y(12) receptor influences the vessel wall response to arterial injury and thrombosis.

BACKGROUND: Platelets are believed to play an important role in atherogenesis and the vessel response to vascular injury. The P2Y(12) receptor (P2Y(12)) plays a central role in amplifying platelet aggregation, dense granule and alpha-granule secretion, P-selectin expression, microparticle formation, and procoagulant membrane changes, regardless of the activating stimulus. We hypothesized that P2Y(12) deficiency might reduce the vessel wall response to vascular injury as well as thrombosis in murine vascular injury models. METHODS AND RESULTS: P2Y(12)-deficient (-/-) mice and littermate controls (+/+) were bred on a C57 BL/6 background. In vivo murine models of arterial injury were employed alone and in combination with bone marrow transplantation to investigate the role of P2Y(12) in the vessel wall response to arterial injury and thrombosis. At 21 days after ferric chloride injury, neointima formation in P2Y(12)(-/-) arteries was significantly less than that observed in control strain arteries (P<0.025). In agreement with this, the intima-media ratio was significantly greater in femoral wire-injured arteries from P2Y(12)(+/+) compared with P2Y(12)(-/-) animals (P<0.05). Bone marrow transplantation was used to examine the importance of vessel wall P2Y(12) versus platelet P2Y(12). Analysis of arterial sections from chimeric animals at 21 days after injury revealed a smaller intima-media ratio in -/- to +/+ animals than in the positive (+/+ to +/+) control group (P<0.01). CONCLUSIONS: These data demonstrate a role for platelet P2Y(12) in the vessel wall response to arterial injury and thrombosis. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis.

Translational mini-review series on immunology of vascular disease: inflammation, infections and Toll-like receptors in cardiovascular disease.

Cardiovascular disease, in which atherosclerosis is the major underlying cause, is currently the largest cause of death in the world. Atherosclerosis is an inflammatory disease characterized by the formation of arterial lesions over a period of several decades at sites of endothelial cell dysfunction. These lesions are composed of endothelial cells, vascular smooth muscle cells, monocytes/macrophages and T lymphocytes (CD4(+)). As the lesions progress some can become unstable and prone to disruption, resulting in thrombus formation and possibly a myocardial infarction or stroke depending upon the location. Although the exact triggers for plaque disruption remain unknown, much recent evidence has shown a link between the incidence of myocardial infarction and stroke and a recent respiratory tract infection. Interestingly, many reports have also shown a link between a family of pattern recognition receptors, the Toll-like receptors, and the progression of atherosclerosis, suggesting that infections may play a role in both the progression of atherosclerosis and in inducing the more severe complications associated with the disease.

Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.

The human malaria parasite, Plasmodium falciparum, degrades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy.

P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.

BACKGROUND AND PURPOSE: The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. EXPERIMENTAL APPROACH: Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. KEY RESULTS: HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. CONCLUSIONS AND IMPLICATIONS: Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.

Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific.

The constant need to respond to changes in the environment is a common feature for all life forms. During evolution, a number of intracellular signal processing systems have evolved to fulfill this requirement. One of the most ancient such systems is the mitogen activated protein kinase (MAPK) signalling network, shared by all eukaryotes. Activation of MAPKs is key to regulation of mitosis and in cellular responses to stress or hormones, for instance. In addition, activity of this signalling system is essential during embryonic development. However, many aspects of MAPK mediated responses are strongly cell-type specific. A family of proteins, called tribbles have recently been described as novel regulators of MAPK function. Our group has previously shown that alterations in tribbles levels lead to profound changes in the activation of the various MAPKs. However, little is known about the cell-type specific aspects of regulation of tribbles expression. Here, we report that expression of all three members of the human tribbles family is dynamically controlled in response to inflammatory stimulation. This regulation, however, is strongly cell-type dependent. Our observations suggest regulation of tribbles expression may play an important role in the cell-type specific cellular responses, mediated by the MAPK network.

The effect of surface-bound protein on the permeability of proteoliposomes.

Proteoliposomes have been prepared from mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol by sonication (SUV) and reverse phase evaporation (REV) and conjugated with succinyl concanavalin A (sConA). The proteoliposomes were characterised in terms of size and composition and covered a range of size (weight-average diameter) from approx. 80 to 300 nm and surface-bound sConA (weight-average number of protein molecules per liposome) from approx. 200 to 1800. The permeabilities of the proteoliposomes to encapsulated D-glucose have been measured and found to increase linearly with protein conjugation. The D-glucose permeability also increases with temperature and passes through a maximum in the region of the gel to liquid-crystalline phase transition temperature. Conjugation has no effect on the chain-melting temperature but slightly decreases the enthalpy of the transition consistent with the withdrawal of some phospholipid participation in chain-melting. The D-glucose permeabilities and thermotropic properties of the proteoliposomes are discussed in terms of the dislocation of the bilayer by the possible off-axis motion of the lipid which anchors the protein to the liposomal surface.

The characterisation of liposomes with covalently attached proteins.

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.

Targeting and delivery of bactericide to adsorbed oral bacteria by use of proteoliposomes.

Proteoliposomes having surface-bound succinylated concanavalin A (s-conA) have been prepared from a range of phospholipid mixtures by sonication (SUV) and reverse phase evaporation (REV) covering a range of size (weight-average diameter (dw)) from approx. 35 to 310 nm and weight-average number of protein molecules per liposomes (Pw) from approx. 50 to 3000. The targeting of the proteoliposomes to adsorbed biofilms of the bacteria Streptococcus sanguis and Streptococcus mutans has been assessed from the extent of inhibition of an enzyme-linked immunosorbent assay (ELISA) for bacterial cell surface antigens. The surface-bound lectin enhances targeting relative to 'naked' liposomes of comparable concentration by factors of 2-50 depending on the liposomal lipid composition and Pw. The effect of the bactericide Triclosan on the thermal properties and permeability characteristics of liposomes has been studied. At and above a molar ratio of Triclosan to lipid of 0.6, Triclosan eliminates the gel to liquid-crystalline phase transition in dipalmitoylphosphatidylcholine (DPPC) containing liposomes and increases the bilayer permeability of both liposomes and proteoliposomes to D-glucose. The proteoliposomes have been used to deliver Triclosan to S. sanguis biofilms and the inhibition of growth of the bacteria after treatment with liposomally delivered Triclosan has been determined using a microtitre plate re-growth assay and compared with growth inhibition by 'free' Triclosan. It is shown that for short exposure times (1 to 2 min) proteoliposomally delivered Triclosan is a more effective growth inhibitor than free Triclosan. The results are discussed in terms of the targeting, retention and subsequent release of Triclosan into the bacterial biofilms.

Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro.

BACKGROUND: Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS: Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS: Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

Effect of selective or combined inhibition of integrins alpha(IIb)beta(3) and alpha(v)beta(3) on thrombosis and neointima after oversized porcine coronary angioplasty.

BACKGROUND: Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective alpha(IIb)beta(3) antagonist (lamifiban), a selective alpha(v)beta(3) antagonist (VO514), and a combined alpha(IIb)beta(3)/alpha(v)beta(3) antagonist (G3580). METHODS AND RESULTS: In vitro, both alpha(v)beta(3) inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective alpha(IIb)beta(3) inhibition had no effect. Intravenous infusions of either alpha(IIb)beta(3) inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective alpha(v)beta(3) inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were administered for 14 days after oversized balloon angioplasty injury. After PTCA, there was regional upregulation of integrin alpha(v)beta(3) in the developing neointima, as assessed by immunohistochemistry. Six hours after PTCA, obstruction of lumen by thrombus was reduced significantly by alpha(IIb)beta(3) inhibition compared with either control or alpha(v)beta(3) inhibition (mean control, 18.7%; VO514, 18.5%; lamifiban, 6.4%; G3580, 7.9%). Twenty-eight days after PTCA, there was a significant reduction of neointima with inhibitors of either integrin (mean intima/media ratio: control, 3.08; VO514, 1.33; lamifiban, 0.97; G3580, 1.32). CONCLUSIONS: We conclude that both integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) participate in neointima development after experimental angioplasty.

Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries.

Previous studies have shown that inhibition of the proto-oncogene c-myb inhibits neointimal formation in various animal models. However, the temporal and spatial expression of c-Myb in the vessel wall after injury is not known, and the mechanism of action of antisense oligonucleotide (AS-ODN-c-myb) inhibition remains unclear. One potential effect of cell cycle dysregulation by inhibition of c-myb is an increase in the rates of apoptosis. In this study, c-Myb expression after percutaneous transluminal coronary angioplasty (PTCA) injury and induction of apoptosis after AS-ODN-c-myb treatment were determined. Immunohistochemistry and cellular phenotyping were used to localize c-Myb expression in porcine coronary arteries at various time intervals after PTCA. In vitro, the effects of AS-ODN-c-myb on the apoptosis of porcine vascular smooth muscle cells (PVSMCs) and endothelial cells were determined by using a cell-death ELISA and time-lapse video microscopy. In vivo, local delivery of AS-ODN-c-myb was performed after PTCA of pig coronary arteries, and apoptosis was quantified at 6 hours. c-Myb is induced in pig coronary arteries after angioplasty, with maximal expression in inflammatory cells at 18 hours and in vascular smooth muscle cells at 3 to 7 days. In vitro, AS-ODN-c-myb enhanced PVSMCs (6.8+/-0.8% [P=<0.001] versus 0.5% serum) but not endothelial cell apoptosis (1.4+/-0.5% [P=NS] versus 0.5% serum). In vivo, 6 hours after porcine coronary angioplasty and delivery of AS-ODN-c-myb, the proportion of apoptotic cells within the media was 4.2+/-0.8% (PTCA alone), 2.3+/-0.2% (PTCA+vehicle), and 9.0+/-1.1% (PTCA+AS-ODN-c-myb; P<0.05 versus PTCA alone and P<0.01 versus PTCA+saline). c-Myb is expressed after PTCA of pig coronary arteries, and AS-ODN-c-myb induces apoptosis of PVSMCs in vitro and medial cells in vivo.

Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein.

OBJECTIVES: Saphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis. METHODS: Surgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37 degrees C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferation cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel was thicknesses. RESULTS: Control, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/microgram DNA, P < 0.05, n = 6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P < 0.05, n = 5. CONCLUSIONS: These findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

Intimal proliferation in an organ culture of human internal mammary artery.

OBJECTIVE: Intimal smooth muscle cell proliferation is an early feature of atherosclerosis. Its progression is difficult to monitor in humans and previous studies have mostly relied on necropsy material. The aim of this study was therefore to establish whether intimal proliferation occurred in an organ culture of human internal mammary artery. METHODS: Segments of freshly isolated internal mammary artery were maintained din standard tissue culture medium containing 30% calf serum for 14 d. Tissue viability (measured by ATP concentration) was maintained during processing and throughout the culture period [211(SEM 28) nmol ATP.g-1 wet weight on d 1 v 208(27) on d 14]. RESULTS: Histological transverse sections of cultured internal mammary artery showed the development of a neointima containing smooth muscle cells identified by immunocytochemistry for alpha actin. Pulse labelling of cultures with [3H]-thymidine showed proliferating cells predominantly in a neointimal layer with few dividing cells in the media. Cultured de-endothelialized vessels showed less neointimal thickening than cultured freshly isolated vessels [16(3) v 36(5) microns, p < 0.0025] as well as a reduced number of dividing cells per mm of neointimal length [3.1(0.6) v 5.5(1.1), p < 0.05]. CONCLUSIONS: Intimal proliferation occurred in organ culture of internal mammary artery. There is evidence for a factor derived from the endothelium, which may be important in the development of intimal proliferation.

Release of platelet derived growth factor in serum-free organ culture of human coronary artery.

OBJECTIVE: The aim was to test the hypothesis that platelet derived growth factor (PDGF) is synthesized in intact coronary arteries and that it is associated with cell proliferation in atherosclerotic plaques. METHODS: Segments of human coronary artery obtained from heart transplant recipients were cultured in serum-free media for 24 h. Tissue viability was assessed by ATP concentration. Cell proliferation was determined by incorporation of [3H] thymidine, autoradiography, and proliferating cell nuclear antigen (PCNA) immunostaining. Coronary artery conditioned media were tested for mitogenic activity using a fibroblast proliferation bioassay. Reverse transcription polymerase chain reaction (RT-PCR) for PDGF A and B was subsequently performed in order to confirm the endogenous nature and isoform of this mitogen. RESULTS: Tissue viability remained unchanged during culture, and cell proliferation was detected by incorporation of [3H] thymidine. Autoradiography and PCNA immunostaining showed proliferating cells within the intimal and medial layers. Coronary artery conditioned media produced significant stimulation of cell growth [127(SEM 29)%, n = 15] above that caused by culture medium alone. This mitogenic activity was inhibited by 42(8)% (n = 8) with a polyclonal neutralising antibody to PDGF. The endogenous nature of this mitogenic activity was confirmed by detection of PDGF-A and PDGF-B mRNA expression using RT-PCR and the identity of the amplified products confirmed by DNA sequencing. CONCLUSIONS: The data provide evidence for active PDGF production and gene expression within cells of the vessel wall. They also suggest that endogenously produced PDGF may play a role in controlling vascular smooth muscle cell proliferation in human coronary arteries.

TGFbeta is active, and correlates with activators of TGFbeta, following porcine coronary angioplasty.

OBJECTIVE: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor beta (TGFbeta) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFbeta: mannose-6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor and thrombospondin-1. METHODS: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFbeta were determined using a modified plasminogen activator-inhibitor/luciferase bioassay. RESULTS: Levels of active TGFbeta significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFbeta in tissues adjacent to the injured artery did not change. Total TGFbeta was significantly higher than controls 2-6 h after injury. M6P/IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. CONCLUSIONS: We conclude that balloon injury causes an early rapid increase in levels of active TGFbeta, that correlates with the expression of TGFbeta activators. Thus, TGFbeta is a good potential target for anti-restenotic therapies.

Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.

A cell kinetic analysis of human umbilical vein endothelial cells.

Cultures of normal human cells 'age' and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43+/-0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47+/-0.3% as assessed by senescence associated beta-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31+/-0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

Kinetic analysis of plasmepsins I and II aspartic proteases of the Plasmodium falciparum digestive vacuole.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Characterization of native falcipain, an enzyme involved in Plasmodium falciparum hemoglobin degradation.

In Plasmodium falciparum, a cysteine protease known as falcipain has been implicated in the essential metabolic process of hemoglobin degradation. Parallel lines of investigation, using native or recombinant enzyme, have led to differing conclusions about the specificity and role of this protease. We have now determined that (1) Native falcipain does not cleave hemoglobin unless this substrate has first been denatured by reducing agents, acid-acetone treatment or plasmepsin action. (2) Reducing agents such as glutathione cannot denature hemoglobin in the presence of catalase, which is accumulated in the digestive vacuole. (3) The purified native enzyme has kinetics similar to those obtained with trophozoite extract, but substantially different from those of recombinant enzyme. (4) Although there are numerous cysteine protease genes in the P. falciparum genome, the falcipain gene is the only one whose transcript can be detected in the early intraerythrocytic parasites. We conclude that falcipain likely works by degrading hemoglobin fragments after initial aspartic protease attack has denatured the substrate. We propose that falcipain inhibitors block the initial steps of degradation indirectly by promoting vacuolar accumulation of osmotically active hemoglobin peptides.