Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [³H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.     

Neosporacaninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-γ production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams’ brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered.     

The effect of surface charges of liposomes in immunopotentiation

The purpose of this study was to establish the effect of surface charges of liposomes on its adjuvant activity to an entrapped protein antigen. The immune responses of rabbits immunized subcutaneously with lysozyme entrapped in neutral negatively and positively charged liposomes and compared with complete Freund's adjuvant (CFA), showed positively charged liposomes to be a better adjuvant than neutral, negatively charged liposomes and even CFA. This was true for solid liposomes also. Interestingly, injection of positively charged liposomes led to the formation of granulomas at the sites of immunization, which was not observed with neutral and negatively charged liposomes.     

Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

The role of apolipoprotein E in the elimination of liposomes from blood by hepatocytes in the mouse

We evaluated the role of apolipoprotein E (apoE) in the clearance of neutral and negatively charged liposomes by hepatocytes in apoE-deficient mice. Negatively charged liposomes were cleared at identical rates in apoE-deficient and wild-type mice; neutral liposomes were cleared at a 3.6-fold slower rate in apoE-deficient mice. ApoE deficiency did not affect hepatic uptake of negatively charged liposomes but lowered that of neutral liposomes >5-fold. Hepatocyte uptake of neutral liposomes was reduced >20-fold in apoE-deficient mice; that of negatively charged liposomes remained unchanged. We conclude that uptake of neutral liposomes by hepatocytes is nearly exclusively apoE-mediated.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Liposome‐Bilirubin Interaction: A Novel Strategy to Eliminate Bilirubin from Systemic Circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin‐liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome‐mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Encapsulation of immunoadjuvant [24C]peptidoglycan monomer into liposomes: Effect on metabolism and immune response in mice

¹⁴C-labeled peptidoglycan monomer was encapsulated into negatively charged, multilamellar liposomes composed of egg phosphatidylcholine, cholesterol and dicetylphosphate. Excretion and tissue distribution of the label in mice were studied after intravenous injections. Encapsulation of peptidoglycan monomer into liposomes as compared to free peptidoglycan monomer, resulted in increased retention of the label, particulary in the liver and to a lesser extent in spleen. The excretion was drastically reduced and delayed even after 4 days when cholesterol-rich (phosphatidylcholine/cholesterol, 7:5 molar ratio) liposomes were used for encapsulation of peptidoglycan monomer. Peptidoglycan monomer and liposomes, when tested separately, stimulate the immune response to sheep erythrocytes in mice. However, there was no significant additive or synergistic effect when peptidoglycan monomer was encapsulated into liposomes.     

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.     

Encapsulation of Enrofloxacin in Liposomes I: Preparation and In Vitro Characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL‐α‐phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of α‐tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 µm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

The mechanism of liposome accumulation in infarction

This paper explores the mechanism(s) whereby liposomes accumulate in chronically ischaemic myocardium and intestine. Plasma prepared from venous blood obtained at sites of myocardial and intestinal infarction does not promote the lysis of positively and negatively charged liposomes in vitro. Albumin-bound lysophosphatidylcholine (≥ 2 mM) lyses positively and negatively charged liposomes in vitro at similar rates. [^99mTc]Diethylenetriamine pentaacetic acid (DTPA) entrapped in positively charged liposomes was accumulated in ischaemic rat caecum/colon 6 and 24 h after mesenteric ligation. Presumably allied to the accumulation of liposomal components, necrotic caecum/colon showed marked Ca²⁺ accumulation and phospholipid depletion. It is postulated that Ca²⁺ and Ca²⁺-activated membrane phospholipases may be implicated in the mechanism of liposome accumulation in chronic ischaemia.     

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.     

Anti-tumor vaccine adjuvant effects of IL-2 liposomes in mice immunized against MCA-102 sarcoma.

MCA-102, a murine sarcoma previously reported to be non-immunogenic in C57/BL6 murine tumor models was used in a tumor vaccine preparation which included liposome encapsulated IL-2 as an adjuvant. C57/BL6 mice were immunized in the right hind footpad with irradiated MCA-102 murine sarcoma cells on days 0, 7, and 21 with or without IL-2 liposome adjuvant at 25,000 IL-2 units/injection. Mice were challenged with live tumor in the right flank on day 35. Survival of mice given IL-2 liposomes with irradiated MCA-102 cells was significantly prolonged over mice given tumor antigen with saline, and non-immunized mice. In addition, mice which received the IL-2 liposome adjuvant also had prolonged survival over those mice immunized with the additional control adjuvants of free IL-2 or dimyristoyl phosphatidyl choline (DMPC) lipid in the form of empty liposomes. IL-2 liposome plus tumor antigen also yielded a significant local protective response against live MCA-102 tumor challenge. When live tumor was injected into the site of previous immunizations on day 21 after two immunizations, the IL-2 liposome adjuvant group showed significantly delayed local growth of tumor compared to animals immunized without adjuvant, or with the adjuvants of empty liposomes or free IL-2. Finally, immunized mice were challenged with irradiated tumor cells and saline intradermally in the ears and delayed type hypersensitivity (DTH), an indicator of helper T cell response, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of conventional or photopolymerized liposomes with platelets in vitro

We have examined the effects of liposomes on in vitro platelet aggregation. Liposomes were prepared from various conventional lipids and from a novel photopolymerizable phosphatidylcholine derivative (DPL, bis[1,2-(methacryloyloxy)dodecanoyl]- -alpha-phosphatidylcholine). None of the liposome preparations studied caused marked platelet aggregation in either plasma or buffer solution. However, positively charged vesicles impaired the ability of platelets in plasma to aggregate in response to ADP, whereas negatively charged vesicles impaired the ability of platelets in buffer to aggregate in response to thrombin. DPL vesicles had only modest effects on platelets in plasma or buffer.