T-lymphocyte coactivator molecules

T-cell recognition and activation occurs within a specialized area of contact known as the immunologic synapse, localized to areas of glycolipid-enriched membrane microdomains. Within this area, T-cell activation is dependent not only upon specific recognition of peptide antigen embedded within molecules of the major histocompatibility complex, but also on a variety of costimulatory receptors and interactions. Engagement of T-cell receptor (TCR) with antigen alone will induce T-cell unresponsiveness; ligation of the coreceptor CD28 will prevent the induction of unresponsiveness. Novel costimulatory molecules belonging to both the CD28 and TNF/TNFR superfamilies have recently been identified. These receptors appear to act at different stages of T-cell differentiation and activation, have been shown to play a role in promoting different T-cell effector functions, and are important for B-cell differentiation and function.     

Liver X receptors regulate dendritic cell phenotype and function through blocked induction of the actin-bundling protein fascin

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data revealed a cross talk between LXR and Toll-like receptor signaling in macrophages, indicating a role in immunity. Here, we show that LXRalpha is expressed in human myeloid dendritic cells (DCs) and induced during differentiation of monocyte-derived DCs, whereas LXRbeta is expressed constitutively at a very low level. LXR activation by 2 different LXR agonists strongly interfered with lipopolysaccharide (LPS)-induced but not with CD40L-induced DC maturation by altering DC morphology and suppressing interleukin-12-but enhancing interleukin-10-secretion. LXR activation in DCs largely blocked their T-cell stimulatory ability despite essentially unaltered expression of various antigen-presenting and costimulatory molecules. Immunologic synapse formation was significantly inhibited by LXR activation along with a complete block in LPS- but not CD40L-induced expression of the actin-bundling protein fascin. Notably, overexpression of fascin in LXR agonist-treated DCs restored immunologic synapse formation and restored their ability to activate T cells. In conclusion, our data reveal LXR as a potent modulator of DC maturation and function mediated in part by blocking the expression of fascin. Due to the central position of DCs in immunity, LXRalpha could be a potential novel target for immunomodulation.     

T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect

Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL-specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8(+) cytotoxic T lymphocytes (CTLs) specific for tumor cells that express the B-cell lineage cell surface molecule CD19. This was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a CD3-zeta intracellular signaling domain. CD19-redirected CTL clones display potent CD19-specific lytic activity and chimeric immunoreceptor-regulated cytokine production and proliferation. Because B-ALL cells can evade T-cell/natural killer- cell recognition by down-regulation of cell surface accessory molecules that participate in the formation of a functional immunologic synapse, we compared the CD19-specific effector function of genetically modified CD8(+) CTLs toward CD19(+) cells with disparate levels of intercellular adhesion molecule 1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after allogeneic HSCT.     

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion

Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules-indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.     

CD28 plays a critical role in the segregation of PKC theta within the immunologic synapse

The signaling pathways that lead to the localization of cellular protein to the area of interaction between T cell and antigen-presenting cell and the mechanism by which these molecules are further sorted to the peripheral supramolecular activation cluster or central supramolecular activation cluster regions of the immunologic synapse are poorly understood. In this study, we investigated the functional involvement of CD28 costimulation in the T cell receptor (TCR)-mediated immunologic synapse formation with respect to protein kinase C (PKC)theta; localization. We showed that CD3 crosslinking alone was sufficient to induce PKC theta; capping in naive CD4(+) T cells. Studies with pharmacologic inhibitors and knockout mice showed that the TCR-derived signaling that drives PKC theta; membrane translocation requires the Src family kinase, Lck, but not Fyn. In addition, a time course study of the persistence of T cell molecules to the immunologic synapse indicated that PKC theta;, unlike TCR, persisted in the synapse for at least 4 h, a time that is sufficient for commitment of a T cell to cell division. Finally, by using TCR-transgenic T cells from either wild-type or CD28-deficient mice, we showed that CD28 expression was required for the formation of the mature immunologic synapse, because antigen stimulation of CD28(-) T cells led to a diffuse pattern of localization of PKC theta; and lymphocyte function-associated antigen-1 in the immunologic synapse, in contrast to the central supramolecular activation cluster localization of PKC theta; in CD28(+) T cells.     

Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer

Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand-induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell-inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.     

Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the "giant substrate problem" because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.     

The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming

We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ～ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-β1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.     

Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection

Although cytomegalovirus (CMV) expresses proteins that interfere with antigen presentation by class I major histocompatibility complex (MHC) molecules, CD8+ cytotoxic T cells (CTLs) are indispensable for controlling infection and maintaining latency. Here, a cytokine flow cytometry assay that employs fibroblasts infected with a mutant strain of CMV (RV798), which is deleted of the 4 viral genes that are responsible for interfering with class I MHC presentation, was used to examine the frequency and specificity of the CD8+ CTLs to CMV in immunocompetent CMV-seropositive individuals. A large fraction of the CD8+ CTL response was found to be specific for viral antigens expressed during the immediate early and early phases of virus replication and presented by fibroblasts infected with RV798 but not wild-type CMV. These results demonstrate that the inhibition of class I antigen presentation observed in CMV-infected cells in vitro is not sufficient to prevent the induction of a broad repertoire of CD8+ CTLs after natural infection in vivo. Thus, reconstitution of T-cell immunity in immunodeficient patients by cell therapy or by vaccination may need to target multiple viral antigens to completely restore immunologic control of CMV.     

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells

Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.     

The c-Abl tyrosine kinase regulates actin remodeling at the immune synapse

Actin dynamics during T-cell activation are controlled by the coordinate action of multiple actin regulatory proteins, functioning downstream of a complex network of kinases and other signaling molecules. The c-Abl nonreceptor tyrosine kinase regulates actin responses in nonhematopoietic cells, but its function in T cells is poorly understood. Using kinase inhibitors, RNAi, and conditional knockout mice, we investigated the role of c-Abl in controlling the T-cell actin response. We find that c-Abl is required for normal actin polymerization and lamellipodial spreading at the immune synapse, and for downstream events leading to efficient interleukin-2 production. c-Abl also plays a key role in signaling chemokine-induced T-cell migration. c-Abl is required for the appropriate function of 2 proteins known to be important for controlling actin responses to T-cell receptor (TCR) engagement, the actin-stabilizing adapter protein HS1, and the Rac1-dependent actin polymerizing protein WAVE2. c-Abl binds to phospho-HS1 via its SH2 domains and is required for full tyrosine phosphorylation of HS1 during T-cell activation. In addition, c-Abl is required for normal localization of WAVE2 to the immune synapse (IS). These studies identify c-Abl as a key player in the signaling cascade, leading to actin reorganization during T-cell activation.     

Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol‐12‐myristate‐13‐acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA‐activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA‐dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.     

Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).     

IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4⁺ T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4⁺ T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag^−/− mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo.Vesicular traffic has emerged as a central player in the assembly and function of the immune synapse (IS), the specialized interface that forms at the T-cell membrane on contact with an antigen-presenting cell (APC) bearing cognate peptide–MHC complexes. Indeed, polarized recycling of several receptors (1, 2), including the T-cell receptor (TCR) (3), has been shown to control the clustering of these receptors at the synaptic zone and to sustain signaling initiated by their engagement. It is now well established that vesicular traffic to the IS is coopted not only by receptors but also by membrane-associated signaling mediators that are required for signal initiation and amplification, the most prominent being the kinase Lck and the adaptor LAT (linker for activation of T cells) (4, 5). These molecules are carried to the IS by recycling endosomes that move along microtubular tracks toward the centrosome, which polarizes just below the IS on contact with the cognate APC (6).We have recently provided evidence that the intraflagellar transport protein 20 (IFT20) (7) and other components of the intraflagellar transport (IFT) system, which regulates the assembly of the primary cilium (8), act as unconventional players in IS assembly by selectively controlling the polarized traffic of recycling TCRs and other recycling receptors, such as the transferrin receptor (TfR) (9, 10). We investigate here the role of IFT20 in T-cell activation, using a conditional knockout mouse carrying a null IFT20 allele in T cells. We show that IFT20 is required for TCR signaling, IS formation, and recruitment to the TCR activation sites of vesicular LAT downstream of centrosome polarization. These defects in TCR-induced signaling of IFT20-deficient T lymphocytes translate to an inability to mount an antigen-specific T-cell response both ex vivo and in vivo. We also provide evidence of the physiological relevance of these findings by showing that IFT20 deficiency affects disease severity and outcome in a mouse model of CD4⁺ T-cell-driven colitis.     

Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P <.000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P <.001) and F cells (P <.01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.     

Interferon γ limits the effectiveness of melanoma peptide vaccines

The development of effective therapeutic vaccines to generate tumor-reactive cytotoxic T lymphocytes (CTLs) continues to be a top research priority. However, in spite of some promising results, there are no clear examples of vaccines that eradicate established tumors. Most vaccines are ineffective because they generate low numbers of CTLs and because numerous immunosuppressive factors abound in tumor-bearing hosts. We designed a peptide vaccine that produces large numbers of tumor-reactive CTLs in a mouse model of melanoma. Surprisingly, CTL tumor recognition and antitumor effects decreased in the presence of interferon γ (IFNγ), a cytokine that can provide therapeutic benefit. Tumors exposed to IFNγ evade CTLs by inducing large amounts of noncognate major histocompatibility complex class I molecules, which limit T-cell activation and effector function. Our results demonstrate that peptide vaccines can eradicate large, established tumors in circumstances under which the inhibitory activities of IFNγ are curtailed.     

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.     

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p/ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.     

Sickled erythrocytes accelerate clotting in vitro: an effect of abnormal membrane lipid asymmetry

A membrane lipid abnormality induced by sickling and found as a permanent alteration in the irreversibly sickled cell (ISC) is the rearrangement of phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) from the inner to the exterior side of the lipid bilayer. Since PS can provide a catalytic surface for the binding of blood coagulation factors and thus can exhibit procoagulant activity, we investigated the influence of oxy and deoxy reversibly sickled cells (RSC) ass well as ISC on clotting in vitro. Red blood cells (RBC), as the source of phospholipid, were added to platelet-poor citrated plasma containing Russell's viper venom (RVV) and clotting time was measured after recalcification. The clotting time after addition of normal RBC and oxy- RSC was similar to the saline blank (100 sec). In contrast, both oxy- ISC and deoxy completely sickled RSC shortened clotting time by 30%. Using liposomes prepared with identical phospholipid composition to the outer lipid leaflet of either normal RBC, RSC or ISC clotting times similar to those with intact cells were achieved. Since the liposomes did not contain protein, accentuation of clotting appears to be related to abnormal phospholipid organization, in particular to the abnormal exposure to aminophospholipids on the outer surface of the membrane. This abnormality may contribute to the pathogenesis of the vaso- occlusive episode in sickle cell anemia.