Exposure Of Anionic Phospholipids Research Articles

Effects of selective serotonin reuptake inhibitors on platelet functions: a literature review.

Many epidemiological studies have evidenced an increased bleeding risk associated with selective serotonin reuptake inhibitors (SSRIs), yet the underlying mechanisms remain unclear. This review summarizes data on SSRIs' effects on platelet functions assessed with assays used in clinical practice and highlights the areas that deserve further investigation. Conflicting results of SSRI effects on platelet aggregation were observed irrespectively of the agonist used, the antidepressant drug or the study type. Alike, discrepant results were reported with flow-cytometry-based assays assessing either platelet surface glycoprotein levels, integrin activation, agonist-induced secretion of intraplatelet granule content or membrane anionic phospholipid exposure. Other tests may have detected a platelet function defect in SSRIs samples, however, results were largely inconsistent. Critical literature examination unveils very low certainty of evidence on potential SSRI effect on platelet functions. Findings are often inconsistent even when similar methods are used, most likely because of differences in study design, included patients (age, comorbid conditions), SSRIs' type and dose, uncontrolled confounding factors, and statistical analysis power. Further studies are needed to disentangle any intrinsic antiplatelet effect of SSRIs and the multiple confounding factors, mainly the depression control itself and the degree of platelet SERT inhibition.

CD36-fibrin interaction propagates FXI-dependent thrombin generation of human platelets.

Thrombin converts fibrinogen to fibrin and activates blood and vascular cells in thrombo-inflammatory diseases. Platelets are amplifiers of thrombin formation when activated by leukocyte- and vascular cell-derived thrombin. CD36 on platelets acts as sensitizer for molecules with damage-associated molecular patterns, thereby increasing platelet reactivity. Here, we investigated the role of CD36 in thrombin-generation on human platelets, including selected patients with advanced chronic kidney disease (CKD). Platelets deficient in CD36 or blocked by anti-CD36 antibody FA6.152 showed impaired thrombin generation triggered by thrombin in calibrated automated thrombography. Using platelets with congenital function defects, blocking antibodies, pharmacological inhibitors, and factor-depleted plasma, CD36-sensitive thrombin generation was dependent on FXI, fibrin, and platelet signaling via GPIbα and SFKs. CD36-deficiency or blocking suppressed thrombin-induced platelet αIIbβ3 activation, granule exocytosis, binding of adhesion proteins and FV, FVIII, FIX, FX, but not anionic phospholipid exposure determined by flow cytometry. CD36 ligated specifically soluble fibrin, which recruited distinct coagulation factors via thiols. Selected patients with CKD showed elevated soluble fibrin plasma levels and enhanced thrombin-induced thrombin generation, which was normalized by CD36 blocking. Thus, CD36 is an important amplifier of platelet-dependent thrombin generation when exposure of anionic phospholipids is limited. This pathway might contribute to hypercoagulability in CKD.

Anticardiolipin from Periodontitis Patients Impact Fetal Loss and Annexin V

Anticardiolipin antibodies, found at elevated serum concentrations in 15% to 20% of individuals with periodontitis, are associated with adverse pregnancy outcomes, thrombotic conditions, and accelerated atherosclerosis in autoimmune disease such as the antiphospholipid syndrome. Our previous studies demonstrated that antibodies raised in mice against Porphyromonas gingivalis caused fetal loss in a mouse pregnancy model due to anticardiolipin antibodies. Such antibodies are induced via molecular mimicry with the serum protein β2-glycoprotein 1 (β2GP1), the target antigen of anticardiolipin. Furthermore, human anticardiolipin IgG is associated with increased serum markers of vascular inflammation, and IgG purified from periodontitis subjects with elevated anticardiolipin stimulates inflammatory cytokine production by endothelial cells and a trophoblastic cell line. Activation of the trophoblastic cells by anticardiolipin occurs through Toll-like receptor 4. In the present study, we observed that IgG anticardiolipin from periodontitis subjects also causes fetal loss in mice. Displacement of the protective 2-dimensional lattice formed by annexin V on trophoblast surfaces by anticardiolipin, via its interaction with its target antigen β2GP1, leading to fibrin clot formation due to exposure of anionic phospholipids to plasma, is a plausible pathogenic mechanism explaining adverse obstetrical outcomes in antiphospholipid syndrome. Therefore, we assessed such interactions in periodontitis. We observed that anticardiolipin from periodontitis subjects competes for annexin V on an artificial phosphatidylserine monolayer, replicating a key activity of autoantibodies found in patients with antiphospholipid syndrome. In addition, we found that anticardiolipin from periodontitis subjects increases annexin V levels on the BeWo choriocarcinoma cell line, consistent with mobilization of annexin V to the cell surface to facilitate repair following membrane damage. The data indicate that sera and IgG from periodontitis subjects with elevated anticardiolipin levels may influence pregnancy outcomes due to interactions with annexin V.

GPIbα Activation Triggers Platelet Tissue Factor/FVIIa-Dependent Procoagulant Activity, Which Is Dampened By Platelet Secreted TFPI and Protein S. Novel Platelet-Based Model of Hemostasis

Human platelets contain Tissue Factor (TF) demonstrated by western blotting (WB), IP, confocal IF microscopy and flow cytometry (FC) using an array of different α-TF antibodies. Moreover, TF is synthesized by platelets and has procoagulant activity (PCA) (Panes et al. Blood 2007). Platelets also contain the full-length α-isoform of TFPI that is exposed on the plasma membrane of activated, coated-platelets. A new role of Protein S (PS) is to function as TFPI co-factor for TF/FVIIa/FXa inactivation. In fact, TFPI function, enhanced by protein S (PS), dampens thrombin generation on the platelet surface (Wood, JP et al, 2014). In endothelial cells TFPI appears associated with the cell surface through glycosylphosphatidyl-inositol-mediated anchorage, suggesting some type of association with cholesterol-rich domains in cell membranes (lipid rafts, LR). Platelets also contain PS, but its functional association with platelet TFPI to inhibit platelet TF-dependent PCA remains still unknown. Aim: to disclose the physiologic role of TFPI and PS on the TF-dependent PCA of human platelets. Results: Stimulation of isolated, washed platelets with VWF-Ristocetin (VWF-R) resulted in 10-fold increase of TF-dependent FXa generation within 2-5min, compared with non-stimulated (N-S) platelets: [median 17(10-37) to 182(43-847) nM FXa/2x107 platelets, n=198]. VWF-R induced anionic phospholipid exposure, but no α-δ-granules release in washed platelets. FX activation could be triggered without external FVII, although this was required to sustain the reaction. FVII/FVIIa was demonstrated in washed platelets by WB, confocal IF microscopy and FC, and its membrane expression augmented after platelet activation. FXa induced by VWF-R was abolished by pre-incubation with TFPI or with several polyclonal or mAbs against to each TF, FVIIa or GPIbα. In contrast, TRAP stimulation (n=84) induced little or no FXa generation [Median 32(11-219) nM FXa/2x107platelets], but FX activation was increased by 54% (range 13-109%, p=0.0039) in platelets pre-incubated with α-TFPI. This increase was more pronounced with pre-incubation with α-PS (90%, range 31-227%, p<0.004). Combination of α-TFPI and α-PS did not enhance further FX activation. The releasate fraction of TRAP-stimulated platelets inhibited the FXa produced by VWF-R-stimulated platelets, supporting that TRAP-induced secretion of TFPI and PS explained this effect. Membrane exposure of platelet TFPI measured by FC was decreased after TRAP stimulation, a paradox explained by the high content of TFPI and PS in microparticles (MP) contained in the TRAP releasates. In contrast, the smaller number of MP released by VWF-R activation contained neither TFPI nor PS (WB assay). IP assays in platelet membrane fractions showed co-precipitation of TF, FVII/FVIIa and GPIbα. All these proteins co-precipitated also with TFPI in lipid raft fractions. Importantly, TFPI was notably augmented in LR fractions after TRAP stimulation, as compared with VWF-R stimulation. Moreover, TRAP stimulation resulted in co-precipitation of TFPI and PS in cytosolic, membrane and released platelet fractions. Conclusions: 1. TF-dependent PCA of washed human platelets is specifically and rapidly induced by GPIbα activation and it´s not accompanied with α-δ-granules release, including TFPI and PS. 2. Platelets contain enough membrane FVII to trigger the FX activation. 3. TRAP induces granule release, including TFPI and PS, which block TF-dependent PCA. 3. This TFPI is predominantly localized in LR fractions and co-precipitates with PS. 4. Secreted protein S likely localizes TFPI to the platelet membrane rich in anionic-rich phospholipids. 5. These results suggest that TF is translocated to LR for its inactivation. 6.These findings lead us to propose a novel model in which clotting is triggered by platelet TF during the first stage of platelet adhesion through GPIbα-VWF interaction; and TF/FVIIa/FXa would be dampened by the secreted TFPI and PS during subsequent platelet activation. Human platelets TF, FVII and the anionic phospholipid surface become central players to assemble and localize the whole clotting process into and around the platelet plug for both hemostasis and atherothrombosis; and platelet TFPI and PS would modulate its growth inactivating TF/FVIIa/FXa on the platelet surface. DisclosuresNo relevant conflicts of interest to declare.

Surface code—biophysical signals for apoptotic cell clearance

Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

Infusible platelet membrane as a platelet substitute for transfusion: an overview.

Platelets are small, irregularly shaped clear cell fragments in circulating blood; they have a biconvex disc structure with an equatorial diameter of 2–3 μm and are anucleate. Platelets play a dual role in haemostasis. First, they adhere to and aggregate at sites of vascular lesion1,2. Second, the exposure of anionic phospholipids on the surface of activated platelets acts as a catalytic site for the initiation of the coagulation cascade3,4 that will strengthen initial haemostatic plug formation. Due to the short life-span of platelet concentrates, units of platelets are generally stored in blood banks for 3–5 days, after which they are discarded. This results in a chronic shortage of fresh platelets. In recent years, the demand for standard platelet concentrates has continued to increase. Various approaches are being taken to develop new human platelet products, as possible alternatives to currently available platelet concentrates5,6. Platelet microparticles are microvesicles of platelet membranes that were first investigated as putative platelet substitutes over 39 years ago. These particles are first observed as vesicles released from platelets after adhesion to vessels walls7–10. Circulation of these microparticles can, therefore, be thought of as a marker of in vivo platelet activity11. Platelet microparticles form spontaneously during the storage of platelets and are found in platelet concentrates12–14, fresh-frozen plasma, and cryoprecipitate15. Platelet microparticles, like intact platelets, have procoagulant activity9,16–21, adhere to the vascular subendothelium, and enhance platelet adhesion22. Because platelet microparticles possess haemostatic properties23,24 similar to those of intact platelets, it was rational for researchers to investigate their potential as a possible platelet substitute. In this regard, a microparticulate human platelet preparation has been developed by Cypress Bioscience (San Diego, CA, USA). This company prepared infusible platelet membrane (IPM) from outdated platelet concentrates. This IPM, which consists of spherical vesicles with a diameter of approximately 0.6 microns, has been shown to have various procoagulant activities25,26. Generally speaking, the most feasible approaches to the development of a platelet substitute are those that use phospholipid vesicles or membranes.

Inhibitory Autoantibodies to Protein Disulfide Isomerase From Systemic Lupus Erythematosus Patients Are Prothrombotic

AbstractAbstract 392Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by loss of self-tolerance, aberrant autoantibody production and multiple organ damage. Treating the disease symptoms (flares) has remarkably improved the survival of SLE patients, but thrombosis remains a major negative prognostic factor. Currently available tests do not predict thrombotic events; hence there is a need for identification of novel biomarkers of thrombosis associated with SLE and characterization of the relevant pathophysiologic mechanisms. We have shown that inhibition of cell-surface protein disulfide isomerase (PDI) can regulate the thrombotic properties of the endothelium in vitro, through exposure of phosphatidylserine (PS). We therefore hypothesized that inhibitory autoantibodies to PDI may promote or enhance the prothrombotic phenotype in SLE.We developed a quantitative ELISA to measure the titers of anti-PDI autoantibodies present in the sera and performed a cross-sectional analysis of sera from 217 SLE patients and 99 healthy controls enrolled in the Oklahoma Lupus Cohort. Low levels of PDI autoreactivity were detected in 54 out of 99 healthy controls tested, with a median of 4.185 AU and an interquartile range (IQR) of 2.25–4.18. Anti-PDI autoantibodies were detected in 107 out of 217 SLE sera tested, with significantly higher PDI autoreactivity (median 5.982, IQR 2.43–11.23, p = 0.042 Mann-Whitney test). We defined elevated anti-PDI titers as those to be higher than the 95th percentile of serum anti-PDI levels in the control group. Thirty-five out of the 217 SLE patients (16%) exhibited anti-PDI titers above the 95th percentile cut-off, an incidence significantly higher (p=0.0057, Fisher’s exact test) than the control group (5/99, 5% by cut-off definition). Interestingly, the incidence of patients with elevated anti-PDI autoreactivity was doubled in SLE patients with secondary antiphospholipid syndrome (APS), 20 out of 89 (22%), as compared to SLE patients without APS (15 out of 128, 11%, p = 0.04 Fisher’s exact test). Accordingly, we found a significant correlation between the titers of anti-PDI autoantibodies and the anti-cardiolipin IgG levels (Spearman r= 0.212, P = 0.003), indicating a link with the presence of antiphospholipid antibodies. Since the APS diagnosis involves both the detection of antiphospholipid antibodies and the presence of clinical thrombotic events, this might be an indication that anti-PDI autoantibodies are associated with thrombosis in SLE.Clinical history revealed that SLE patients with elevated PDI autoreactivity had higher SLE Disease Activity Index (SLEDAI) scores than those with normal anti-PDI levels (P = 0.0256, Mann-Whitney test), indicating a correlation between anti-PDI levels and disease activity. We observed significant inverse correlations between anti-PDI titers and complement levels as measured by the total hemolytic complement assay (Spearman r = −0.134, P = 0.049), C3 levels (Spearman r = −0.169, P = 0.013) and C4 levels (Spearman r = −0.215, P < 0.001). While the biological significance of this correlation is under investigation, activation of the classical complement pathway on the surface of PS-exposing cells has been documented in the literature.We have shown that PDI inhibition on endothelial surface promotes PS exposure and a thrombotic phenotype. We tested the inhibitory properties of anti-PDI autoantibodies and found that 50% of the SLE sera with elevated anti-PDI titers inhibited PDI reductase activity in vitro, thus indicating that these autoantibodies can modulate PDI function. Furthermore, affinity-purified anti-PDI autoantibodies from SLE sera were able to induce a prothrombotic phenotype on endothelial cells in vitro as evidenced by tissue factor decryption and enhanced prothrombinase activity, a direct indicator that anti-PDI autoantibodies induce anionic phospholipid exposure in vascular cells.In conclusion, we found an increased incidence of elevated anti-PDI titers in SLE, especially in patients with antiphospholipid syndrome, that correlate with disease activity. These autoantibodies inhibit PDI enzymatic activity and are prothrombotic in vitro. To our knowledge this is the first report of autoantibodies directly inducing the exposure of anionic phospholipids on vascular cells with mechanistic implications for the pathophysiology of SLE and APS. Disclosures:No relevant conflicts of interest to declare.

Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of tumor epithelial cell AP might provide a target for 2aG4 and contribute to the increased effectiveness of such combination therapy in vivo. We conclude that the combined targeting of mtp53 and the tumor vasculature is a novel effective strategy for combating advanced breast tumors.

Reduction of Phosphatidylserine Exposure through Copper Chelation Leads to Reduced Fibrin Deposition After Laser Induced Vascular Injury In Vivo.

Abstract 3049Poster Board II-1025Colocalization and assembly of blood coagulation factors in the presence of negatively charged phospholipids leads to a 1,000-fold increase in the rate of thrombin generation compared to the solution reaction. We have established prothrombin fragment 1, the region of prothrombin containing the γ-carboxy-glutamic acid domain, as a probe for anionic phospholipids including phosphatidylserine. Prothrombin fragment 1 binds with high affinity to phosphatidylserine-containing membranes in vitro and identifies phosphatidylserine exposure relevant for the site of assembly of coagulation complexes in vivo. In order to determine the effect of phosphatidylserine exposure on thrombus formation during the laser injury model in vivo, we treated mice orally with the Cu2+ chelator tetrathiomolybdate for one week prior to study. This treatment has been shown to suppress phosphatidylserine exposure in rats (PNAS, 100: 6700-05, 2003). After copper chelator treatment, normal partial thromboplastin times (39 sec vs 42 sec, p=0.5) and whole blood counts in treated versus untreated mice were similar, suggesting that copper chelation did not affect the function of coagulation factors or total blood cell counts. Annexin V and Prothrombin fragment 1 were also used to measure phosphatidylserine exposure after thrombin (1 U/ml) stimulation of washed platelets using flow cytometric analysis. Platelets from untreated mice exhibited 2-fold increase in binding of both Annexin V and Prothrombin fragment 1 after thrombin stimulation; these values are similar to those previously reported. In contrast, the platelets of treated mice did not expose phosphatidylserine upon thrombin stimulation. Treatment with copper chelator did not affect platelet degranulation, as determined by surface exposure of P-selectin in flow cytometry. In addition, total phospholipid content and the ratio of outer to inner membrane phospholipids was not affected by treatment with copper chelator, suggesting that any reduction in detection of phosphatidylserine was due to reduction in exposure on the cell surface in response to an appropriate stimulus rather than reduced biosynthesis. Fluorescently conjugated Prothrombin fragment 1 or fluorescently conjugated antibody directed against phosphatidylserine were used as probes to follow the kinetics of phosphatidylserine exposure after the laser injury of cremaster muscle arterioles of a living mouse using high speed fluorescence intravital microscopy. Endogenous platelets were labeled with a fluorescently conjugated Fab fragment of an anti-CD41 antibody and fibrin deposition was measured using a fluorescently conjugated antibody that recognizes fibrin but not fibrinogen. We observed a 42% reduction (median of 18 thrombi, p=0.02) in Prothrombin fragment 1 binding and a 60% reduction (median of 27 thrombi, p=0.0002) in anti-phosphatidylserine binding after laser injury compared to untreated animals (n=58 thrombi). The accumulation of platelets during thrombus formation was not affected by the treatment when compared to untreated mice (p=0.4). On the other hand, fibrin deposition was reduced by 64% in treated mice (median of 38 thrombi, p=0.001) when compared to untreated animals (39 of thrombi). These data suggest that suppression of phosphatidylserine exposure reduces assembly of coagulation complexes resulting in a suboptimal concentration of thrombin for full fibrin generation but sufficient thrombin to activate platelets to yield a normal platelet thrombus. This emphasizes the importance of the exposure of anionic phospholipids as the surface for the colocalization of the coagulation complexes in vivo. DisclosuresNo relevant conflicts of interest to declare.

Platelet Tissue Factor: Fast and Specific Clotting Activation Pathway Mediated by VWF-GPIba Interaction and Platelet Membrane FVII. Human Platelets Contain All the Components for Assembling the Prothrombinase Complex and Their Procoagulant Function Is Independent of Aggregation/Secretion and GPVI Function.

Abstract 3000Poster Board II-977Human platelets synthesize and store functionally silent tissue factor (TF) which expresses procoagulant activity (PCA) after platelet activation. Fast activation of TF was elicited by VWF-Ristocetin (VWF-R) through GPIbαa activation and Src-Lyn transduction pathway (Blood, Nov 2008; 112:113). Given that GPVI, along with GPIb and TF have been found in “lipid rafts”, and the activated form of GPVI signals through Fyn, another member of the Src family, we tested if GPVI was involved in TF-initiated PCA. We also studied the time-course and pathway specificity of TF activation and the role of platelet FVII in PCA. Weak TF immunofluorescence and co-localization with GPIba were observed in non stimulated washed platelets. A mild increase of TF fluorescence was detected 2 min after TRAP activation, which augmented when the stimulus was VWF-R. Furthermore, striking enhancement of TF fluorescence occurred 2 min after depositing platelets over a VWF-coated surface, but not over fibrinogen or albumin. Platelets adherent to VWF matrix showed GPIb clustering and loss of co-localization with TF. Externalization of TF was confirmed by immunoprecipitation (Ip) of biotinylated membranes before and after platelet activation. Concomitantly, TF-dependent FXa generation increased 5-10-fold shortly after VWF stimulus. Washed platelets stimulated with VWF-R agglutinated normally when stirred in an aggregometer, and the fraction of platelets exposing anionic phospholipids (annexin V binding) was similar to parallel samples stimulated with TRAP. However, VWF-R induced null 14C-serotonin secretion and P-selectin exposure (flow cytometry) in washed platelets. In contrast, TRAP, collagen, ADP and convulxin induced full platelet aggregation, 14C-serotonin and P-selectin secretion at 2-5 min, but with no increase in FXa generation. Platelet PCA was inhibited by antibodies against TF, GPIba, FVIIa, as well as by SU6656 and PP2 (Src pathway inhibitors), but not by Gö6850 (a PKC inhibitor) or exogenous TFPI. p85, a subunit of PI-3K constitutively associated with GPIb complex, becomes strongly associated with TF after stimulation with VWF-R, though only weakly after TRAP activation, confirming the coordinate activation of GPIb and TF.FVII and FX were revealed in platelet membrane fractions by immunoblotting and both co-precipitate with TF in non-stimulated platelets. Two min after activation with VWF-R striking co-precipitations of TF with FVII and FX light chains were evidenced, denoting activation of platelet FVII and FX. When exogenous FX was added to the assay, the amount of FXa generated after 1 and 2 min stimulation was similar whether or not exogenous FVIIa was added.Platelets from four non-related patients with bleeding related to hereditary defect of GPVI had null aggregation and secretion with convulxin and collagen, less than 7% labeling of GPVI by flow cytometry and an immunoreactive membrane GPVI of Mr≈40kDa (native GPVI Mr=62kDa). All of them had normal agglutination with VWF-R and normal FXa generation.In summary, GPIb activation by VWF constitutes a unique and fast inducer of platelet TF-dependent PCA. This process requires anionic phospholipid exposure, but is independent of platelet GPIIb/IIIa and GPVI function. Platelet FVII can initiate FXa generation without need of plasma FVII. The associations of platelet FVII and FX with TF on membrane fractions, together with the large amount of FV in platelets, indicate that human platelets provide not just TF and a PCA phospholipid platform, but also all the components of the prothrombinase complex to trigger the clotting process. Taken together, our results underline the central role of platelets in the whole hemostatic process, unifying primary and secondary hemostasis and circumscribing thrombin generation and fibrin deposition where platelet plug is being formed. Platelet PCA should become a pharmacological target for preventing or managing bleeding and thrombotic disorders. Disclosures:No relevant conflicts of interest to declare.

Targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases

SummaryTargeting exposed anionic phospholipids on a spectrum of virus-infected cells can protect against lethal virus infections in vivo.

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.

Plasmin Enhances Cell Surface Tissue Factor Activity without Increasing Tissue Factor Antigen Levels at the Cell Surfaces of Mesothelial and Endothelial Cells.

Mesothelial cells that line the thoracic and peritoneal cavities play an important role in protecting the heart, lungs and internal organs and keeping the surfaces free of friction and non-adhesive. Although human mesothelial cells in culture express low levels of tissue factor (TF), TF expression was not detected in vivo in the mesothelial lining of normal pleura but was detected in the mesothelium overlying injured or inflamed lung tissue. Inflammation in the lung parenchyma predisposes to the development of inflammatory exudative pleural effusions and pleural loculation, but the mechanism(s) responsible for TF expression in mesothelial cells under such conditions remains unclear. In the present study, we investigated whether plasmin and thrombin, two major proteases present in exudative pleural effusions, induce TF expression in cultured primary human pleural mesothelial cells (HMC). Confluent monolayers of HMC were treated with plasmin (50 nM) and thrombin (5 nM) for varying time periods. TF expression was analyzed by measurement of cell surface TF activity in FX activation assay, TF antigen by Western blotting and TF mRNA by Northern blot analysis. Both plasmin and thrombin increased cell surface TF activity by 3–4 fold. Thrombin markedly increased TF antigen levels. Surprisingly, only a minimal increase in TF antigen levels was seen in the plasmin-treated cells and no increase in TF mRNA was observed in these cells. Interestingly, 75% of the increase in TF activity was observed within 30 min of plasmin treatment. In thrombin-stimulated cells, TF activity increased slowly, reaching maximum at 4–6 h. Treatment of HMC with actinomycin-D or cycloheximide prior to the addition of the protease failed to inhibit the plasmin-induced TF activity whereas they completely attenuated the thrombin-induced TF activity. These data suggest that plasmin and thrombin enhance TF activity in HMC by different mechanisms, and that plasmin-induced TF activity is independent of de novo synthesis of TF. We also examined the effect of plasmin on TF activity in human umbilical vein endothelial cells (HUVEC). Although plasmin had a minimal effect on naive HUVEC, 30 min plasmin treatment increased cell surface TF activity of thrombin-stimulated endothelial cells by more than two-fold. In our earlier studies, we showed that proteases could mobilize intracellular TF to the cell surface of fibroblasts and thus enhance TF activity independent of de novo synthesis of TF. However, FACS analysis and 125I-FVIIa binding to cell surface TF revealed no significant differences in TF antigen levels at the surfaces of control and plasmin-stimulated HMC. Since exposure of anionic phospholipids at the outer cell surface membrane can increase cell surface TF activity, we next sought to determine if plasmin increases anionic phospholipids on the outer bilayer of the HMC cell membrane. Prothrombin activation assays showed no increase in anionic phospholipids at the surface in plasmin-treated cells. Although recent studies suggest that disulfide bond formation of cysteines in TF enhances TF procoagulant activity, our recent studies in other cell model systems suggest that this mechanism is unlikely to account for plasmin-mediated induction of cell surface TF activity in HMC. While further studies are required to exclude this possibility, our present data indicate that plasmin enhances cell surface TF activity of HMC and HUVEC by a novel mechanism that is yet to be elucidated.

Imaging Acute Cardiac Cell Death: Temporal and Spatial Distribution of 99mTc-Labeled C2A in the Area at Risk After Myocardial Ischemia and Reperfusion

The exposure of anionic phospholipids is a near-universal molecular signature for cell death. Based on our prior finding that the (99m)Tc-labeled C2A domain of synaptotagmin I accumulates intensely in the area at risk, this study quantitatively characterized the temporal and spatial distribution of the radiotracer in a rat model of myocardial ischemia and reperfusion. Myocardial ischemia and reperfusion were induced by occlusion of the left anterior descending coronary artery in rats. The temporal uptake of the labeled fusion protein of C2A and glutathione-s-transferase (C2A-GST) in the area at risk was investigated by intravenously injecting the radiotracer at 0, 1, 3, 6, and 24 h after reperfusion, and the radioactivity uptake was quantified by gamma-counting of infarcted and ischemic noninfarcted cardiac tissues. Alternatively, the radiotracer was injected at 2 h after reperfusion, and the uptake was measured at 1, 3, 6, and 24 h after injection. In vivo planar imaging was performed on a gamma-camera using a parallel-hole collimator. The distribution of radioactivity was qualitatively examined by autoradiography. The relationship between the uptake of the radiotracer in the area at risk and the ischemic duration was examined by gamma-counting. Temporally, the radioactivity uptake in the area at risk maximized when the radiotracer was injected before 3 h after reperfusion. Injections at 6 and 24 h after reperfusion resulted in a 30% and 50% reduction in uptake, respectively. However, when the injection was done early (2 h after reperfusion), the tracer was retained in the area at risk with little washout for at least 24 h. Spatially prominent hot-spot uptake was seen in all cases of planar imaging. In autoradiography, the distribution of radioactivity predominantly coregistered with the infarcted regions. This distribution profile was confirmed by direct gamma-counting. In addition, the absolute radiotracer uptake in the infarcted and ischemic noninfarcted tissues, in terms of percentage injected dose per gram, was independent of the ischemic duration. (99m)Tc-C2A-GST has an uptake profile in the area at risk that is appropriate for imaging cardiac cell death in the acute phase.

ANTIBODY-MEDIATED TARGETING OF “INSIDE-OUT” ANIONIC PHOSPHOLIPIDS IN VIRAL DISEASE (47.21)

Abstract The anionic phospholipid phosphatidylserine (PS) is found exclusively in the inner leaflet of the plasma membrane of resting mammalian cells. We hypothesized that certain events that occur during virus replication (eg cell activation or membrane rearrangement) would trigger the exposure of anionic phospholipids on the outer surface of virus- infected cells and subsequently on the enveloped viruses that bud out of these virus- infected cells. We further hypothesized that these exposed anionic phospholipids would serve as targets for anti-viral therapy. We demonstrate here that anionic phospholipids become exposed on the enveloped Pichinde Virus (a model virus for Lassa Fever virus, a potential bioterrorism agent) and on Pichinde virus-infected cells. To detect anionic phospholipids, we used a chimeric monoclonal antibody, bavituximab, that binds anionic phospholipids in a B2-glycoprotein I dependent manner. We show that bavituximab treatment is able to cure overt disease in guinea pigs lethally infected with Pichinde virus. Bavituximab treatment reduced the amounts of virus in multiple tissues and caused direct clearance of virus from the blood. Direct clearance of free virus and antibody-dependent cellular cytotoxicity of virus-infected cells appear to be the major mechanisms that contribute to the anti-viral effect of bavituximab. Bavituximab-treated survivors were immune to reinfection. Furthermore, the murine version of bavituximab, 3G4, shows therapeutic efficacy in a lethal murine model for human cytomegalovirus. Our study demonstrates the promise of anionic phospholipids as targets for new broad-spectrum anti-viral drugs.

Tissue Factor Activation: Is Disulfide Switching a General Regulatory Mechanism?.

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces trigger the coagulation cascade and cell signaling. It is a well-known fact that only a small fraction of TF at the cell surface is coagulantly active whereas a majority of TF on the cell surface is non-functional (cryptic). However, it is unclear, at present, how the coagulant active TF differs from the cryptic form, and mechanisms involved in TF activation. Recent studies show that a thiol oxidizing agent, HgCl2, increases TF coagulant activity on the surface of HL-60 cells by several fold (Chen et al., Blood vol 106, abstract #684, 2005). Further, TF is shown to associate with protein disulfide isomerase (PDI) in HaCaT cells (Ahamed et al., Blood vol 106, abstract #685, 2005). Based on these and other observations, it has been proposed that switching between cryptic and coagulant TF involves cleavage and formation of allosteric disulfide bond (Cys186-Cys209) and PDI has been implicated in controlling the conversion of cryptic TF to the coagulant form and to act as a switch between TF-mediated signaling and coagulation. Although these data are interesting and novel, there is no fail-proof evidence that disulfide switching alone and not other potential changes, such as exposure of anionic phospholipids, at the cell surface is responsible for the TF activation associated with various treatments. Therefore we have examined the effect of HgCl2 and other treatments on TF activation in MDA 231 cells in relation to anionic phospholipids and also characterized the cellular expression of PDI in this and other cell types. As reported earlier, the HgCl2 treatment increased the cell surface TF coagulant activity (5-fold or higher). However, the HgCl2 treatment also increased the prothombinase activity by 3-fold. More importantly, annexin V, which binds to anionic phospholipids, markedly reduced the increased TF coagulant activity associated with the HgCl2 treatment whereas it had only minimal and insignificant effect on TF activity of the control cells. Further, pretreatment of cells with 5,5′-dithio-bis(2-nitronezoic acid) (DTNB), a sulfhydryl reagent that reacts with thiol groups and thus can block disulfide switching, failed to prevent the increase in TF activity associated with the HgCl2 treatment. Interestingly, we found that treatment of MDA 231 cells with glutathione (5 to 100 mM), a cell impermeable reducing agent, also increased the surface TF activity by about 2- to 3-fold. In additional studies, we found that PDI antibodies had no effect on either the TF coagulant activity or TF-mediated cell signaling. Further, we found no evidence for the expression of PDI at the cell surface in immunofluorescence confocal microscopy as both monoclonal and polyclonal PDI antibodies failed to stain nonpermeabilized cells whereas they brightly stained intracellular PDI in permeabilized cells. In contrast, TF antibodies stained intensely the surface of both nonpermeabilized and permeabilized cells. Exposure of tumor cells to various proteases failed to transport the intracellular PDI to the cell surface. The present data raise a valid question whether disulfide switching by PDI plays the predominant and general regulatory role in controlling the TF coagulant activity and signaling functions. Our data also suggest that other cellular changes, including increase in anionic phospholipids, may be responsible for increased TF coagulant activity associated with the thiol oxidizers and other treatments.

Serotonergic Mechanisms in Haemostasis: A Connection between Major Depression and Thrombotic Risk.

Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p<0.05 vs. controls). Treatment of patients with the SSRI normalized the expression of these antigens vs. control samples. Studies performed under flow conditions only showed a significant increase of the fibrin formation on vascular damaged vessels of patients with major depression vs. controls (71.1±9.5 % vs. 45.8±5.3 %, p<0.05) with corresponding elevations in F1+2 levels. In vitro incubation of blood samples with a SSRI was followed by a 30% of reduction in fibrin formation. Similar tendencies were confirmed in studies performed along clinical treatment of patients with SSRI. A decrease of 20% and 80% of covered surface by fibrin was observed after 8 and 24 weeks of treatment respectively. Levels of whole blood TF of patients suffering depression were found elevated vs. control samples (1.3±0.4 ng/ml vs. 1.0± 0.4 ng/ml respectively) and tended to decrease during the treatment (p<0.05). Our results reveal a more thrombogenic state in patients diagnosed of major depression associated to serotonergic mechanisms. Strategies aimed to control serotonergic mechanisms in platelets may offer a potential therapy for the regulation of the prothrombotic state of patients with major depression, but also in patients with cardiovascular risk.

Tissue Factor Enriched Microvesicles Plus Factor VIIa Induce Platelet Aggregation in the Absence of Coagulation Proteins: Evidence for the Involvement of Internalization and Trafficking Mechanisms.

The favorable action of FVIIa in critically deterred hemostasis is not completely elucidated though it has been suggested that FVIIa could activate platelets through mechanisms independent of TF. Standard aggregometry, ultrastructural, and immunocytochemical methods were combined with other techniques to assess the interaction of human TF (hTF) enriched microvesicles (MVs) and rFVIIa in a system with repeatedly washed platelets in absence of plasma proteins. Sequential analysis of morphology and immunolocalization of TF on platelet cryosections were also performed. Modifications in the expression of activation antigens and exposure of anionic phospholipids (Annexin V binding) were evaluated by flow cytometry. Activation of tyrosine phosphorylation mediated mechanisms was also monitored. Several inhibitory strategies were used to characterize mechanisms involved in these interactions. After exposure to hTF bearing MVs (hTF-MVs), platelets became reversibly activated reaching a maximum peak in the first minute (41 ± 3% maximal aggregation). Ultrastructural studies revealed that hTF-MVs became rapidly internalized by platelets. This process induced changes in platelet shape, organelle centralization and enhanced platelet-platelet interactions, but did not result in the formation of compact aggregates. Immunocytochemical studies at ultrastructural level demonstrated hTF-MVs trafficking from the external membranes to the alpha granules via the open canalicular system. Flow cytometry studies detected a moderate increase in the expression of platelet activation antigens. In the absence of rFVIIa platelets always return to an apparent resting state in 5 min, recovering their discoid shape, though immunocytochemical studies still demonstrate TF presence in platelets. When rFVIIa was present together with hTF-MVs, platelets became irreversibly aggregated (81% ± 6%). Ultrastructural studies confirmed internal contraction, degranulation and formation of perfectly structured platelet aggregates. Flow cytometry confirmed a marked expression of activation dependent antigens and a significant enhancement of annexin V binding which was never detected in studies with hTF-MVs alone. Antibodies to GPIIb-IIIa blocked reversible and irreversible aggregation steps. Heparins or hirudin inhibited the irreversible aggregation that occurred in the presence of rFVIIa, but did not prevent the initial reversible aggregation caused by hTF-MVs. Antibodies to P-selectin reduced the initial reversible activation of platelets in response to hTF-MVs, but did completely block the aggregation that occurred in the presence of rFVIIa. This study demonstrates that platelets internalize TF when it is exposed on MVs and confirms the existence of TF trafficking from external sources into internal platelet storage pools. Our results prove that the complex rFVIIa-hTF activates platelets in the practical absence of plasma proteins. If TF can be stored in platelets, the hypothesis of a direct action of rFVIIa on platelets independent of TF should be reconsidered, since internal pools of TF could be exposed on the surface upon activation. Our studies indirectly suggest that TF stored in platelets could enhance the potential thrombogenic capacity of platelets under special pathologic situations.

A Monoclonal Antibody that Binds Anionic Phospholipids on Tumor Blood Vessels Enhances the Antitumor Effect of Docetaxel on Human Breast Tumors in Mice

Anionic phospholipids, principally phosphatidylserine, become exposed on the external surface of viable vascular endothelial cells in tumors, providing an excellent marker for tumor vascular targeting. We recently raised an IgG monoclonal antibody, 3G4, which binds to anionic phospholipids in a beta2-glycoprotein I-dependent manner. It inhibited tumor growth in a variety of rodent tumor models by stimulating antibody-dependent cellular cytotoxicity toward tumor vessels. In the present study, we tested the hypothesis that docetaxel, which is known to have antivascular effects on tumors, might induce exposure of anionic phospholipids on tumor vasculature and, thus, enhance the antitumor activity of 3G4. Treatment of human umbilical vascular endothelial cells with subtoxic concentrations of docetaxel (20 pmol/L) in vitro caused anionic phospholipids to be externalized without inducing apoptosis. Docetaxel treatment of mice increased the percentage of tumor vessels that expose anionic phospholipids from 35% to 60%. No induction of phosphatidylserine was observed on vessels in normal tissues even after systemic treatment with docetaxel. Treatment of mice bearing orthotopic MDA-MB-435 human breast tumors with 3G4 plus docetaxel inhibited tumor growth by 93%. Treatment of mice bearing disseminated MDA-MB-435 tumors with 3G4 plus docetaxel reduced the average number of tumor colonies in the lungs by 93% and half the animals did not develop tumors. In both tumor models, the antitumor effect of the combination was statistically superior (P < 0.01) to that of docetaxel or 3G4 alone. Combination therapy reduced the tumor vessel density and plasma volume in tumors to a greater extent than did the individual drugs. The combination therapy was no more toxic to the mice than was docetaxel alone. These results indicate that, as an adjuvant therapy, 3G4 could enhance the therapeutic efficacy of docetaxel in breast cancer patients.

Stored platelets contain residual amounts of tissue factor: evidence from studies on platelet concentrates stored for prolonged periods

The advent of new strategies for pathogen reduction has raised the question of whether platelet (PLT) concentrates (PCs) exposed to longer periods of storage retain adequate hemostatic function. The effects of prolonged storage on adhesive and procoagulant functions of PLTs in PCs have been analyzed. The ability of PLTs to interact with surfaces was assessed by en face electron microscopy. Exposure of anionic phospholipids or tissue factor (TF) antigen on PLTs was assessed by flow cytometry and by immunocytochemical methods at the ultrastructural level. Studies were performed in six different PCs followed 0, 3, 5, 7, and 11 days of storage. A progressive impairment of PLT-adhesive functions was observed after 5 days of storage. A progressive increase in expression of anionic phospholipids and development of procoagulant activity (PCA) measured by a modified prothrombin time (mPT) was observed along the storage. Incubation of PLTs with a specific anti-TF resulted in prolongation of the mPT by approximately 10 to 15 percent. Flow cytometry revealed minimal TF expression at later storage times. Immunocytochemical studies showed minimal TF labeling when studies were performed on PLT whole mounts. Labeling was markedly improved when PLTs were previously exposed to sonication. Prolonged storage of PCs was associated with decreased PLT-adhesive capacities and enhanced PCA. Current preparation procedures and storage media have important limitations for preserving PCs for longer than 1 week. PLTs in PCs retain residual amounts of TF as assessed by immunocytochemical and functional assays. The origin and hemostatic significance of this TF should be investigated further.