Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis.

ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 µM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 µM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway.

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21).

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite.

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.

Disruption of multivesicular body vesicles does not affect major histocompatibility complex (MHC) class II-peptide complex formation and antigen presentation by dendritic cells.

The antigen processing compartments in antigen-presenting cells (APCs) have well known characteristics of multivesicular bodies (MVBs). However, the importance of MVB integrity to APC function remains unknown. In this study, we have altered the ultrastructure of the MVB by perturbing cholesterol content genetically through the use of a deletion of the lipid transporter Niemann-Pick type C1 (NPC1). Immunofluorescence and electron microscopic analyses reveal that the antigen processing compartments in NPC1(-/-) dendritic cells (DCs) have an abnormal ultrastructure in that the organelles are enlarged and the intraluminal vesicles are almost completely absent and those remaining are completely disorganized. MHC-II is restricted to the limiting membrane of these enlarged MVBs where it colocalizes with the peptide editor H2-DM. Curiously, proteolytic removal of the chaperone protein Invariant chain from MHC-II, degradation of internalized foreign antigens, and antigenic-peptide binding to nascent MHC-II are normal in NPC1(-/-) DCs. Antigen-pulsed NPC1(-/-) DCs are able to effectively activate antigen-specific CD4 T cells in vitro, and immunization of NPC1(-/-) mice reveals surprisingly normal CD4 T cell activation in vivo. Our data thus reveal that the localization of MHC-II on the intraluminal vesicles of multivesicular antigen processing compartments is not required for efficient antigen presentation by DCs.

Platelet and red cell responses to three North American pit vipers.

We investigated the hemotoxic effects of three North American pit vipers in healthy human donor blood. Using experiments focusing on platelet and red blood cell activity, we found differential effects of these venoms on these cellular components. Platelet aggregation was most induced by C. adamanteus. Platelet activation was highest with C. atrox. Red blood cells had calcium expression and erythrocyte formation most induced by C. adamanteus and A. piscivorus. These results demonstrate the complex interplay of individual cellular effects with clinical presentations seen in envenomings from these species.

Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

Heparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4-polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4-polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

Modulation of ultralarge immune complexes in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. OBJECTIVES: To determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti-PF4 antibody (hRTO). METHODS: Results: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. CONCLUSIONS: HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

A Double in vivo Biotinylation Technique for Objective Assessment of Aging and Clearance of Mouse Erythrocytes in Blood Circulation.

SUMMARY: We have recently developed a new technique to objectively identify erythrocyte cohorts of defined age in mouse blood. The technique (termed double in vivo biotinylation, DIB) involves an initial biotinylation of all erythrocytes in circulation, followed after a few days by a second biotinylation, at a lower density, that labels the biotin-negative erythrocytes that have entered since the first biotinylation. The proportions of biotin(high), biotin(low), and biotin(negative) erythrocytes are enumerated by flow cytometry. The DIB technique allows us to track age-related changes on erythrocyte cohorts (Protocol A), and to simultaneously identify very young and older erythrocyte populations in the blood (Protocol B). Using this technique, we have reexamined: i) the relationship between age and buoyant density of erythrocytes, ii) erythrocyte destruction through a random removal mechanism, and iii) the expression of phosphatidylserine on aging erythrocytes. We have also used the DIB technique to study age-related changes in the expression of various markers like CD47 and CD147 and green autofluorescence in aging erythrocyte populations.

Minimal role for the alternative pathway in complement activation by HIT immune complexes.

BACKGROUND: Anti-platelet factor 4 (PF4)/heparin immune complexes that cause heparin-induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle. OBJECTIVES: These studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies. METHODS: Using IgG monoclonal (KKO) and/or patient-derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1-esterase inhibitor [C1-INH]), alternative pathway (anti-factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma. RESULTS: Classical pathway inhibitors BBK32 and C1-INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex-induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti-dinitrophenyl (DNP) antibody and the multivalent DNP--keyhole limpet hemocyanin antigen. CONCLUSIONS: Collectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG-containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.

Neutrophil functional heterogeneity is a fixed phenotype and is associated with distinct gene expression profiles.

Differences in the ability of neutrophils to perform relevant effector functions has been identified in a variety of disease states. Although neutrophil functional heterogeneity is increasingly recognized during disease, few studies have examined neutrophil functional heterogeneity during periods of health. In this study, we systematically characterize neutrophil functional heterogeneity in a cohort of healthy human subjects using a range of biologically relevant agonists including immune complexes, bacterial ligands, and pathogens. With repeated testing over several years, we show that neutrophil functional capability represents a fixed phenotype for each individual. This neutrophil phenotype is preserved across a range of agonists and extends to a variety of effector functions including degranulation, neutrophil extracellular trap release, reactive oxygen species generation, phagocytosis, and bacterial killing. Using well-phenotyped healthy human subjects, we demonstrate that neutrophil functional heterogeneity is characterized by differences in neutrophil gene expression patterns. Altogether, our findings demonstrate that while neutrophil function is highly heterogeneous among healthy subjects, each individual's functional capability represents a fixed phenotype defined by a distinct neutrophil gene expression profile. These findings may be relevant during disease states where the ability to perform relevant neutrophil effector functions may impact disease course and/or clinical outcome.

Distinct MHC class II molecules are associated on the dendritic cell surface in cholesterol-dependent membrane microdomains.

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl ß-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

Heterogeneity in neutrophil responses to immune complexes.

Immune complexes (ICs) can trigger inflammation and thrombosis, in part, by activating neutrophils. Much attention has focused on the serologic characteristics of ICs and Fc receptors associated with cellular activation, but few studies have examined host susceptibility to neutrophil activation by ICs. Here, we use a novel whole blood system to investigate the ability of ICs to cause neutrophil activation and degranulation. Using monoclonal anti-platelet factor 4/heparin (PF4/heparin), anti-protamine/heparin antibodies, patient-derived anti-PF4/heparin antibodies, and heat-aggregated immunoglobulin G as model ICs, we demonstrate that heparin-containing ICs cause robust, heparin-dependent neutrophil activation and degranulation which is mediated by both FcγRIIa and complement. Longitudinal testing over a 1-year period shows that an individual's neutrophil response to ICs represents a fixed phenotype resulting in high, intermediate, or low reactivity. Examination of individuals at the extremes of reactivity (high vs low) shows that phenotypic variation resides in the cellular compartment and is correlated with host white blood cell count and absolute neutrophil count, but not age, sex, race, polymorphisms in neutrophil Fcγ receptors, or CR1, CR3, and Fcγ receptor expression on neutrophils. Together, these studies demonstrate that susceptibility to neutrophil activation by ICs is intrinsic to the host and is likely genetic in origin. These findings may be relevant to the heterogeneous clinical outcomes seen in patients with heparin-induced thrombocytopenia and other IC-mediated disorders and could potentially identify patients at high risk for thrombotic and inflammatory complications.

A role of phosphatidylserine externalization in clearance of erythrocytes exposed to stress but not in eliminating aging populations of erythrocyte in mice.

Age dependent changes in phosphatidylserine (PS) externalization were studied in mouse erythrocytes of different age groups (range 1-55 days) by using a newly developed double in vivo biotinylation (DIB) technique. Around 3-4% of the erythrocytes freshly released in the circulation were PS(+) but this proportion fell rapidly to 1% or less and did not increase at later time points. Blocking erythrocyte clearance from the circulation by in vivo depletion of macrophages (by treatment with clodronate loaded liposomes) for up to 7 days did not result in accumulation of PS(+) erythrocytes in the circulation indicating that the low percentage of PS(+) cells within old erythrocytes (age >40 days) was not related to the clearance of PS(+) erythrocytes by macrophages. In vitro treatment with stress inducing agents like deoxyglucose or Ca(++)/calcium ionophore resulted in a marked induction of PS externalization in mouse erythrocytes and this effect was most prominent in the youngest erythrocyte population (age <10 days). Kinetics of clearance of different age groups of stress exposed erythrocytes after intravenous infusion into recipient mice indicated that the young erythrocytes were cleared at fastest rate from the circulation as compared to erythrocytes of older age groups. Within young erythrocytes exposed to stress, PS(+) erythrocytes were preferentially cleared. Taken together our results suggest that PS externalization is unlikely to have a role in the removal of old erythrocytes from blood circulation but may have a role in the clearance of stressed and damaged young erythrocytes in blood circulation.

Age-dependent increase in green autofluorescence of blood erythrocytes.

Green auto-fluorescence (GAF) of different age groups of mouse blood erythrocytes was determined by using a double in vivo biotinylation (DIB) technique that enables delineation of circulating erythrocytes of different age groups. A significant increase in GAF was seen for erythrocytes of old age group (age in circulation more than 40 days) as compared to young erythrocytes (age less than 15 days). Erythrocytes are removed from blood circulation by macrophages in the reticulo-endothelial system and depletion of macrophages results in an increased proportion of aged erythrocytes in the blood. When mice were depleted of macrophages for 7 days by administration of clodronate loaded liposomes, the overall GAF of erythrocytes increased significantly and this increase could be ascribed to an increase in GAF of the oldest population of erythrocytes. Using the DIB technique, the GAF of a cohort of blood erythrocyte generated during a 5 day window was tracked in vivo. GAF of the defined cohort of erythrocytes remained low till 40 days of age in circulation and then increased steeply till the end of the life span of erythrocytes. Taken together our results provide evidence for an age dependent increase in the GAF of blood erythrocytes that is accentuated by depletion of macrophages. Kinetics of changes in GAF of circulating erythrocytes with age has also been defined.

Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies.

Anti-protamine (PRT)/heparin antibodies are a newly described class of heparin-dependent antibodies occurring in patients exposed to PRT and heparin during cardiac surgery. To understand the biologic significance of anti-PRT/heparin antibodies, we developed a murine monoclonal antibody (ADA) specific for PRT/heparin complexes and compared it to patient-derived anti-PRT/heparin antibodies, as well as comparing polyclonal and monoclonal antibodies with anti-platelet factor 4 (PF4)/heparin. Using monoclonal antibodies and polyclonal patient-derived antibodies, we show distinctive binding patterns of anti-PRT/heparin antibodies as compared with PF4/heparin antibodies. Whereas heparin-induced thrombocytopenia (HIT) antibody binding to PF4/heparin is inhibited by relatively low doses of heparin (0-1 U/mL), anti-PRT/heparin antibodies, including ADA, retain binding to PRT/heparin over a broad range of heparin concentrations (0-50 U/mL). Unlike PF4/heparin antibodies, which recognize PF4 complexed to purified or cell-associated glycosaminoglycans (GAGs), anti-PRT/heparin antibodies show variable binding to cell-associated GAGs. Further, binding of anti-PRT/heparin antibodies to PRT/dextran complexes correlates closely with the ability of antibodies to bind to cell-surface PRT. These findings suggest that antibody binding to PRT/dextran may identify a subset of clinically relevant anti-PRT/heparin antibodies that can bind to cell-surface GAGs. Together, these findings show important serologic differences between HIT and anti-PRT/heparin antibodies, which may account for the variability in disease expression of the two classes of heparin-dependent antibodies.

Assessment of survival of aging erythrocyte in circulation and attendant changes in size and CD147 expression by a novel two step biotinylation method.

Three intravenous injections (1mg each) of biotin-X-NHS (BXN) given at 24h intervals labeled all circulating erythrocytes with biotin in C57Bl/6 mice. After 5 days, administration of another i.v. injection of BXN (0.6mg) resulted in the labeling of erythrocytes released in blood circulation after the first biotinylation step, with a lower intensity of biotin. The older erythrocyte population with high intensity of biotin (biotin(high) population) and the later population of newly formed erythrocytes with lower intensity of biotin (biotin(low) population) could be stained with streptavidin-APC (SAv) and identified by flow cytometry. Using the double biotinylation technique, we could examine the survival and age related changes in biotin(low) population of erythrocytes that was released in circulation during a defined time period (5 days). Our results indicate that the percentage of Biotin(low) erythrocytes in circulation remained static for 10 days after the second biotinylation step and than started to decline steadily with time. Mean fluorescence intensity of biotin label on surviving biotin(low) population of erythrocytes however remained stable. These results suggest that after 15 days of release in blood, erythrocytes may undergo random destruction. Furthermore, forward scatter as well as CD147 expression of Biotin(low) population also declined with age. Double biotinylation technique described in this communication offers an easy method for tracking age related changes in populations of erythrocytes released in circulation during a defined period of time.