S E McKenzie

Thomas Jefferson University, Philadelphia, Pennsylvania, United States

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Publications (84)352.47 Total impact

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    ABSTRACT: IntroductionPlatelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)-mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or β2 Glycoprotein I (β2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild-type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG-GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs.Methods Pre-formed anti-CD40L or anti-β2GPI ICs were injected into hFcR/Caldaggef1+/+ or hFcR/Caldaggef1-/- mice, with or without clopidogrel pre-treatment. Animals were observed for symptoms of shock for 30 minutes, during which time core body temperature was monitored. Platelet counts were obtained before and 30 minutes after IC injection. Lungs were assessed for thrombosis by histology or near-infrared imaging.ResultsBoth CD40L and β2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1+/+ mice. hFcR/Caldaggef1-/- mice were protected from CD40L and β2GPI IC-induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC-induced ITT. Consistent with these findings, IC-induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC-challenged mice was strongly dependent on CalDAG-GEFI.Conclusions Our studies demonstrate that CalDAG-GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG-GEFI may be a promising target for the intervention of IC-associated, FcγRIIa-mediated thrombotic conditions.This article is protected by copyright. All rights reserved.
    Journal of Thrombosis and Haemostasis 10/2014; · 6.08 Impact Factor
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    ABSTRACT: Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis which is often life-threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. Here we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacological inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. 12-LOX was shown to be essential for FcγRIIa-induced PLCγ2 activity leading to activation of calcium mobilization, Rap1 and PKC activation, and subsequent activation of the integrin αIIbβ3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbβ3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identify 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.
    Blood 08/2014; · 9.78 Impact Factor
  • Steven E McKenzie, Bruce S Sachais
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    ABSTRACT: To review the recent developments in understanding the pathophysiology of heparin-induced thrombocytopenia (HIT) and in applying this knowledge to the treatment of patients with suspected and proven HIT.
    Current opinion in hematology. 07/2014;
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    ABSTRACT: For the anucleate platelet it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA profiling platforms, and what the transcriptomes' relationship is with the platelet proteome. We profiled the platelet transcriptome of 10 healthy young males (5 white and 5 black) with no notable clinical history using RNA sequencing and by Affymetrix microarray. We found that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, independently of race and of the employed technology. Our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. Pseudogenes represented a notable exception by exhibiting a difference in expression by race. Comparison of our mRNA signatures a publicly available quantitative platelet proteome showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. However, a high number of mRNAs that were present in the transcriptomes of all 10 individuals had no representation in the proteome. Spearman correlations of the relative abundances for those genes represented by both an mRNA and a protein showed a weak (~0.3) yet significant (P = 5.0E-16) connection. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified gene groups corresponding to distinct cellular processes. The results of our analyses provide novel insights for platelet biology, and indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease.Reviewed by: This article was reviewed by Dr Mikhail Dozmorov (nominated by Dr Yuri Gusev), Dr Neil Smalheiser and Dr Eugene Koonin.
    Biology Direct 02/2014; 9(1):3. · 2.72 Impact Factor
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    ABSTRACT: There is little data considering relationships among human RNA, demographic variables and primary human cell physiology. The Platelet RNA And eXpression-1 (PRAX1) study measured platelet aggregation to arachidonic acid, ADP, PAR1-AP and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. 5911 uniquely mapped mRNAs and 181 miRNAs were expressed commonly and validated in a separate cohort. 129 mRNAs and 15 miRNAs were differentially expressed (DE) by age, and putative targets of these miRNAs were over-represented among these mRNAs. 54 mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship between the RNA types in these "pairs" suggests miRNAs regulate mRNA levels upon aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future association studies between RNAs and clinical end points or ex vivo platelet function must account for age and gender.
    Blood 02/2014; · 9.78 Impact Factor
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    ABSTRACT: Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.
    PLoS ONE 01/2014; 9(7):e102259. · 3.53 Impact Factor
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    ABSTRACT: Our goal was to identify a unique gene-expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene-expression pattern for a known SC-enriched region - the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (~18,500 genes) to compare gene-expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, 131 in the top. Among genes upregulated in the bottom, ~30% were classified as growth and/or developmental genes including several in the the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOX) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.
    Stem cells and development 08/2013; · 4.15 Impact Factor
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    ABSTRACT: Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Y323, Y352 and Y525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Y525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases (SFKs). Phosphorylation of Lat Y191 and PLCγ2 Y759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-induced Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Since potentiation of Syk phosphorylation is not observed in murine platelets, PKC deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyper-phosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk kinase regulation by PKCβ in human platelets.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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    ABSTRACT: Recent work by the Encyclopedia of DNA Elements project showed that non-protein-coding RNAs account for an unexpectedly large proportion of the human genome. Among these non-coding RNAs are microRNAs (miRNAs), which are small RNA molecules that modulate protein expression by degrading mRNA or repressing mRNA translation. MiRNAs have been shown to play important roles in hematopoiesis including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. Additionally, disordered miRNA biogenesis and quantitative or qualitative alterations in miRNAs and their targets are associated with hematological pathologies. Platelets contain machinery to process pre-miRNAs into mature miRNAs, and specific platelet miRNA levels have been found to correlate with platelet reactivity. This review summarizes the current state of knowledge of miRNAs in megakaryocytes and platelets, and the exciting possibilities for future megakaryocyte–platelet transcriptome research.
    Journal of Thrombosis and Haemostasis 06/2013; 11:340-350. · 6.08 Impact Factor
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    ABSTRACT: BACKGROUND: Human blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts. RESULTS: From each of four healthy individuals we generated long RNA (>=40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (>=16 PCR cycles beyond beta-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts. CONCLUSIONS: Our analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and, thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at: http://cm.jefferson.edu/platelets_2012/.
    BMC Genomics 01/2013; 14(1):1. · 4.40 Impact Factor
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    ABSTRACT: The integrin family is comprised of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand binding-induced signals into cells. In human platelets, FcγRIIa has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbβ3 - the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for α(IIb)β(3)-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.
    Blood 12/2012; · 9.78 Impact Factor
  • Steven E McKenzie
    Blood 05/2012; 119(22):5065-6. · 9.78 Impact Factor
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    ABSTRACT: Abstract 3298 The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. While the repertoire of platelet transcripts is a reflection of the megakaryocyte at the time of platelet differentiation, post-transcriptional events are known to occur. Furthermore, a strong correlation between the expressed mRNAs and proteome has been identified. Having a complete understanding of the platelet transcriptome is important for generating insights into the genetic basis of platelet disease traits. To capture the complexity of the platelet transcriptome, we performed RNA sequencing (RNA-seq) in leukocyte-depleted platelets from 10 males, with median age of 24.5 yrs and unremarkable medical history. Their short and long RNA platelet transcriptomes were analyzed on the SOLiD 5500xl sequencing platform. We generated [~]3.5 billion sequence reads [~]40% of which could be mapped uniquely to the human genome. Our analysis revealed that [~]9,000 distinct protein-coding mRNAs and [~]800 microRNAs (miRNAs) were present in the transcriptome of each of the 10 sequenced individuals. Comparison of the levels of mRNA expression across the 10 individuals showed an exceptional level of consistency with pair-wise Pearson correlation values [≥]0.98. The miRNA expression profiles across the 10 individuals showed a similar consistency with pair-wise Pearson correlation values [≥]0.98. Surprisingly, we found that these mRNAs and miRNAs accounted for a little over 1/2 of all of the uniquely mapped sequence reads suggesting the abundant presence of additional non-protein coding RNA (ncRNA) transcripts. Using the annotated entries of the latest release of the ENSEMBL database, we investigated the genetic make-up of these other transcripts. We found that [~]25% of each individual's uniquely mapped reads corresponded to non-protein coding transcripts from mRNA-coding loci. These reads accounted for more than 10,000 distinct such transcripts. In addition, each of the individuals in our cohort expressed an average of [~]1,500 pseudogenes and [~]200 long intergenic non-coding RNAs (lincRNAs). The short RNA profiles of the ten individuals revealed an abundance of diverse categories of ncRNAs including the signal recognition particle RNA (srpRNA), small nuclear RNA (snRNA) and small cytoplasmic RNAs (scRNA). These ncRNAs are involved in the processing of pre-mRNAs and their presence and prevalence in the anucleate platetet suggests the existence of a complex network of mRNA processing that persists after the megakaryocyte fragmentation. We also investigated the RNA-omes of the ten individuals for evidence of transcription of the pyknon category of ncRNAs. Pyknons are of particular interest because each has numerous intergenic and intronic copies whereas nearly all known human protein-coding genes contain one or more pyknons in their mRNA. Recent experimental work has shown that intergenic instances of the pyknons are transcribed in a tissue- and cell-state specific manner. An average of [~]100,000 pyknons are transcribed in each of the 10 sequenced individuals suggesting the possibility of a far-reaching network of interactions that link exonic space to distant non-exonic regions and are active in platelets. Lastly, we found that a large variety of distinct repeat element categories are expressed in the RNA-omes (both short and long) of these individuals. Among the most abundantly represented categories of repeat elements were DNA transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons such as long interspersed elements (LINEs) and short interspersed elements (SINEs). In summary, our RNA-seq analyses have revealed a spectrum of platelet transcripts that transcends protein-coding genes and miRNAs. Indeed, the transcripts that have their source in genomic features not previously discussed or analyzed in the platelet context represent a very significant portion of all platelet transcripts. This in turn suggests an unanticipated richness, and presumably commensurate complexity, for the pl telet transcriptome. While the role of these novel non-protein coding RNAs is currently unknown it is expected that at least some of them may be of functional significance which will in turn permit a better understanding of the molecular mechanisms that regulate platelet physiology and may contribute to processes beyond thrombosis and hemostasis. DisclosuresNo relevant conflicts of interest to declare.
    Blood (ASH Annual Meeting Abstracts). 01/2012; 120(21):Abstract 3298.
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    ABSTRACT: Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called "tissue-specific" promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be "tissue specific," such claims have generally not been rigorously validated. The long-term goal of this work is identifying "cell preferential" miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression ("selectively reduced [SR]") in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. DisclosuresNo relevant conflicts of interest to declare.
    ASH Annual Meeting Abstracts. 01/2012; 120(21):3296-.
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    ABSTRACT: Platelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI(-/-) (hFcR/CDGI(-/-)) were generated. Compared with controls, aggregation of hFcR/CDGI(-/-) platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI(-/-) platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT.
    Blood 06/2011; 118(4):1113-20. · 9.78 Impact Factor
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.
    Blood 11/2010; 117(7):2241-6. · 9.78 Impact Factor
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    ABSTRACT: To explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.
    Blood 11/2010; 117(3):971-4. · 9.78 Impact Factor
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    ABSTRACT: Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4(+) and CD8(+) T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcgammaR expression (FcgammaRI, III, IV) and the dominant expression of the inhibitory receptor FcgammaRIIB. Consistent with this idea, transgenic expression of the activating human FcgammaRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of FcgammaRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcgammaRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.
    The Journal of Immunology 11/2009; 183(11):7129-39. · 5.52 Impact Factor
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    Steven E McKenzie, Michael P Reilly
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    ABSTRACT: In this issue of Blood, Gratacap and colleagues analyze the effects of the multikinase inhibitor dasatinib on platelets, helping to explain the occurrence of bleeding related to its use for CML patients resistant to or intolerant of imatinib.(1) In addition, by virtue of careful experimentation using multiple functional platelet assays, these authors are paving the way for the development of kinase inhibitors as antithrombotic agents.
    Blood 09/2009; 114(9):1722-3. · 9.78 Impact Factor

Publication Stats

1k Citations
352.47 Total Impact Points

Institutions

  • 1998–2014
    • Thomas Jefferson University
      • • Division of Hospital Medicine
      • • Department of Pediatrics
      Philadelphia, Pennsylvania, United States
  • 2013
    • Thomas Jefferson University Hospitals
      Philadelphia, Pennsylvania, United States
  • 2002
    • Christiana Care Health System
      Wilmington, Delaware, United States
  • 1992–2000
    • The Children's Hospital of Philadelphia
      • • Department of Pediatrics
      • • Division of Hematology
      Philadelphia, PA, United States
  • 1999
    • Nemours
      • Department of Pediatrics
      Jacksonville, Florida, United States
  • 1996–1999
    • University of Pennsylvania
      • School of Engineering and Applied Science
      Philadelphia, PA, United States
  • 1992–1995
    • Hospital of the University of Pennsylvania
      Philadelphia, Pennsylvania, United States