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Mortimer Poncz

Publications and source records attributed to Mortimer Poncz.

At least 19 recordsLinked to original sources
PubMed2006

Maturation stage-specific regulation of megakaryopoiesis by pointed-domain Ets proteins.

Numerous megakaryocyte-specific genes contain signature Ets-binding sites in their regulatory regions. Fli-1 (friend leukemia integration 1), an Ets transcription factor, is required for the normal maturation of megakaryocytes and controls the expression of multiple megakaryocyte-specific genes. However, in Fli-1-/- mice, early megakaryopoiesis persists, and the expression of the early megakaryocyte-specific genes, alphaIIb and cMpl, is maintained, consistent with functional compensation by a related Ets factor(s). Here we identify the Ets protein GABPalpha (GA-binding protein alpha) as a regulator of early megakaryocyte-specific genes. Notably, GABPalpha preferentially occupies Ets elements of early megakaryocyte-specific genes in vitro and in vivo, whereas Fli-1 binds both early and late megakaryocyte-specific genes. Moreover, the ratio of GABPalpha/Fli-1 expression declines throughout megakaryocyte maturation. Consistent with this expression pattern, primary fetal liver-derived megakaryocytes from Fli-1-deficient murine embryos exhibit reduced expression of genes associated with late stages of maturation (glycoprotein [GP] Ibalpha, GPIX, and platelet factor 4 [PF4]), whereas GABPalpha-deficient megakaryocytes were mostly impaired in the expression of early megakaryocyte-specific genes (alphaIIb and cMpl). Finally, mechanistic experiments revealed that GABPalpha, like Fli-1, can impart transcriptional synergy between the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 (friend of GATA-1). In concert, these data reveal disparate, but overlapping, functions of Ets transcription factors at distinct stages of megakaryocyte maturation.

Animals↗
PubMed2005

Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications.

Heparin-induced thrombocytopenia (HIT) antibodies recognize complexes between heparin and platelet factor 4 (PF4). Heparin and PF4 bind HIT antibodies only over a narrow molar ratio. We explored the involvement of platelet surface-bound PF4 as an antigen in the pathogenesis of experimental HIT. We show that cell-surface PF4 complexes are also antigenic only over a restricted concentration range of PF4. Heparin is not required for HIT antibody binding but shifts the concentration of PF4 needed for optimal surface antigenicity to higher levels. These data are supported by in vitro studies involving both human and murine platelets with exogenous recombinant human (h) PF4 and either an anti-PF4-heparin monoclonal antibody (KKO) or HIT immunoglobulin. Injection of KKO into transgenic mice expressing different levels of hPF4 demonstrates a correlation between the severity of the thrombocytopenia and platelet hPF4 expression. Therapeutic interventions in this model using high-dose heparin or protamine sulfate support the pathogenic role of surface PF4 antigenic complexes in the etiology of HIT. We believe that this focus on surface PF4 advances our understanding of the pathogenesis of HIT, suggests ways to identify patients at high risk to develop HIT upon heparin exposure, and offers new therapeutic strategies.

Animals↗
PubMed2005

In vivo characterization of the integrin beta3 as a receptor for Hantaan virus cellular entry.

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha(v)beta(3) integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta(3) is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta(3) integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta(3) or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P <0.01) and 18.4 +/- 0.9 days (P < 0.01), respectively. XT-199, a chemical blocker of the alpha(v)beta(3) receptor also prolonged survival to 19.5 +/- 1.3 days (P < 0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta(1) antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta(1) receptors as well as through beta(3) receptors in vivo. Our data demonstrate that the beta(3) receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta(3) for cellular entry within an organism.

Animals↗
PubMed2005

PF4/heparin complexes are T cell-dependent antigens.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, thrombotic disorder associated with development of anti-platelet factor 4 (anti-PF4)/heparin autoantibodies. Little is known about the antigenic and cellular requirements that initiate the immune response to these complexes. To begin to delineate mechanisms of autoantibody formation in HIT, we studied the immunizing effects of murine PF4 (mPF4)/heparin in mice with and without thymic function. Euthymic mice were injected with mPF4/heparin complexes, mPF4, heparin, or buffer. Mice injected with mPF4/heparin, but not mPF4 or heparin alone, developed heparin-dependent autoantibodies that shared serologic and functional characteristics of human HIT antibodies, including preferential binding to mPF4/heparin complexes and causing heparin- and FcRgammaIIA-dependent platelet activation. In contrast, athymic mice did not develop HIT-like antibodies. Taken together, these studies establish that PF4/heparin complexes are highly immunogenic and elicit self-reacting anti-PF4/heparin antibodies in a T cell-dependent manner.

Animals↗
PubMed2005

Intracellular interaction of von Willebrand factor and factor VIII depends on cellular context: lessons from platelet-expressed factor VIII.

We have previously reported that ectopically expressed factor VIII (FVIII) is stored within platelets and is released upon platelet activation. Studies by others in various cell lines have suggested that having von Willebrand factor (VWF) coexpression is necessary for FVIII granular storage and for its secretion. We tested the importance of VWF coexpression for ectopic storage of FVIII in platelets and for its bioavailability. Transgenic mice expressing platelet-specific FVIII were crossed onto a VWF-/- background. Antigenic levels of platelet FVIII in these mice were nearly unchanged whether VWF was present or not. Whole-blood clotting times and FeCl3 carotid artery injury correction demonstrated that platelet FVIII demonstrably improved the bleeding diathesis in FVIIInull mice independent of the platelets' VWF status. Immunogold electron microscopy demonstrated that platelet FVIII is stored in platelet alpha-granules independent of the presence of VWF. It appears that FVIII's interaction with VWF and its intracellular transportation, storage, and secretion differ greatly depending on the cell type. The molecular basis for these differences now needs to be elucidated.

Animals↗
PubMed2005

The relative role of PLCbeta and PI3Kgamma in platelet activation.

Stimulation of platelet G protein-coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP(2)) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCbeta). It also results in the phosphorylation of PIP2 by the gamma isoform of phosphatidylinositol 3-kinase (PI3Kgamma) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCbeta (PLCbeta2 and PLCbeta3) or lacking the G(betagamma)-activated isoform of PI3K (PI3Kgamma). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCbeta2/beta3-/- platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kgamma disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCbeta2/beta3-/- and PI3Kgamma-/- platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCbeta2/beta3 and PI3Kgamma play vital roles in platelet cytoskeletal dynamics.

Actins↗
PubMed2005

Mechanistic basis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) occurs in 1% to 5% of patients treated with unfractionated high-molecular weight heparin and often results in limb- and/or life-threatening thrombotic complications. This disorder involves the formation of antibodies to a complex of Platelet Factor 4 (PF4) with the heparin. We believe that the pathogenesis of this disease begins with having an excess of platelet PF4 release. The freed PF4 is bound to platelet and other vascular membrane surfaces and has three critical roles in the initiation of HIT. (1) Infused heparin neutralizes a portion of excess surface PF4, directly enhancing local thrombosis. (2) The excess PF4 is mobilized into PF4/heparin complexes that stimulates HIT antibody production. (3) The remaining PF4 complexed to heparanoids and heparin on the vascular surfaces now bind to these HIT antibodies and through surface Fc gammaRII receptors leads to more platelet activation and removal, thrombus formation, and inflammation. These events, in turn, further stimulate PF4 release, perpetuating repetitive cycle that results in the clinical manifestations of this disorder.

Dose-Response Relationship, Drug↗
PubMed2005

Megakaryocyte biology and related disorders.

Platelets, derived from megakaryocytes, have an essential role in thrombosis and hemostasis. Over the past 10 years, a great deal of new information has been obtained concerning the various aspects of hematopoiesis necessary to maintain a steady-state platelet level to support physiologic hemostasis. Here we discuss the differentiation of HSCs into megakaryocytes, with emphasis on the key cytokine signaling pathways and hematopoietic transcription factors. Recent insight into these processes elucidates the molecular bases of numerous acquired and inherited hematologic disorders. It is anticipated that the growing knowledge in these areas may be exploited for new therapeutic strategies to modulate both platelet numbers and their thrombogenicity.

Animals↗
PubMed2004

Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia and thrombosis (HITT) is a severe complication of heparin therapy caused by antibodies to complexes between unfractionated heparin (UFH) and platelet factor 4 (PF4) that form over a narrow molar range of reactants and initiate antibody-induced platelet activation. We observed that UFH and tetrameric PF4 formed ultralarge (> 670 kDa) complexes (ULCs) only over a narrow molar range with an optimal ratio of PF4 to heparin of approximately 1:1. These ULCs were stable and visible by electron microscopy, but they could be dissociated into smaller complexes upon addition of heparin. ULCs formed inefficiently when PF4 was incubated with low-molecular-weight heparin, and none formed with the pentasaccharide fondaparinux sodium. In addition, mutation studies showed that formation of ULCs depended on the presence of PF4 tetramers. The ULCs were more reactive as determined by their capacity to bind to a HITT-like monoclonal antibody and showed greater capacity to promote platelet activation in an antibody- and FcgammaRIIA-dependent manner than were the smaller complexes. The capacity of PF4 to form ULCs composed of multiple PF4 tetramers arrayed in a lattice with several molecules of UFH may play a fundamental role in autoantibody formation, antibody-dependent platelet activation, and the propensity for thrombosis in patients with HITT.

Animals↗
PubMed2004

Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin.

The platelet-specific chemokine platelet factor 4 (PF4) is released in large amounts at sites of vascular injury. PF4 binds to heparin with high affinity, but its in vivo biologic role has not been defined. We studied the role of PF4 in thrombosis using heterozygote and homozygote PF4 knock-out mice (mPF4(+/-) and mPF4(-/-), respectively) and transgenic mice overexpressing human PF4 (hPF4(+)). None of these lines had an overt bleeding diathesis, but in a FeCl(3) carotid artery thrombosis model, all showed impaired thrombus formation. This defect in thrombus formation in the mPF4(-/-) animals was corrected by infusing hPF4 over a narrow concentration range. The thrombotic defect in the mPF4(+/-) and mPF4(-/-) animals was particularly sensitive to infusions of the negatively charged anticoagulant heparin. However, the same amount of heparin paradoxically normalized thrombus formation in the hPF4(+) animals, although these animals were anticoagulated systemically. Upon infusion of the positively charged protein, protamine sulfate, the reverse was observed with mPF4(+/-) and mPF4(-/-) animals having improved thrombosis, with the hPF4(+) animals having worsened thrombus formation. These studies support an important role for PF4 in thrombosis, and show that neutralization of PF4 is an important component of heparin's anticoagulant effect. The mechanisms underlying these observations of PF4 biology and their clinical implications remain to be determined.

Animals↗
PubMed2004

Interactions of platelet factor 4 with the vessel wall.

Platelet factor 4 (PF4) is a platelet-specific protein that is stored in platelet alpha granules and released following platelet activation. PF4 was the first chemokine that was isolated, but unlike other chemokines, it may not have a clear role in inflammation. Gathering evidence suggests that unlike other chemokines that bind to specific receptors, PF4's biology depends on its unusually high affinity for heparan sulfates and other negatively charged molecules at concentrations attained in the immediate vicinity of activated platelets. There has been one report that PF4 binds to CXCR3B, a chemokine receptor isoform that may be present in some vascular beds, but the biological relevance of this single observation is not clear. We propose that the main biological role of PF4 and the basis for its presence in the alpha granules of all known mammalian platelets is to neutralize surface heparan sulfate side-chains of glycosaminoglycans and to optimize thrombus development at sites of vascular injury. In addition, the binding of PF4 to surface glycosaminoglycans may also underlie its angiostatic and proatherogenic properties. Additionally, PF4 binds to several other proteins that are central to thrombosis, angiogenesis, and atherogenesis. These interactions may also contribute to its biological and pathobiological effects. Certainly, future studies using in vivo models to test biological relevance of each of these proposed mechanisms by which PF4 interacts with the vasculature are needed, as are studies to define the importance of PF4 binding to CXCR3B.

Animals↗
PubMed2003

Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment.

Activated platelets release their granule content in a concentrated fashion at sites of injury. We examined whether ectopically expressed factor VIII in developing megakaryocytes would be stored in alpha-granules and whether its release from circulating platelets would effectively ameliorate bleeding in a factor VIIInull mice model. Using the proximal glycoprotein 1b alpha promoter to drive expression of a human factor VIII cDNA construct, transgenic lines were established. One line had detectable human factor VIII that colocalizes with von Willebrand factor in platelets. These animals had platelet factor VIII levels equivalent to 3% to 9% plasma levels, although there was no concurrent plasma human factor VIII detectable. When crossed onto a factor VIIInull background, whole blood clotting time was partially corrected, equivalent to a 3% correction level. In a cuticular bleeding time study, these animals also had only a partial correction, but in an FeCl3 carotid artery, thrombosis assay correction was equivalent to a 50% to 100% level. These studies show that factor VIII can be expressed and stored in platelet alpha-granules. Our studies also suggest that platelet-released factor VIII is at least as potent as an equivalent plasma level and perhaps even more potent in an arterial thrombosis model.

Animals↗
PubMed2003

Antithrombotic thrombocytes: ectopic expression of urokinase-type plasminogen activator in platelets.

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Animals↗
PubMed2003

Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV.

OBJECTIVE: Under some circumstances the HIV virus may infect cells that do not express receptors essential to HIV-entry. We hypothesized that platelet- and megakaryocyte-derived microparticles (MP) could play a role in such infections. MP are circular membrane fragments shed from the surface of eukaryotic cells. After adhesion to target cells, MP may transfer membrane-associated proteins to these cells. We found that peripheral blood platelet- (PMP) and megakaryocyte-derived MP (MegaMP) that highly express CXCR4 may transfer this receptor from the surface of platelets or megakaryocytes to the surface of CXCR4-null cells. DESIGN: Since this mechanism could potentially allow CD4+/CXCR4-null cells to become infected by T-tropic HIV, we incubated several human CD4+/CXCR4-null cells such as normal erythroblasts, glioblastomas U87, MAGI and hematopoietic cell lines UT-7, HEL and TF-1 with PMP or MegaMP. We found that these cells became CXCR4+. We next exposed these cells to X4-HIV (IIIB) and evaluated their susceptibility to infection by PCR, ELISA, and morphological analysis. RESULTS: We observed in all instances that after CD4+/CXCR4-null cell lines 'acquired' CXCR4 from PMP or MegaMP, they could became infected by X4 HIV. CONCLUSIONS: We postulate that both PMP and MegaMP may play a novel and important role in spreading HIV-1 infection by transferring the CXCR4 co-receptor to CD4+/CXCR4-null cells.

Blood Platelets↗
PubMed2003

Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters.

Emerging evidence supports a role for platelets in the progression of atherosclerosis in addition to an involvement in thrombotic vascular occlusion. Platelet Factor 4 (PF4), a chemokine released by activated platelets, stimulates several pro-atherogenic processes. Therefore, we examined the localization of PF4 and the homologous protein, Neutrophil Activating Protein-2 (NAP-2) in lesions representing the evolution of human atherosclerotic plaques. Carotid plaques from 132 patients with critical carotid stenosis and 6 autopsy specimens were studied. Clinical, histologic and immunohistochemical data were analyzed using a chi(2)-test. PF4 was detected in the cytoplasm of luminal and neovascular endothelium, in macrophages and in regions of plaque calcification. The presence of PF4 in macrophages and neovascular endothelium correlated with lesion grade (p = 0.004; p = 0.044). Staining of macrophages for PF4 correlated with the presence of symptomatic atherosclerotic disease (p = 0.028). In early lesions, PF4 was commonly found in macrophages of early lesions (Grade I/II), whereas NAP-2 was rarely present. In conclusion, correlation between PF4 deposition, lesion severity and symptomatic atherosclerosis suggests that persistent platelet activation may contribute to the evolution of atherosclerotic vascular lesions. These studies support the rationale for the chronic use of anti-platelet therapy in patients at risk for developing symptomatic atherosclerosis.

Adult↗
PubMed2003

Hematology grants workshop.

Uppermost among the many concerns of young researchers is acquiring funding for beginning a career as a clinician-scientist. This chapter is targeted specifically at those individuals considering an academic physician-scientist career and those on the verge of becoming independent researchers. In Section I, Drs. Poncz and Iannone discuss the Mentored Career Development Award (K08). They summarize the application process, highlighting the critical components of a successful application and what the review process entails. In Section II, Dr. Werner discusses what applicants need to know about the NIH Institutes' program, review, and grants management function; the different NIH staff whom applicants should contact during the various stages of the grants process; and the important sections and key phrases in NIH Program Announcements for career development awards.

Consensus Statements as Topic↗
PubMed2002

Platelet factor 4 enhances the binding of oxidized low-density lipoprotein to vascular wall cells.

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an approximately 10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.

Amino Acid Substitution↗