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Biomedical subjects

A H Lazarus

Publications and source records attributed to A H Lazarus.

At least 19 recordsLinked to original sources
PubMed2003

von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function.

The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF-dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF-dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF-dependent platelet activation was compared to their ability to inhibit thrombin-dependent activation. The results demonstrate that VWF-dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin-sensitive phosphatidylinositide 3-kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin-dependent platelet activation. In addition, thrombin-dependent but not VWF-dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin- and VWF-dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin-dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF-dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF-dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.

Adenosine Diphosphate↗
PubMed2002

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C).

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.

Adolescent↗
PubMed2001

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab')2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti-idiotype interactions are not required.

Adsorption↗
PubMed2000

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets.

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine-threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.

Animals↗
PubMed2000

Monoclonal antibody-mediated inhibition of the human HLA alloimmune response to platelet transfusion is antigen specific and independent of Fcgamma receptor-mediated immune suppression.

Presensitization of donor platelets with allo-specific immunoglobulin (Ig)G results in a diminished immune response against subsequent transfusions of platelets. To understand better the mechanism of how alloantibody presensitization results in a decreased alloimmune response, we have used murine monoclonal antibodies directed to polymorphic and non-polymorphic regions of human leucocyte antigen (HLA) as well as platelet-specific molecules. Here, we demonstrated that presensitization with anti-human HLA class I antibodies, as well as beta2-microglobulin-specific antibody, protected against alloantibody production to five subsequent untreated platelet challenges. Use of complement fixing, non-fixing or F(ab')2 fragments of HLA-specific antibody also resulted in complete inhibition of alloantibody production. This protection was not seen when the platelets were presensitized with monoclonal antibodies to CD42a (GPIX), CD32 (low-affinity IgG/Fcgamma receptor) or murine IgG and was thus independent of B-cell FcgammaRII-mediated immune suppression. The inhibition observed was independent of HLA alloantigenic specificity as antibodies directed at the beta2-microglobulin portion of HLA class I were as effective as antibodies against any of the HLA-alpha regions (either polymorphic or non-polymorphic) of class I. This work demonstrates that monoclonal antibody-mediated suppression of the human HLA alloimmune response to platelet transfusion is antigen specific and is independent of FcgammaRII-mediated immune regulation, complement fixing or HLA alloantigenic specificity.

Animals↗
PubMed1999

Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity.

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2(d)) SCID or BALB/c (H-2(d)) mouse platelets were transfused weekly into fully allogeneic CBA (H-2(k)) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 +/- 0.6/microL and SCID mouse platelets could be prepared to have significantly lower (<0. 05/microL) MC numbers. Transfusions with 10(8) BALB/c platelets (containing approximately 100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 10(8) SCID mouse platelets (containing approximately 5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/microL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/microL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0. 05/microL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.

Animals↗
PubMed1999

Antibody-mediated inhibition of the human alloimmune response to platelet transfusion in Hu-PBL-SCID mice.

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA-mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu-PBL-SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti-HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet-specific anti-HPA-1a (PL(A1)) sera did not induce a decrease in the anti-HLA alloantibody response. This demonstrated that platelet-induced HLA alloimmunization can be blocked by anti-HLA antibody-sensitized cells.

Animals↗
PubMed1999

p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation.

We have investigated the role of p38 mitogen-activated protein kinase (MAPK) in von Willebrand factor (VWF)-dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF-dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post-addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S-transferase-MAPK activated protein kinase-2. To determine if p38 MAPK was necessary for porcine VWF-induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF-induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580-inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

Humans↗
PubMed1999

Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

Adenosine↗
PubMed1999

Inhibition of a secondary human alloimmune response via the soluble active component of CD154 (CD40L) in severe combined immune-deficient mice engrafted with human lymphocytes.

BACKGROUND: Alloimmunization requires a process known as co-stimulation. An important co-stimulatory pathway for most immune responses is mediated by the interaction of CD40 on antigen-presenting cells with CD154 (CD40L) on host T cells. Blockade of this co-stimulatory pathway simultaneous with exposure to challenge with HLA-incompatible cells is hypothesized to inhibit alloimmunization. STUDY DESIGN AND METHODS: Severe combined immune-deficient (SCID) mice were reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID mice) from a subject primed to HLA antigens and challenged with HLA-incompatible lymphocytes. Mice were challenged in the presence or absence of an 18-kDa soluble recombinant active form of human CD154 (18-kDa CD154). Human IgG production, alloimmunization, and in vitro T-cell responsiveness were assessed. RESULTS: There was no significant effect of 18-kDa CD154 on human IgG levels in these mice, but it inhibited the development of HLA-specific alloantibody in this model to five subsequent untreated white cell challenges. In vitro T-cell proliferation in a mixed lymphocyte culture was also prevented by 18-kDa CD154. CONCLUSION: The recombinant protein 18-kDa CD154 inhibited the ability of the Hu-PBL-SCID mice to mount a secondary immune response to allostimulation. This implies that transfusion-induced alloimmunization utilizes CD40-CD154 co-stimulation and that blockade of this pathway can inhibit T-cell function and interfere with the development of alloimmunization.

Animals↗
PubMed1998

Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor zeta chain and other intracellular proteins in lymphocyte subpopulations.

Expression of the T-cell receptor (TCR) zeta chain in normal individuals was studied by two-color flow cytometric analysis using digitonin-permeabilized human peripheral blood lymphocytes. Optimal detection of the TCR zeta chain involved fixation of cells in 0.25% paraformaldehyde for 2 min and permeabilization with 500 microg/ml of digitonin at 4 degrees C. Permeabilized lymphocytes and monocytes displayed decreased forward light scatter properties. Neutrophils did not survive this permeabilization/fixation/washing procedure. Permeabilization did not affect the ability of antibodies to CD3, CD4, CD8, CD16, and CD19 to detect CD antigens on lymphocytes, and the percentage of lymphocytes reacting with these antibodies was comparable between untreated and permeabilized cells. Staining of the TCR zeta chain was accomplished by incubating permeabilized cells with unconjugated antibody to the zeta chain, followed by an FITC-conjugated F(ab')2 goat anti-IgG. Following TCR zeta chain staining, cells were blocked with 25 microg/ml of mouse IgG for 20 min to saturate the goat anti-mouse antibody and then incubated with a phycoerythrin-conjugated mouse antibody to a variety of lymphocyte surface antigens. The TCR zeta chain was observed in lymphocytes displaying CD3, CD4, CD8, CD16, or TCRgammadelta markers. This permeabilization/fixation/washing procedure was also validated for detection of another intracellular T-cell protein known as the cytolytic granule-associated protein, recognized by the TIA-1 antibody. Thus the technique can be applied to detection of at least two different intracellular T-cell markers.

Antibodies, Monoclonal↗
PubMed1998

Platelet activation induced by porcine factor VIII (Hyate:C).

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.

Animals↗
PubMed1998

The cellular immunology associated with autoimmune thrombocytopenic purpura: an update.

Chronic autoimmune thrombocytopenic purpura (AITP) is an organ specific autoimmune bleeding disease in which autoantibodies are directed against the individual's own platelets, resulting in increased Fc-mediated platelet destruction by macrophages in the reticuloendothelial system. Although AITP is primarily mediated by IgG auto-antibodies, their production is regulated by the influence of T lymphocytes and antigen presenting cells (APC). This review argues that enhanced T helper cell/antigen presenting cell interactions in patients with AITP may be responsible for IgG anti-platelet auto-antibody production. Understanding these cellular immune responses in AITP may lead to the development of more immune specific therapies for the management of this disease.

Antigen-Presenting Cells↗
PubMed1998

Intravenous immunoglobulin and anti-D in idiopathic thrombocytopenic purpura (ITP): mechanisms of action.

Infusion of large amounts of intravenous immunoglobulin (IVIG) or anti-D can reverse the low platelet count in patients with ITP within hours of the initiation of treatment. In some cases, the effects of IVIG appear to far outlast several half-lives of the product. Several mechanisms have been proposed to explain these rapid and long term effects and these will be discussed in this review.

Antibodies, Anti-Idiotypic↗
PubMed1998

Analysis of transmembrane signalling and T cell defects associated with idiopathic thrombocytopenic purpura (ITP).

Adult chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by production of autoreactive antibodies to platelet antigens. It is now becoming clear that autoantibody production, in general, is regulated by T helper (Th) cells. Several recent studies have examined potential defects in T cell function in this disease and have demonstrated that patients with ITP possess abnormal lymphocyte activation and Th1/Th2-mediated cytokine production. Although the underlying cause(s) of aberrant T cell function in this disease are not known, studies from other models of autoimmune disease indicate that defects in T cell transmembrane signalling can be causally linked to abnormal T cell activation and cytokine production. This review will present some of the major T cell signalling pathways and discuss how altered T cell signalling may be linked to autoimmunity with an emphasis on ITP. Recent preliminary findings of a potential defect in the signal transduction apparatus in lymphocytes from three patients with ITP will also be presented.

Autoimmunity↗
PubMed1997

Flow cytometric analysis of platelets from children with the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure.

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

Actins↗
PubMed1996

Regulation of BCR- and PKC/Ca(2+)-mediated activation of the Raf1/MEK/MAPK pathway by protein-tyrosine kinase and -tyrosine phosphatase activities.

Ligation of the B cell Ag receptor (BCR) activates a protein-tyrosine kinase (PTK) and CD45 protein-tyrosine phosphatase (PTPase)-dependent signaling cascade that results in the activation of Ras. This pathway of Ras activation can operate independently of protein kinase C (PKC) activity. Activation of Ras may lead to two distinct Ras-dependent pathways involving either a Raf1/MEK/MAPK module or a MEKK/SEK/SAPK module; however, it is unclear as to how Ras controls the independent activation of either of these pathways. We have used genistein and phenylarsine oxide (PAO) as inhibitors of PTK and PTPase, respectively, to investigate whether they regulate the BCR- and Ca2+/PKC-dependent activation of the Ras/Raf1/MEK/MAPK module. Assays of phosphotransferase activities conducted with Ag (TNP6-OVA)-specific 7.9 murine B lymphoma cells demonstrated that BCR-mediated stimulation of the Raf1/MEK/MAPK module is controlled by PTK and PTPase activities. An elevation in [Ca2+]i was required to optimally activate Raf1 and MEK through the BCR. However, when signaling through the BCR was bypassed by direct stimulation of the Raf1/MEK/MAPK module via a rise in [Ca2+]i and phorbol ester-induced PKC activation, the phosphotransferase activities of Raf1, MEK and MAPK were still regulated in a PTK-dependent manner that was also partially sensitive to the PTPase inhibitor PAO. Thus, at least two alternate routes, i.e. a BCR/PTK/Ras-dependent route and another PKC/Ca(2+)-dependent route, may converge at the level of Raf1 for activation of the Raf1/MEK/MAPK module in B cells.

Animals↗