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Autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder in which autoantibodies are directed against an individual's own platelets, leading to enhanced clearance through Fc receptor (R)-mediated phagocytosis by macrophages residing in the reticuloendothelial system (RES), particularly in the spleen. The production of IgG autoantibodies is critically dependent on cellular immune mechanisms particularly relating to T cells. We review the recent literature of the cell-mediated immunology of AITP focusing on platelet phenotype, genetics, T-cell reactivities, and cytokine profiles in patients with AITP. Understanding the interaction between these cell-mediated mechanisms is vital for developing antigen specific immunotherapies to treat this autoimmune disease.

Clinical studies have convincingly demonstrated that WBC reduction of blood components can reduce the incidence of primary HLA antigen alloimmunization at least in patients with acute myeloid leukemia.1 However, despite receiving WBC‐reduced platelets, approximately 19 percent of acute myeloid leukemia patients still became alloimmunized.1 In addition, it is not known whether WBC reduction will prevent HLA antigen alloimmunization among those recipients who have an intact immune system (e.g., general surgery patients). The biologic mechanisms responsible for immune stimulation by blood components are still incompletely understood but are probably related to both donor and recipient factors. Animal models have proven indispensable for elucidating the basic mechanisms of immunity against foreign antigens and several such models have been developed to study various aspects of transfusion immunobiology.2-4 Our laboratory has developed a murine transfusion model that has allowed us to focus on recipient factors affecting the immune response to WBC‐reduced platelets. This article will describe how the murine immune response to platelet transfusion is critically dependent on recipient antigen‐processing and‐presentation pathways and how these pathways can be exploited to modulate immunization against WBC‐reduced platelets. (Less)

The role of plasma fibronectin (pFn) in thrombosis/hemostasis is controversial. We previously demonstrated that pFn supports thrombosis in pFn-/- mice. Interestingly, depletion of pFn in fibrinogen (Fg)/VWF-/- mice resulted in enhanced thrombus formation, revealing a functional switch of pFn in the absence of Fg/VWF. However, the mechanism controlling this switch is unknown. Furthermore, the hemostatic function of pFn is unclear. To address these questions, we established Fg/pFn-/- and VWF/pFn-/- mice in addition to triple deficient mice (Fg/VWF/pFn-/-, TKO). TKO mice had significantly higher mortality (23.1% vs. 9.1%) and longer tail bleeding time than their pFn+ Fg/VWF-/- littermates. Autopsy revealed severe subcutaneous or abdominal bleeding. pFn depletion in Fg-/- mice also increased mortality from 5.5% to 25.6% and prolonged bleeding times; the latter effect was reversed with pFn infusion. Using intravital microscopy, we found that pFn deposition at the site of injury preceded significant platelet accumulation, suggesting that pFn is an efficient and rapid hemostatic factor. Based on thromboelastography, fibrin clots from pFn+ mice were significantly stronger than those from pFn-/- mice. Consistent with this, inferior vena cava occlusion was significantly delayed in pFn-/- mice. Under confocal microscopy, fluorescently-labeled pFn was actively recruited into the fibrin network in mouse and human plasma. Under electron microscopy, physiological levels of pFn doubled the diameter of fibrin fibers, suggesting that pFn contributes to lateral aggregation of fibrin protofibrils. Interestingly, pFn enhances platelet aggregation when linked with fibrin and inhibits aggregation when fibrin is absent. pFn therefore gradually switches from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data established pFn as an important supportive factor in hemostasis and a unique self-limiting regulator of thrombosis, suggesting the therapeutic potential of pFn transfusion in controlling bleeding complications, particularly for those receiving antithrombotic therapy for heart attack and stroke.

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI). Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential. Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation in vitro and ex vivo and inhibited murine thrombus formation in vivo without significantly changing bleeding time or platelet count. In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet β3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Summary. 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 0 )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.

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.

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.

Recipient IgG immunity against leukoreduced donor platelets is dependent on indirect T-cell allorecognition and is suppressed in vivo by inhibitors (aminoguanidine, AMG) of inducible nitric oxide synthase (iNOS). To examine recipient processing pathways of donor platelet antigens, enriched macrophages (antigen-presenting cells [APC]) from BALB/c (H-2d) mice were pulsed with allogeneic C57BL/6 (H-2b) platelets and transfused weekly into naive BALB/c mice. Platelet-pulsed APC stimulated IgG antidonor antibody production in 45% of recipients by the second transfusion and in 100% by the sixth transfusion; this response was enhanced by pulsing in the presence of interferon-γ. By the sixth transfusion, high-titer IgG1 (mean titer 4990) and IgG2a (1933) isotypes specific for donor major histocompatibility complex (MHC) class I antigens were detected. Platelet pulsing in the presence of AMG or colchicine significantly inhibited the ability of APC to stimulate IgG alloantibodies; only 50% (P

Idiopathic thrombocytopenic purpura (ITP) is characterized by the development of a specific anti-platelet autoantibody immune response mediating the development of thrombocytopenia. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autoantibodies. In 15-20% of SLE cases, patients develop thrombocytopenia which appears to be autoimmune in nature (SLE-TP). To better understand the pathogenesis of the thrombocytopenia associated with SLE, we investigated the overlapping platelet and cellular immune features between SLE and ITP. Thirty-one patients with SLE, eight with SLE-TP, and 17 with ITP, were studied and compared to 60 healthy controls. We evaluated platelet-associated IgG, platelet microparticles, reticulated platelets, platelet HLA-DR expression, in vivo cytokine levels, lymphocyte proliferation, and the T lymphocyte anti-platelet immune response in these patients. Patients with SLE-TP and those with ITP had increased platelet-associated IgG, an increased percentage of platelet microparticles, a higher percentage of reticulated platelets and larger platelets, suggesting antibody-mediated platelet destruction and increased platelet production. More than 50% of patients with ITP had increased HLA-DR on their platelet surface whereas subjects with SLE-TP did not. Analysis of serum cytokines demonstrated increased levels of IL-10, IL-15 and TNF-alpha in patients with SLE, but in those with ITP, only increased levels of IL-15 were seen, no increases in any of these cytokines were observed in patients with in SLE-TP. The ability of lymphocytes to proliferate in response to phorbol myristate acetate (PMA) stimulation was increased in SLE-TP, but was normal in both SLE and ITP. Lymphocytes from subjects with ITP displayed an increased ability to proliferate on exposure to platelets, in contrast, those with SLE-TP did not. While the number of subjects evaluated with SLE-TP was small, these data reveal a number of differences in the immunopathogenesis between SLE-TP and ITP.

Anton Chekhov was not the only classic author to hold Russian theatres captive for much of the 1990s. At the beginning of 1999, a careful, though inexact, count of Moscow shows based on works by Chekhov, Alexander Ostrovsky, Nikolay Gogol, and Fedor Dostoevsky revealed some interesting numbers. The leader, Ostrovsky, who wrote or co-wrote over fifty plays, had thirty-five productions of his plays running at various houses thJoughout the capital. Right behind him, with thirty productions of plays or dramatized stories, was Chekhov. (If we include the ten one-act plays and count separately the early works that later evolved into others, Chekhov can generously be considered the author of eighteen dramatic works.) Gogol's plays and prose works formed the basis of fifteen productions, and Dostoevsky's novels and stories had been adapted for twelve stage productions.

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.

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 (PLA1) 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.

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-2d) SCID or BALB/c (H-2d) mouse platelets were transfused weekly into fully allogeneic CBA (H-2k) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 ± 0.6/μL and SCID mouse platelets could be prepared to have significantly lower (

Platelets express Toll-like receptor 4 (TLR4), and this has been shown to be responsible for the thrombocytopenia induced by lipopolysaccharide (LPS) administration in vivo. We studied the role of LPS in mediating platelet phagocytosis by THP-1 cells in vitro by flow cytometry. Opsonization of platelets with an IgG monoclonal (W6/32) antibody or with IgG autoantibody-positive sera from patients with autoimmune thrombocytopenia (AITP) significantly enhanced platelet phagocytosis (P < .001). In contrast, platelet phagocytosis did not occur if platelets were bound with only LPS. If, however, the LPS-bound platelets were also opsonized with either W6/32 or autoantibody-positive sera with titers greater than 4, there was a significant and synergistic increase in Fc-dependent platelet phagocytosis (P < .001, P = .003, P = .048, and P = .047). These results suggest that, in the presence of antiplatelet antibodies, bacterial products can significantly alter platelet phagocytosis, and this may have relevance to how Gram-negative infections enhance platelet destruction in some patients with AITP.

Sera from HIV-1-infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate, HIV-p24 and HIV-gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid-sensitive and alkaline-sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV-lysate columns; 96% (25/26) of the acid-eluted antibodies were HIV-specific but 48% (12/25) of the alkaline-eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline-eluted HIV-p24 antibodies, 40% (8/20) reacted specifically with HIV-p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline-eluted HIV-gpl20 antibodies (n=17), 88% (15/17) were HIV gpl20-specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti-p24 antibodies recognized three bands with approximate molecular weights of 72 000 to 95 000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti-HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline-sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV-infected haemophiliacs.

Purpura, initially recognized in ancient times, was defined into clinical syndromes in the 16th, 17th and 18th centuries. With advances in microscope science in the nineteenth century, the platelet was identified, leading to the recognition of the thrombocytopenic component of idiopathic thrombocytopenic purpura (ITP). The 20th century brought recognition of the pathophysiology of the disorder and the clinical states were refined and treatments for ITP developed. The latter half of the 20th century has focused on the autoimmune components of ITP, attempting to develop diagnostic tests, apply new therapies, and elucidate the immune dysregulation associated with, and underlying, the disorder.

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.

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.

The PFA-100® system provides an in-vitro method of assessing primary platelet-related haemostasis by measuring the time (the closure time, or CT) taken for a platelet plug to occlude a microscopic aperture cut into a membrane coated with collagen and either epinephrine or ADP. We used the system to establish normal ranges for CTs in healthy children, adults and neonates. Mean CTs of healthy children were independent of the needle gauge used (21G or 23G) for blood sampling; they were very similar to the mean CTs of healthy adults, but longer than mean CTs of healthy neonates. Although children with haemophilia had normal CTs, the PFA-100 system was found to be potentially useful in screening for von Willebrand disease in children.

The past is prologue. Reviewing the history of transfusion tells us how far we have come, but also where we need to go. The past has been filled with innovation and important discoveries, but is also fraught with stumbling blocks and unintended side effects. Although much has been achieved and transfusion is safer today than ever, nonetheless we are recognizing new potential concerns with transfusion and we are undergoing a paradigm shift in our attitudes, approach and patient management in regard to blood transfusion.

Blood conservation, or Patient Blood Management (PBM), is a paradigm shift in transfusion practice. Recognizing the potential adverse effects associated with blood transfusion, PBM emphasises the use of alternatives to transfusion in order to minimize unnecessary or inappropriate blood transfusion. The ONTraC program is a network of transfusion coordinators in 25 Ontario hospitals with the focus of implementing PBM. The program has been highly successful in reducing transfusion rates and improving clinical outcomes, and has proven very cost-effective. This paper summarizes results of the program from its inception in 2002-2011.

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. (Less)

Objective: To examine the effectiveness of cyclic oral high-dose (HD) dexamethasone therapy in pediatric patients with chronic immune thrombocytopenic purpura (ITP), which has been reported to cause complete remission in adults with chronic ITP. Study design: Eleven children with primary chronic ITP, with a median disease duration of 28 months (range, 6 to 120 months), were treated with cycles of HD dexamethasone therapy. Results: Excellent short-term responses (initial platelet counts ≤50 × 10 9 /L, increasing to >100 × 10 9 /L within 72 hours of completion of an HD dexamethasone cycle) were observed in 78% of 41 cycles. Long-term effects include one complete response (platelet count ≥150 × 10 9 /L) and three partial responses (platelet count ≥50 and Conclusions: Dexamethasone, given orally in high doses, is an effective drug in achieving short-term platelet responses, but it induced long-term remissions in fewer than half of the children with well-established chronic ITP. Its effect on B lymphocytes requires further elucidation. A prospective, controlled study will be needed to establish whether cyclic HD dexamethasone therapy can alter the natural history of children with early chronic ITP and thus avoid splenectomy. (J Pediatr 1997;130:17-24)

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with >/=0.5 nM alpha-thrombin for 10-60 s at 37 degrees C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 10(11) platelets/liter) after their incubation with 10 nM alpha-thrombin for 10 s. Parallel binding of alpha-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated alpha-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by

Platelet transfusions are life-saving treatments for many patients with thrombocytopenia; however, their use is generally discouraged in the autoimmune disorder known as immune thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility complex (MHC) class I transfusions affected antiplatelet CD61-induced ITP. BALB/c CD61 knockout mice (CD61(-)/H-2(d)) were immunized against platelets from wild-type syngeneic BALB/c (CD61(+)/H-2(d)), allogeneic C57BL/6 (CD61(+)/H-2(b)), or C57BL/6 CD61 KO (CD61(-)/H-2(b)) mice, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP. When nondepleted splenocytes were transferred to induce antibody-mediated ITP, both CD61(+) platelet immunizations generated immunity that caused thrombocytopenia independently of allogeneic MHC molecules. In contrast, when B-cell-depleted splenocytes were transferred to induce T-cell-mediated ITP, transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP development. In addition, allogeneic platelet transfusions into SCID mice with established CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice, bone marrow histology in the rescued mice showed normalized megakaryocyte morphology, and in vitro CD61-specific T-cell cytotoxicity was significantly suppressed. These results indicate that antibody-mediated ITP is resistant to allogeneic platelet transfusions, while the T-cell-mediated form of the disease is susceptible, suggesting that transfusion therapy may be beneficial in antibody-negative ITP.

Background Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation. Objectives To elucidate this relationship in human platelets. Materials and Methods Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis. Results We found that at optimal treatment conditions (90 min, 37 °C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P Conclusions These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis.

SummaryFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies against fetal/neonatal platelets. FNAIT is also linked with miscarriages, although the incidence and mechanisms of fetal death have not been well studied. Integrin αIIbβ3 (GPIIbIIIa) and the GPIbα complex are major glycoproteins expressed on platelets and are also major antigens targeted in autoimmune thrombocytopenia (ITP), but reported cases of anti-GPIb-mediated FNAIT are rare. Bacterial and viral infections have been causally linked with the pathogenesis of immune-mediated thrombocytopenia (ITP); however, it is unknown whether these infections contribute to the severity of FNAIT. Here, immune responses against platelet antigens were examined by transfusing wild-type (WT) mouse platelets into β3-/- or GPIbα-/- mice. To mimic bacterial or viral infections, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) were injected intraperitoneally following platelet transfusions. The FNAIT model was established by breeding the immunised female mice with WT male mice. We demonstrated for the first time that the platelet GPIbα has lower immunogenicity compared to β3 integrin. Interestingly, co-stimulation with LPS or Poly I:C markedly enhanced the immune response against platelet GPIbα and caused severe pathology of FNAIT (i.e. miscarriages). LPS or Poly I:C also enhanced the immune response against platelet β3 integrin. Our data suggest that bacterial and viral infections facilitate the anti-platelet GPIbα response, which may lead to a severe non-classical FNAIT (i.e. miscarriage but not neonatal bleeding) that has not been adequately reported in humans.

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.

Thrombin accelerates its own production by activating platelets (and perhaps other cells) to provide coagulant surfaces on which prothrombinase, the enzyme complex that activates prothrombin in plasma and consists of equimolar factor Xa (serine protease) and factor Va (cofactor), assembles in a Ca(2+)-dependent reaction. Thrombin also activates factor V to provide the cofactor for factor Xa in prothrombinase, and factor VIII to provide the cofactor for factor X activation by factor IXa. Even when factor X activation is initiated by tissue factor, efficient propagation of factor X activation is critically dependent on intrinsic tenase (the factor IXa and factor VIIIa enzyme complex). These procoagulant actions of thrombin explain why good antithrombins can inhibit both intrinsic and extrinsic prothrombin activation. Because of the low level of thrombin (1 nM) required to initiate platelet activation and the activation of platelet-bound factor V, only highly efficient antithrombins, such as hirudin, can abrogate platelet-dependent prothrombinase assembly.

Flow cytometric analysis of blood leukocytes is currently used for both routine clinical measurements as well as for cutting edge research applications. This technology has enabled rapid and accurate determination of leukocyte antigens and quantitative analysis of leukocyte subsets, tests of leukocyte function, determination of the presence of antineutrophil and antilymphocyte antibodies in plasma and on cells, measurement of CD34+ hematopoietic stem cells in peripheral blood and bone marrow samples, measurement of apoptosis, and detection of virusinfected leukocytes. This review will focus on the use of the flow cytometer for investigations of blood leukocytes in transfusion medicine.

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.

Recent studies suggest that patients undergoing warm heart surgical procedures have reduced postoperative bleeding. To determine if this is due to differences in platelet activation, we measured platelet membrane glycoproteins (GPIb, GPIIb/IIIa, GMP 140), platelet fragments, and platelet counts before, during, and after normothermic (37 degrees C) or hypothermic (28 degrees to 30 degrees C) cardiopulmonary bypass. Cardiopulmonary bypass was associated with a significant decrease in platelet count, platelet membrane GPIb, and platelet fragments, and an increase in GMP 140 (p0.05). Normothermic cardiopulmonary bypass induced an early significant increase in granulocytes, whereas this was delayed until after rewarming in the hypothermic group. Mean 24-hour postoperative blood loss was 786 +/- 226 mL in the cold group versus 547 +/- 56 mL in the warm group (p = not significant). We conclude that cardiopulmonary bypass affects platelet activation and integrity and that these changes are similar in direction and magnitude for hypothermic and normothermic techniques.

Platelet apoptosis and activation have been studied in human platelets treated with BH3-only mimetic ABT-737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis-associated caspases, initiator caspase-9 of intrinsic apoptosis pathway, executioner caspase-3 and initiator caspase-8 of extrinsic death receptor pathway, and platelet activation was detected by P-selectin (CD62) exposure on the platelet surface. We found that ABT-737 predominantly induced activation of caspases-9, -3 and -8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase-8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase-8 activation is triggered in ABT-737- and A23187-treated anucleate platelets through the mitochondria-initiated caspase activation cascade bypassing the death receptors, and (ii) ABT-737-treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival.

Immune thrombocytopenia (ITP) is a bleeding disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. Several reports have observed that ITP is associated with a peripheral deficiency of tolerance-inducing CD4+CD25+FoxP3+ T regulatory cells (Tregs). Using a murine model of ITP, we analyzed Tregs in the spleen and thymus. CD61 knockout mice were immunized against wild-type (CD61+) platelets, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice. Compared with SCID mice receiving naive splenocytes, within 2 weeks after transfer, the ITP SCID mice became thrombocytopenic (< 200 × 109 platelets/L) and had increased serum anti-CD61 antibodies. The quantity of thymic Tregs by 2 weeks after transfer was significantly elevated, whereas Tregs in the spleens were significantly reduced. Treatment of the ITP mice with 2 g/kg intravenous immunoglobulin raised the platelet counts, reduced antibody production, and normalized the thymic and splenic Treg populations. Compared with thymocytes from ITP mice treated with intravenous immunoglobulin, thymocytes from untreated ITP mice delayed the onset of ITP when administered before engraftment with immune splenocytes. These results suggest that ITP in mice is associated with a peripheral Treg deficiency because of thymic retention and therapy normalizes the Tregs.

Anemia in both acute and chronic conditions is associated with an increased risk of organ injury (brain, heart, kidney) and mortality. Thus, anemia is not "safe". Impairment of tissue oxygen delivery likely contributes as a central mechanism; however, the existing treatments for anemia (i.e., transfusion, erythropoiesis stimulating agents, blood substitutes) have not produced a demonstrable improvement in patient outcomes despite their efficacy to increase blood oxygen content. Indeed, transfusion of red blood cells (RBCs) has been attributed to increase mortality in non-bleeding patients. Thus, the pathophysiology of anemia-induced morbidity and mortality and its treatments are complex and incompletely understood. New knowledge continues to emerge regarding the cellular mechanisms that maintain oxygen homeostasis during anemia. Nevertheless, the application of this knowledge has not yet led to improvements in patient outcomes. As both anemia and transfusion are associated with increased mortality, utilization of multimodal patient blood management strategies may be effective in avoiding both of these predictors of adverse outcomes. We propose to review new strategies to avoid both anemia and transfusion with the goal of improving patient outcomes and safety.We reviewed several approaches that utilize patient blood management to improve patient outcomes, including 1) characterization of biomarkers of anemia-induced tissue hypoxia to identify appropriate patient-specific treatment thresholds or hemoglobin (Hb) triggers; 2) development of adequately powered clinical trials that will help to define appropriate guidelines for the perioperative treatment of anemia and optimal Hb thresholds for transfusion of RBCs in specific patient populations; and 3) demonstration that an established blood conservation program (ONTraC) can reduce RBC transfusion and its associated adverse outcomes.Anemia is associated with increased morbidity and mortality. Ongoing initiatives to treat anemia and optimize patient blood management may improve patient outcomes. A broader application of these approaches may improve the overall safety of anesthesia and surgery for patients with anemia.

Approximately five decades ago, alloimmunization to human leukocyte antigens (HLA) and platelet refractoriness were recognized as potentially serious complications of platelet transfusions. The mechanisms that result in stimulating immunity against blood products are still incompletely understood but are related to both the composition of the donor product transfused and the immune status of the recipient. Based on murine studies of platelet immunity, platelets are inherently immunogenic and there are at least two independent levels of immunoregulation against platelet transfusions. The first level resides within the recipient and is related to antigen processing/presentation events and CD8+ T cell-mediated immunosuppression. The second level relates to the donor product and includes donor antigen presenting cells (APC) levels as well as age-induced changes in donor APC and/or platelets. Implementation of pre-storage leukoreduction of cellular blood components led to a marked reduction in platelet alloimmunization and its dreaded complication, platelet refractoriness. Platelet refractoriness is usually managed by transfusion of matched platelets, selected according to one of the many published methods. It is unclear which of these methods is superior, and given the difficulty of obtaining a perfectly matched product, perhaps the most logical approach is to use a combination of selection strategies. This review discusses the various aspects of platelet alloimmunization and the clinical consequences that may result. It highlights how animal studies have shed light on the immune mechanisms responsible for allogeneic platelet immunity and immunomodulation and reviews relevant literature on clinical and laboratory manifestations of immune platelet refractoriness.

Apoptosis, or programmed cell death, is a physiological mechanism that serves for controlled deletion of damaged cells. While long attributed exclusively to nucleated cells, over recent years it has been recognized that apoptosis also occurs in anucleate platelets. We describe here experiences of determining markers of apoptosis in human platelets treated in vitro with pro-apoptotic chemical and physical stimuli. These include depolarization of mitochondrial inner membrane, cytochrome c release, expression of pro-apoptotic and anti-apoptotic proteins of Bcl-2 family, activation of apoptosis executioner caspase-3, exposure of phosphatidylserine, platelet shrinkage, fragmentation to microparticles, blebbing and filopod extrusion on the platelet surface. These assays serve to characterize platelet apoptosis in different cellular compartments (mitochondria, cytosol and plasma membrane) and at the whole-cell level. Methods commonly employed in studies of platelet apoptosis markers include flow cytometry, Western blot analysis and electron microscopy. An integrated methodological approach, based on determination of different platelet apoptosis markers, represents a useful tool for examining platelet apoptosis in various physiological and pathological settings.

The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.

Intravenous anti-D is often used in the treatment of autoimmune thrombocytopenic purpura (AITP), but little is known about its mechanisms of action. To investigate anti-D's potential in vivo mechanism(s) of action, a small group (N = 7) of children with chronic AITP was studied. The children initially received either 25 or 50 microg/kg of WinRho-SD in a four-cycle cross-over trial, and peripheral blood samples from the first and third cycles were assessed for cytokine levels at pre-treatment, 3 hr, 1 day, and 8 days post-treatment. Results showed that platelet counts significantly increased in all the children by day 8 post-treatment. Analysis of serum by ELISA showed that there was a significant but transient rise in both pro- and anti-inflammatory cytokine/chemokine levels (e.g., IL1RA, IL6, GM-CSF, MCP-1 alpha, TNF-alpha and MCP-1) by 3 hr post-treatment in both cycles which returned to baseline levels by 8 days post-treatment. These results suggest that anti-D administration may initially activate the RES in the form of cytokine/chemokine secretion, which is subsequently followed by an increase in platelet counts. It is possible that the induced cytokine/chemokine storm may have an effect on several physiological processes such as those mediating either adverse effects or potentially RES phagocytic activity.

Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunologically savvy than we once thought.

Treatment with human platelet antigen (HPA)-matched platelets (PLTs) is the optimal therapy for bleeding secondary to neonatal alloimmune thrombocytopenia. Recent advances in high-throughput DNA-based blood group and PLT antigen genotyping have made it possible to screen plateletpheresis donors for potential HPA-matched PLT transfusion.This prospective study evaluated genomic DNA from plateletpheresis donors for single-nucleotide polymorphisms (SNPs) associated with HPA-1, -2, -3, -4, -5, and -15 to determine whether high-throughput multiplex genomic DNA PCR and oligonucleotide extension technology can be used for mass-scale PLT antigen genotyping. Genotyping using SNP technology was confirmed using sequence-specific polymerase chain reaction (SSP-PCR).Of the 748 donors screened, 277 were found to be negative for antigens implicated in alloimmune thrombocytopenia. In addition, two donors were homozygous for HPA-1b/b and -2b/b, six donors for HPA-1b/b and -3b/b, one for HPA-2b/b and -3b/b, one for HPA-1b/b and -5b/b, 10 for HPA-1b/b and -15 b/b, four for HPA-5b/b and -15b/b, and one for HPA-2b/b and -15b/b. Retesting using SSP-PCR was conducted for 60 donors. Discrepant results occurred between SNP and SSP-PCR in less than 20% of samples for HPA-1b/1b/HPA-3b/3b, HPA-5b/5b, and HPA-15b/b.High-throughput multiplex PCR SNP and confirmatory molecular genotyping are useful for mass-scale screening of apheresis PLT donors to provide antigen-negative genotypes. Refinements to mass-scale multiplex analysis technology would reduce further the confirmatory testing needed.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder caused by maternal antibody–mediated destruction of fetal/neonatal platelets. It is the most common cause of severe thrombocytopenia in neonates, but the frequency of FNIT-related miscarriage is unknown, and the mechanism(s) underlying fetal mortality have not been explored. Furthermore, although platelet αIIbβ3 integrin and GPIbα are the major antibody targets in immune thrombocytopenia, the reported incidence of anti-GPIbα–mediated FNIT is rare. Here, we developed mouse models of FNIT mediated by antibodies specific for GPIbα and β3 integrin and compared their pathogenesis. We found, unexpectedly, that miscarriage occurred in the majority of pregnancies in our model of anti-GPIbα–mediated FNIT, which was far more frequent than in anti-β3–mediated FNIT. Dams with anti-GPIbα antibodies exhibited extensive fibrin deposition and apoptosis/necrosis in their placentas, which severely impaired placental function. Furthermore, anti-GPIbα (but not anti-β3) antiserum activated platelets and enhanced fibrin formation in vitro and thrombus formation in vivo. Importantly, treatment with either intravenous IgG or a monoclonal antibody specific for the neonatal Fc receptor efficiently prevented anti-GPIbα–mediated FNIT. Thus, the maternal immune response to fetal GPIbα causes what we believe to be a previously unidentified, nonclassical FNIT (i.e., spontaneous miscarriage but not neonatal bleeding) in mice. These results suggest that a similar pathology may have masked the severity and frequency of human anti-GPIbα–mediated FNIT, but also point to possible therapeutic interventions.

Rapid purification of human lymphocyte subpopulations is an essential step in order to elucidate their interactions and/or contributions in various disease states. Cell purification using a Magnetic Activated Cell Sorter (MACS) is a relatively new technology which has been shown to be rapid and yield highly purified populations of cells. This report describes both a simple one-step positive selection method using the MACS to purify either human CD4+ or CD19+ lymphocytes from PBMC and a sequential separation of both CD4+ and CD19+ cell populations. These methods can separate the cell populations in approximately 4 h with yields > 90% and purity of 97 +/- 3% for CD4+ T cells and 92 +/- 5% for CD19+ B cells. In functional studies, purified CD19+ B cells secreted 13- and 24-fold more IgM and IgG, respectively, than the CD19- cell fraction in 10 day B cell stimulation assays. Purification of the two cell types did not cause any significant activation as shown by proliferation. Both cell types, however, were able to proliferate upon stimulation with interleukin-2.

Plasma levels of lipoprotein(a) [Lp(a)] correlate directly with the risk of atherosclerosis. In four patients who under-went plasma exchange (PE), we measured plasma Lp(a) and report a significant acute reduction after two treatments. In two subjects who subsequently underwent 7 weeks of PE treatments, we report a tendency of plasma Lp(a) levels to return to baseline values. This possible escape of plasma Lp(a) from ongoing PE treatment, that removes it from plasma, has not previously been reported and suggests that plasma Lp(a) is under considerable pressure to be maintained at genetically determined levels.