Serum Antiplatelet Antibody Research Articles

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

Platelet Antibodies in Infertility Patients.

Platelet antigens can stimulate the body to produce platelet alloimmune antibodies through blood transfusion, pregnancy, and autoimmunity. In the blood of pregnant women, anti-platelet antibodies can cause embryo implantation failure, abortion, etc. if they are present. The platelet antibody was screened in 326 infertile patients (282 primary infertility and 44 secondary infertility) and 522 healthy controls in the physical examination center of our hospital by solid phase agglutination of red blood cells. The positive rate of anti-platelet antibody was 9.51% in the infertility group and 2.30% in the healthy control group. There was a significant difference between them (χ2 = 4.51, p < 0.05). The positive rate of anti-platelet antibody in the infertility group was significantly higher than that in the control group. The positive rate of anti-platelet antibody in the secondary infertility patients was significantly higher than that in the primary infertility patients (χ2 = 1.62, p < 0.05), and the positive rate of serum anti-platelet antibody increased gradually with the increase of infertility years. The positive rate of anti-platelet antibody is closely related to infertility and gradually increases with the age of infertility.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Thrombopoietin Can Induce Tolerogenic Responses Via the Modulation of Thymic and Splenic T Lymphocyte Populations in a Murine Model of Immune Thrombocytopenia (ITP)

AbstractAbstract 1088Clinical trials using thrombopoietin (TPO) mimetics have clearly shown their benefit in raising platelet counts in patients with immune thrombocytopenia (ITP), however, little is known whether TPO may affect immunologic responses. It has been demonstrated that, like other therapies, TPO treatment can rescue the peripheral CD4+CD25+FoxP3+ T regulatory cell (Tregs) deficiency that is commonly observed in patients with ITP (Bao W et al Blood 116;4639, 2010). Of interest, an earlier murine study also suggested that TPO could affect the production of T cells, particularly CD4+ T cells (Zhao JZ et al Haematologica 83:572, 1998). Using a murine model of ITP that demonstrates both antibody and T cell mediated thrombocytopenia, we studied T cell responses in ITP mice treated with TPO. ITP was induced in severe combined immunodeficient (SCID) mice by administration of 2×104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 and the mice where either treated or not with weekly subcutaneous injections of 1ug of murine TPO. Mice were bled weekly to count platelets and obtain sera for anti-platelet antibody production and at 3 weeks after the splenocyte transfer, they were sacrificed and their spleens and thymi were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry to quantify CD4+CD25+FoxP3+ (Tregs) and CD8+CD25+FoxP3+ (Tc-regs). We observed that TPO treatment caused an expected significant increase in platelet counts, however, the treatment also caused a significant reduction in the titre of serum anti-platelet antibodies. This was associated with significant changes in the proportions of both CD4+ Tregs and CD8+ Tc-regs within the thymus and spleen. TPO treatment rescued the peripheral splenic Treg and Tc-reg deficiencies and the cells ability to functionally suppress the proliferation of conventional CD4+CD25- T cells. The data suggests that in addition to its megakaryocyte-stimulating properties, TPO treatment has additional tolerance-inducing effects associated with anti-platelet antibody reduction and CD4+ Treg and CD8+ Tc-reg stimulation. Disclosures:No relevant conflicts of interest to declare.

Platelet production and destruction in liver cirrhosis

Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Antiplatelet antibodies in WASP(−) mice correlate with evidence of increased in vivo platelet consumption

To study the role of antiplatelet antibodies in the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). A flow cytometric method was developed for detection of serum antiplatelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with 5-chloromethylfluorescein diacetate (CMFDA) in order to track their clearance from the circulation. WASP(-)muMT(-/-) mice were generated by standard breeding methods. Serum antiplatelet antibodies were detected in approximately 40% of WASP(-) males. The mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(-) males show an approximately 50% reduction in platelet counts, 5% to 10% show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies. In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily immunoglobulin G, and are also detected in >30% of CD47(-/-) males. WASP(-)muMT(-/-) males, which demonstrate no serum- or platelet-associated antibodies, show a degree of thrombocytopenia similar to that of WASP(-) males. Their platelet clearance rates remain accelerated--more so in WASP(-)muMT(-/-) than WASP(+)muMT(-/-) recipients. These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms: an antibody-independent mechanism that largely requires WASP deficiency in trans, and an antibody-dependent mechanism that does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody-dependent clearance of WASP(-) platelets.

Idiopathic Thrombocytopenic Purpura in a Patient with Carcinoma of the Uterine Cervix

We describe here the case a patient with advanced cervix carcinoma and who developed idiopathic thrombocytopenic purpura (ITP). A 63-year-old woman with stage IV squamous cell carcinoma of the uterine cervix and that was complicated by hydronephrosis was treated palliatively with 45Gy of external beam radiation to the pelvis. About 3 years later, she developed hematochezia and severe thrombocytopenia. The laboratory examinations showed no evidence of thrombotic thrombocytopenic purpura or disseminated intravascular coagulopathy, and she was positive for serum anti-platelet antibodies. On the bone marrow examination, there was a normal number and morphology of megakaryocytes with no evidence of malignant cell infiltration. We made the clinical diagnosis of ITP, and the intravenous immunoglobulin and steroid therapy was successful. This case suggests the possibility that ITP can occur in association with advanced cervix carcinoma.

Antiplatelet Antibodies in WASP(−) Mice Correlate with Accelerated in Vivo Platelet Consumption Rates.

The Wiskott-Aldrich Syndrome is a congenital X-linked immunodeficiency caused by mutations in the WASP gene. The most common clinical presentation is recurrent infection, eczema, and thrombocytopenia. Autoimmune disease occurs in as much as 70% of WAS patients. WASP functions to transmit and integrate signals that originate at the cell membrane and result in actin polymerization. The latter in turn augments signal transduction downstream of cell surface ligands such as the T-cell receptor and macrophage integrins. Dysfunction of these signal transduction pathways is thought to result in the immunodeficiency. The mechanism by which WASP deficiency results in thrombocytopenia, however, is not well understood. Although WASP(−) platelets are smaller than normal, no cytoskeletal defect has been consistently observed in them. While platelet aggregation abnormalities have been reported, it is not known whether they contribute significantly to the hemorrhagic complications seen in severely thrombocytopenic WAS patients. WASP(−) platelets are consumed more rapidly in vivo than are normal platelets, both in normal volunteers and in WAS patients. Platelet production rate may be reduced as well. Splenectomy improves platelet counts in WAS patients, but the subsequent incidence of ITP is high (23% in one study). WAS patients with low but detectable levels of WASP can show a fluctuating thrombocytopenia similar to the course of ITP. These findings suggest that autoimmunity could contribute to the thrombocytopenia of WAS. A murine model of WAS shows a milder thrombocytopenia (approximately 50% of normal) and normal platelet size. We have shown that WASP(−) murine platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. Their in vivo consumption rate is more affected by antibody opsonization than is that of WT platelets, a finding we have corroborated with ex vivo phagocytosis studies. A subset of WASP(−) mice show a more severe thrombocytopenia with an increased fraction of reticulated platelets (RP), suggesting the presence of antiplatelet antibodies. To test this, we have optimized a flow cytometric assay for serum antiplatelet antibodies byusing target platelets that are briefly formalin fixed, andusing target platelets from muMT(−/−) mice, which lack background levels of surface antibodies.Using this assay, we have detected serum anti-platelet antibodies in a subset of WASP(−) and WASP(−)CD47(−/−) mice. We have not detected antibodies in WT(B6) mice or in CD47(−/−) mice. We are unable to detect these antibodies with non-fixed target platelets. Use of fixed WT target platelets significantly reduces the sensitivity of the assay. The antibodies are predominantly of the IgG class. Their in vivo significance is supported by the finding that they are more frequent in WASP(−) mice with increased RP (4 of 5 tested) than in those with normal RP (1 of 7 tested). Also, antibody expressing (Ab+) mice show more rapid consumption of CMFDA-labeled WASP(−) platelets than do Ab-mice. To determine whether these antibodies have the differential effect on in vivo WASP(−) platelet consumption that we previously observed after ex vivo opsonization, we simultaneously quantified the consumption rate of WT and WASP(−) platelets in antibodyexpressing (Ab+) and antibody-negative (Ab−) WASP(−) mice. We did this by labeling the two platelet preparations with different fluorescent markers (CMFDA and BMQC). We verified that the fluorescent markers had no differential effect on platelet consumption in vivo. We then found more rapid consumption of both WT and WASP(−) platelets in Ab(+) mice. In some of the latter, WASP(−) platelet consumption is enhanced relative to that of WT platelets. These results suggest that WASP deficient mice, and WAS patients, may be both more prone to develop antiplatelet antibodies and, in some cases, more susceptible to their effects on platelet consumption.

Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). Consumption rates of WAS protein (WASP)(-) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(-) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. CMFDA-labeled WASP(-) platelets are consumed more rapidly than WT platelets in either WT or WASP(-) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(-) reticulated platelets. The number of reticulated platelets is reduced in WASP(-) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(-) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(-) platelets are more accelerated by opsonization than are those of WT platelets. Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(-) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies.

Rapid In Vivo Consumption and Ex Vivo Phagocytosis of WASP(−) Platelets.

Patients with the Wiskott-Aldrich Syndrome (WAS) have thrombocytopenia and increased platelet consumption rates, and may have reduced platelet production rates. WASP(−) mice have been reported to have only a mild thrombocytopenia. We find that when fully crossed onto the C57Bl/6J background, WASP(−) mice have a >50% reduction in platelet counts. Ex vivo labeled WASP(−)platelets are consumed 2 to 4-fold faster than are WT platelets in WT mice, and with exponential rather than linear kinetics. Clearance rates of WASP(−) platelets in WT mice, and vice versa, indicate that rapid consumption is due to factors both intrinsic and extrinsic to platelets. In vivo biotinylation demonstrates consumption rates comparable to those seen with ex vivo labeling, and shows a normal rate of consumption of WASP(−) reticulated (immature) platelets. Reticulated platelet counts are reduced, indicating that their production rate is reduced. Megakaryocytes are increased in spleen and bone marrow, and in the latter their ploidy distribution is normal, suggesting that impaired platelet production occurs at the level of thrombopoiesis. The absolute turnover rates of mature and reticulated platelets, however, indicate that maturation of the latter can account for only a fraction of the former's turnover in either WT or WASP(−) mice. A subset of WASP(−) mice show an increased fraction of reticulated platelets and more severe thrombocytopenia, and some members of this subset also express serum anti-platelet antibodies. CMFDA-labeled WASP(−) platelets opsonized with anti-CD61(IgG1) antibody are more susceptible to ex vivo phagocytosis by bone marrow derived macrophages (BMDM) than WT platelets, and as susceptible as CD47(−/−) platelets. After opsonization with 6A6(IgG2b) antibody, WASP(−) platelets are also taken up more rapidly than WT platelets by BMDM, but less rapidly than are CD47(−/−) platelets. The in vivo consumption rate of WASP(−) platelets in WT recipients is more accelerated by opsonization with anti-CD61 antibody than is that of WT platelets. Increased phagocytosis is not due to (A) altered levels of the targeted antigens or of CD47 (B) increased exposure of phosphatidyl serine, or (C) antibody-induced activation as assayed by CD62P expression. Increased phagocytosis is not due to selective sensitivity to IgG1 vs IgG2b antibodies, as opsonization with an engineered 6A6(IgG1) antibody leads to reduced phagocytosis for both CD47(−/−) and WASP(−) platelets. Anti-CD61 opsonized platelets deficient in both WASP and CD47 show markedly increased ex vivo phagocytosis compared to platelets deficient in either protein, suggesting that platelet WASP does not function to amplify signals from platelet CD47 through macrophage SIRP-alpha. These results raise the possibility that the binding of low affinity or low titer antibodies that might have no effect on WT platelets could cause thrombocytopenia when the platelets lack WASP. Alternatively, rapid phagocytosis of opsonized WASP(−) platelets could promote a self-reinforcing cycle of increased host antigen presentation and increased immune responses to host antigens.

The disulfide-rich region of platelet glycoprotein (GP) IIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets. Platelet glycoprotein (GP) IIb/IIIa is a common target for antiplatelet autoantibodies. The present studies were undertaken (1) to confirm whether the disulfide rich repeat region of GPIIIa contains target epitopes for antiplatelet antibodies in patients with ITP; (2) to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification; and (3) to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex, the vitronectin receptor, α Vβ 3. Recombinant fusion proteins of four GPIIIa extracellular sequences were prepared and purified. Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti-GPIIb/IIIa antibodies with the fusion protein containing the GPIIIa sequence of residues from 468 to 691. These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of GPIIIa isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients. Smaller peptides including residues 446–501 and residues 593–691 each reacted with only 5 of the 24 patient sera; furthermore all but 3 of these interactions were very weak. Visualization of the conformation of the extracellular portion of α Vβ 3 reveals the location of the 222-residue antigenic GPIIIa (β 3) peptide ‘B’ at the immediately extracellular region of the protein that includes a β-tail domain and several integrin-EGF domains. In summary, predictions of hydrophilicity, surface accessibility and antigenicity and the three dimensional structure of the β 3 integrin correlate with autoantibody binding to a recombinant GPIIIa peptide ‘B’ containing residues 468–691.

Characterization of HIV‐1‐specific antibodies and HIV‐1‐crossreactive antibodies to platelets in HIV‐1‐infected haemophiliac patients

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-gp120 antibodies (n=17), 88% (15/17) were HIV gp120-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 72000 to 95000. 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.

Kinetics of serum antiplatelet antibodies in experimental acute canine ehrlichiosis

The pattern of appearance of serum antiplatelet antibodies during the acute phase of experimental canine ehrlichiosis ( Ehrlichia canis) was investigated in six beagles and correlated with the development of thrombocytopenia. The earliest detection of serum antiplatelet antibodies was made on Day 7 post-inoculation in one dog, on Day 13 in three out of six dogs, and on Day 17 post-inoculation in the remaining two dogs. Thrombocytopenia developed in all infected dogs. The results of this study suggest that antiplatelet antibodies play a role in the destruction of platelets in the acute phase of the disease. It is proposed that E. canis infection in dogs alters the immune system resulting in the overproduction of natural antiplatelet antibodies.

Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis

This report presents evidence for the presence of antiplatelet antibodies in sera of dogs experimentally infected with Ehrlichia canis, during the acute phase of the disease. Six healthy adult male beagle dogs were inoculated intravenously with blood from a longstanding infected dog with the Israel strain 611 of E. canis. Thrombocytopenia and concurrent increase in mean platelet volume were the most consistent haematological signs of the disease. The dogs developed an antibody titre to E. canis from Day 15 postinoculation. All dogs were antiplatelet antibody negative before inoculation. Twenty-four days postinoculation with E. canis, when the platelet count was at its lowest, antibodies to platelets were demonstrated, in the sera of five of the six dogs.

Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies

OBJECTIVE: Our purpose was to determine platelet kinetics in pregnancy by means of noninvasive reticulated platelet counts and to examine in a pilot study whether increased reticulated platelet values were associated with preeclampsia and pregnancy-induced hypertension. STUDY DESIGN: Nulliparous women had blood samples drawn at enrollment (first prenatal visit) and at 28 and 36 weeks' gestation. The percent of reticulated platelets (an index of marrow platelet release correlating with increased thrombopoiesis), platelet-associated immunoglobulin, and serum antiplatelet antibody were measured and correlated with the clinical course for each patient. RESULTS: In 31 normal pregnancies the percent of reticulated platelets was never significantly higher than the values for normal nonpregnant women (5.8% ± 2.2%) in spite of a significant decrease in platelet count by 36 weeks. By contrast, the percent of reticulated platelets in four women with preeclampsia rose significantly to 13.9% ± 11.2% at 28 weeks, before the onset of clinical signs. No women had evidence of immune platelet destruction. CONCLUSION: In normal pregnancy the decline in platelet count is not accompanied by an increase in marrow platelet production, suggesting that the platelet decrease is dilutional without a compensatory thrombopoietic response or alternatively that thrombopoiesis is down-regulated during normal pregnancy. However, platelet production does increase before the onset of symptoms in a small number of women in whom preeclampsia or pregnancy-induced hypertension subsequently develops. These findings may justify a larger prospective study to determine whether noninvasive serial measurement of the percent of reticulated platelets can predict those pregnant women at risk for hypertension and preeclampsia. (AM J OBSTET GYNECOL 1994;170:117-22.)

Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies

<b>Objective:</b> Our purpose was to determine platelet kinetics in pregnancy by means of noninvasive reticulated platelet counts and to examine in a pilot study whether increased reticulated platelet values were associated with preeclampsia and pregnancy-induced hypertension. <b>Study Design:</b> Nulliparous women had blood samples drawn at enrollment (first prenatal visit) and at 28 and 36 weeks' gestation. The percent of reticulated platelets (an index of marrow platelet release correlating with increased thrombopoiesis), platelet-associated immunoglobulin, and serum antiplatelet antibody were measured and correlated with the clinical course for each patient. <b>Results:</b> In 31 normal pregnancies the percent of reticulated platelets was never significantly higher than the values for normal nonpregnant women (5.8% ± 2.2%) in spite of a significant decrease in platelet count by 36 weeks. By contrast, the percent of reticulated platelets in four women with preeclampsia rose significantly to 13.9% ± 11.2% at 28 weeks, before the onset of clinical signs. No women had evidence of immune platelet destruction. <b>Conclusion:</b> In normal pregnancy the decline in platelet count is not accompanied by an increase in marrow platelet production, suggesting that the platelet decrease is dilutional without a compensatory thrombopoietic response or alternatively that thrombopoiesis is down-regulated during normal pregnancy. However, platelet production does increase before the onset of symptoms in a small number of women in whom preeclampsia or pregnancy-induced hypertension subsequently develops. These findings may justify a larger prospective study to determine whether noninvasive serial measurement of the percent of reticulated platelets can predict those pregnant women at risk for hypertension and preeclampsia.

Determination of serum antiplatelet antibodies in patients with idiopathic thrombocytopenic purpura by ELISA

Determination of serum antiplatelet antibodies in patients with idiopathic thrombocytopenic purpura by ELISA

An epidemic of maternal thrombocytopenia associated with elevated antiplatelet antibody: Platelet count and antiplatelet antibody in 116 consecutive pregnancies: Relationship to neonatal platelet count

Twenty-eight (24%) of 116 pregnant women studied prospectively during an 8-month period in 1983 had platelet counts of <150,000/mm3 at least once during pregnancy. Thirteen of these were thrombocytopenic in both the prenatal and the peripartum period. Eighteen were restudied 3 to 12 months after delivery. One woman, who was pregnant again, had a platelet count of 140,000/mm3. In the others, platelet counts were in the normal range. Platelet-associated immunoglobulin G and serum antiplatelet antibody levels were elevated in 79% and 61%, respectively, of these 28 women on at least one occasion. However, 59% of 73 pregnant nonthrombocytopenic women had increased platelet-associated immunoglobulin G levels and 59% had positive serum antiplatelet antibody test results. Twenty women who had increased platelet-associated immunoglobulin G levels and positive serum antiplatelet antibody test results were normal 6 to 10 months after delivery. Of 105 infants studied, 10 were thrombocytopenic. Neonatal thrombocytopenia was not predicted by maternal platelet count, platelet-associated immunoglobulin G, or serum antiplatelet antibody. By the fall of 1984, the incidence of thrombocytopenia had dropped to two in 280 consecutive pregnancies. We conclude that (1) epidemics of thrombocytopenia can occur in pregnant women and (2) if a women is found to be thrombocytopenic for the first time during pregnancy, she should not be subjected to the measures advocated for the management of pregnancy in women with autoimmune thrombocytopenic purpura.

945 HIGH DOSE STEROIUS (HDS) IN CHILDHOOD ACUTE IDICPATHIC THROMBOCYIOPENIA PURPURA

The use of steroids in childhood ITP is ccntroversial. High dose intravenous gammegloblin (IVG) has become an accepted treatment in ITP. HDS in children with ITP has also been proposed; we utilized a regimen incorporating HDS (prednisone 4-8 mgAg/d) rased en initial platelet count in nine newly diagnosed untreated children with ITP severely thrombocytopenic (nean platelets 5 × 109/L)- All cases had positive iiminolcgic evidence of ITP (platelet associated antibodies, serum antiplatelet antibodies), bone marrow compatible with ITP and no clinical or laboratory evidence of other disease. All patients are presently in remission; none require treatment. For the total group it took 1.9 days (range 1-3) to reach a platelet count of 20 × 109/L and 7.1 days (range 2-23) to reach 100 × 109/L. Only one case had a relapse (platelet count 18 × 109/L). This happened en day 14 of therapy while en 2 mg/kg/d prednisone and respondad to an increase to 4 mg/kg/d within 5 days. No serious toxicity was observed except for weight gain ranging fron 3-10% (mean 8%) and 5/9 patients showed periods of hyperactivity and poor discipline. HDS is effective in severely thromoytopenic children with acute ITP, carries few side effects, influences the natural course of the disease, and is at least as effective as IVG and comparatively more cost effective.

Enhancement of the indirect anti-platelet antibody test and its application to platelet immunology.

Comparisons in the same patients of platelet-bound anti-platelet antibody (APA) levels (direct test) with serum APA levels (indirect test) frequently do not give the same results. Indirect test results frequently are negative or marginally elevated even though platelet-bound antibody is greatly increased on direct testing. The most likely cause for these differences between the two tests is insufficient binding of serum antibody to test platelets in vitro. In an effort to enhance in vitro antibody binding, we examined four test modifications affecting platelet attachment. Rabbit anti-human platelet antisera (absorbed with their specific human donor leukocytes to remove HLA- and granulocyte-specific antibodies) were combined under various conditions with human platelets and tested in a modified antiglobulin consumption test utilizing rabbit IgG. The test conditions varied: incubation time of the serum APA-platelet mixture at 37 degrees C; age of the test platelet pool; ionic strength of the mixture (varied through incorporation of a low-ionic-strength solution (LISS); and vortex agitation for 30 seconds prior to the addition of sera. Optimal attachment in standard phosphate-buffered saline took place in 60 minutes using test platelets stored for either 3 or 30 days, and yielded mean values for rabbit immune sera of 9.9 fg of IgG per platelet. Preincubation controls yielded a mean of 0.7 to 1.5 fg per platelet. Incorporation of LISS or vortexing of test platelets did not affect the results of control, but both modifications substantially increased test values of immune sera to 15.8 and 17.3 fg of IgG per platelet, respectively. Combining LISS with vortexing did not further increase values.