Immunoenzyme assay of glycocalicin, platelet glycoprotein Ib fragment. Evaluation of platelet turnover in circulation and differential diagnosis of thrombocytopenia

We propose a method for measurement of plasma glycocalicin, a fragment of platelet membrane glycoprotein Ib. The concentration of glycocalicin is elevated in thrombocythemia and reduced in thrombocytopenia caused by insufficient platelet production, but not in immune thrombocytopenia due to enhanced platelet degradation. Thus, plasma content of glycocalicin is an indicator of platelet turnover. The proposed assay can be used for differential diagnostics of thrombocytopenia. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 476–479, October, 1999     

The platelet volume-number relationship in patients infected with the human immunodeficiency virus

The pathophysiology of thrombocytopenia in the acquired immune deficiency syndrome has not been elucidated completely. Many findings in these patients are identical to those with immune thrombocytopenic purpura. However, recent findings in acquired immune deficiency syndrome patients including the effect of zidovudine on platelet count and the demonstration of ultrastructural changes and viral RNA in megakaryocytes, have suggested that the human immunodeficiency virus may directly infect megakaryocytes, and play a role in acquired immune deficiency syndrome-related thrombocytopenia. To investigate further the mechanism of decreased platelet counts in human immunodeficiency virus-infected patients, the platelet volume-number relationship and corresponding bone marrow findings in 34 patients infected with human immunodeficiency virus were studied. Parameters evaluated included platelet count and mean platelet volume; bone marrow cellularity, megakaryocyte number, and number and percentage of denuded megakaryocyte nuclei. Two thirds of the platelet counts were low, and of these 92% had an inappropriately low mean platelet volume. These individuals had a platelet-volume number relationship that is very similar to that seen in myelosuppressive disorders. In addition, more than 90% of the bone marrows from thrombocytopenic patients had either normal or decreased numbers of megakaryocytes. These observations provide additional evidence to support the hypothesis that the pathophysiology of human immunodeficiency virus-associated thrombocytopenia may be due, at least in part, to a direct effect on the megakaryocytes.     

Thrombocytopenia in a retrovirally-induced murine erythroleukemia.

A variant strain of Rauscher leukemia virus (RLV-A) obtained from a transplantable murine monomyelocytic leukemia causes a disease characterized by frank anemia, wasting, hepatosplenomegaly and erythroblastosis. The involvement of platelets in this disease are reported here. The RLV-A induced a severe thrombocytopenia (25 percent of control level) at the terminal stage of disease. This thrombocytopenia was not associated with disseminated intravascular coagulopathy since the prothrombin times were always within normal limits. The partial thromboplastin time was elevated in the terminal stages of disease and was found to be associated with factor deficiencies, possibly owing to the presence of anti-factor antibodies, in the intrinsic coagulation pathway, especially factor VIII. Further, splenectomy did not abolish the thrombocytopenia, since splenectomized, virally infected animals also developed severe thrombocytopenia (29 percent of control levels). The ensuing splenomegaly during progression of disease was not the cause of the thrombocytopenia. A physiological response to the severe thrombocytopenia was the production of larger size platelets. At terminal stages of the disease, platelet volume increased to 4.2 mu 3 (normal is 3.0 mu 3). An increase in platelet volume was also observed in splenectomized, virally infected animals. Electron microscopy indicated that these circulating platelets contained c-type viral particles. Viral infection was associated with decreased life span of circulating platelets, as measured by 75Se-methionine at mid and terminal stages of the disease. Our results suggest that direct viral infection of platelets and/or megakaryocytes with subsequent cell lysis is a possible cause of the observed thrombocytopenia observed in RLVA-induced disease and may also occur in other retrovirally-induced diseases.     

Bone marrow biopsy in HIV-positive patients with thrombocytopenia. Light and electron microscopy

Isolated thrombocytopenia (platelets less than 100,000 mmc) may be the first clinical symptom in HIV positive patients or occur in all the evolutive phases up to overt AIDS. In this paper bone marrow lesions are evaluated at light and electron microscopy in 32 HIV positive patients with isolated thrombocytopenia (group II and III CDC 1986). At light microscopy an increase in megakaryocytes with small dysplastic changes, plasmacytosis and hypereosinophilia were the bone marrow lesions detected. Electron microscoy revealed megakaryocytes with focal nuclear alterations (hypolobation and dilatation of the perinuclear cisternae) and abnormalities in the maturation of platelets associated with cytoplasmic micro-macrovacuolation, absence of viral particles or of virus correlated structures. About 9% of HIV positive patients presented with isolated thrombocytopenia as a first clinical symptom: thrombocytopenia is not believed to have unfavourable prognostic significance in the evolution to overt AIDS.     

Soluble platelet glycoprotein V in distinct disease states of pathological thrombopoiesis

Quantitative platelet disorders (i.e., thrombocytosis or thrombocytopenia) may also be associated with qualitative platelet alterations. Clonal thrombocythemia (CT), reactive thrombocytosis (RT), immune thrombocytopenic purpura (ITP), and thrombocytopenia of aplastic pancytopenia (AA) or infiltrative bone marrow disorders represent the major classes of pathological thrombopoiesis. Glycoprotein V may serve as an in vivo marker of platelet activation in thrombotic and hemorrhagic states. The aim of this study was to assess circulating plasma soluble platelet glycoprotein V (sGPV) concentrations in distinct disease states of pathological thrombopoiesis. The whole study group comprised 20 patients with thrombocytopenia, 32 patients with thrombocytosis and 14 healthy adults as the control group. sGPV was significantly increased in the group of thrombocytosis patients in comparison to the thrombocytopenic group and the healthy control groups. When sGPV levels were corrected according to platelet number (sGPV/tr), this ratio was very high in patients with thrombocytopenia compared to patients with thrombocytosis and the control group. Our results suggest that there is an ongoing platelet activation associated with thrombocytosis regardless of its origin is either CT or RT. Therefore, glycoprotein V system may serve to activate residual platelets in thrombocytopenia regardless of its origin is either ITP or AA.     

Antigenic differences between human platelets and megakaryocytes.

To investigate the apparent paradox in the observation that most patients with immune thrombocytopenias have normal or increased numbers of megakaryocytes (MKs), the extent of antigenic cross-reactivity between normal platelets and MK was examined. Indirect immunofluorescence and ultrastructural studies were carried out by means of four antisera specific for platelets: anti-GpIb, anti-GpIIb/IIIa, anti-PLA1, and an antiserum from a patient with quinidine-induced thrombocytopenia. Following incubation of freshly collected marrow with these antisera, MK were first identified by phase-contrast microscopy and then inspected for fluorescence. Almost all MKs were found reactive with the last three antisera, albeit to a variable extent. In contrast, only 24% reacted with anti-GpIb. The pattern of fluorescence, ie, rim, partial or cytoplasmic, appeared to be related to the extent of MK fragmentation. Only rim fluorescence of living MKs could be interpreted to indicate that the platelet epitope was exposed on the surface of the precursor cell. The observations suggest that platelet antigens are variably expressed on the plasma membranes of MKs. In a clinical setting, the heterogeneity among platelet target antigens and the extent to which these are exposed on MKs at various stages of maturation may dictate the severity of the thrombocytopenia and degree of ineffective thrombocytopoiesis.     

DNA content and nuclear size of megakaryocytes in thrombocythaemia

Total nuclear DNA content and nuclear size of megakaryocytes were studied in biopsies of the iliac bone marrow of individuals with normal or increased platelet counts. The DNA content was determined using Feulgen cytophotometry of bone marrow smears and the nuclear area by morphometric analysis of megakaryocytes of bone marrow sections. The mean DNA content and the mean nuclear area were both significantly larger in megakaryocytes of patients with thrombocytosis as a result of myeloproliferative disease than in patients with secondary thrombocytosis as well as in two control groups of individuals with normal platelets counts, one comprising healthy volunteers, the other with various non-haematological disorders. There was a statistically significant correlation between the DNA content and nuclear area of the megakaryocytes (r = 0.92) in the entire group of bone marrows studied.     

Immunopathology of thrombocytopenia in experimental malaria.

An early thrombocytopenia was observed in CBA mice during acute infection with Plasmodium berghei. This was associated with an increase in bone marrow megakaryocytes and a reduction of normal syngeneic 111Indium-labelled platelet life span. Malaria-induced thrombocytopenia was thus considered to be the result of increased peripheral platelet destruction rather than central hypoproduction. The occurrence of thrombocytopenia was modulated by T-cell depletion. Indeed, thymectomized, irradiated or anti-CD4 monoclonal antibody-treated mice failed to develop thrombocytopenia, although they were infected to the same extent. Conversely, a significant thrombocytopenia was observed in thymectomized mice reconstituted with CD4+ T cells. During the course of infection, a significant inverse correlation was found between platelet counts and platelet-associated IgG. Normal mice passively transferred with serum from syngeneic malaria-infected mice developed thrombocytopenia. The possibility to raise monoclonal anti-platelet antibodies from P. berghei-infected animals further suggested a role for an antibody-mediated platelet destruction during acute murine malaria infection. These results indicate that in murine malaria, thrombocytopenia is mediated by immune mechanisms and that CD4+ T cells might be significantly involved.     

Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection.

BACKGROUND. Isolated thrombocytopenia accompanied by increased amounts of platelet-associated antibody is a common manifestation of human immunodeficiency virus (HIV) infection, and the thrombocytopenia often improves with zidovudine. It is not clear whether the mechanism of HIV-related thrombocytopenia primarily involves autoimmune destruction of platelets or reduced platelet production by megakaryocytes. METHODS. We studied the survival of 111In-labeled autologous platelets and performed platelet imaging in 24 men with isolated HIV-related thrombocytopenia (16 who received no treatment and 8 who received zidovudine). We also studied 20 HIV-infected men with normal platelet counts (10 who received no treatment and 10 who received zidovudine) and studied 12 healthy seronegative men as controls. RESULTS. Mean (+/- SD) platelet survival was significantly decreased in both the untreated and the zidovudine-treated patients with HIV-related thrombocytopenia (to 92 +/- 33 and 129 +/- 44 hours, respectively; both P < 0.001), as compared with the normal controls (198 +/- 15 hours). Mean platelet survival was also significantly decreased in the HIV-infected patients with normal platelet counts (untreated, 162 +/- 23 hours, P < 0.01; zidovudine-treated, 166 +/- 35 hours, P < 0.05). Imaging studies, however, revealed no evidence of increased clearance of autologous platelets in the liver or spleen in any of these groups. Mean platelet production was significantly depressed in the untreated patients with thrombocytopenia (23,000 +/- 11,000 platelets per cubic millimeter per day, P < 0.001) as compared with the healthy controls (45,000 +/- 6,000 per cubic millimeter per day). Mean platelet production was significantly increased, however, in the men treated with zidovudine, both in those with thrombocytopenia (60,000 +/- 31,000 platelets per cubic millimeter per day, P < 0.01 vs. controls) and in those without thrombocytopenia (68,000 +/- 22,000 per cubic millimeter per day, P < 0.01). CONCLUSIONS. Although there was a moderate reduction in platelet survival in HIV-infected persons, these patients, regardless of platelet counts, also had decreased production of platelets, possibly due to viral infection of the megakaryocytes. Zidovudine appears to improve platelet production.     

Suspects in the tale of lupus-associated thrombocytopenia

Immunologically mediated thrombocytopenia is a frequent clinical manifestation in patients with systemic lupus erythematosus (SLE). Autoantibodies targeting platelet membrane glucoproteins have a central role in peripheral platelet destruction. Autoantibodies against thrombopoietin are also present in about one‐third of patients, but their pathogenetic role is obscure. Thirty‐eight serum samples from SLE patients were tested for anti‐platelet antibodies, anti‐thrombopoietin antibodies and levels of circulating thrombopoietin. Bone marrow histology was also assessed. Thirty‐nine per cent of sera displayed anti‐thrombopoietin antibodies and 29% had circulating anti‐platelet antibodies. Anti‐thrombopoietin antibodies were associated with lower thrombopoietin concentrations, and lower mean platelet values in long‐term follow‐up. Anti‐platelet antibodies were present in about 40% of thrombocytopenic and non‐thrombocytopenic individuals but were absent in patients who had recovered from thrombocytopenia, supporting their pathogenetic role. Both autoantibodies were absent in control sera from patients with rheumatoid arthritis and primary Sjögren’s syndrome. Decreased bone marrow cellularity, normal or low number of hypolobulated, pyknotic megakaryocytes and stromal alterations were prominent findings in thrombocytopenic SLE patients, suggesting a defect in megakaryopoiesis. These findings were not evident in specimens from patients with idiopathic thrombocytopenic purpura who had increased megakaryocytes, normal cellularity and absence of stromal alterations. In conclusion, peripheral destruction due to platelet autoantibodies, anti‐thrombopoetin antibodies, lower effective circulating thrombopoetin and impaired compensatory response due to bone marrow damage interact in SLE and thrombocytopenia ensues.     

Diagnostic value of tests for reticulated platelets, plasma glycocalicin, and thrombopoietin levels for discriminating between hyperdestructive and hypoplastic thrombocytopenia

We measured reticulated platelets (RPs) and plasma glycocalicin (GC) and thrombopoietin (TPO) levels simultaneously in 107 thrombocytopenic patients to clarify the diagnostic value of these tests for discriminating hyperdestructive from hypoplastic thrombocytopenia. The percentage of RPs and GC index (plasma GC level normalized for the individual platelet count) were markedly elevated in patients with idiopathic thrombocytopenic purpura (ITP) but normal or slightly elevated in patients with aplastic anemia (AA) or chemotherapy-induced thrombocytopenia (ChemoT). For RP percentage for diagnosing hyperdestructive thrombocytopenia the sensitivity and specificity were excellent but were lower for the GC index. Absolute RP counts and plasma GC levels were markedly decreased and plasma TPO levels markedly elevated in patients with AA or ChemoT, but absolute RP counts and plasma GC levels were moderately decreased and plasma TPO levels only slightly elevated in patients with ITP. The sensitivity and specificity of plasma TPO levels for diagnosing hypoplastic thrombocytopenia were excellent. Using the RP percentage and plasma TPO levels in combination improved specificities. Simultaneous measurement of RP percentage and plasma TPO level may help discriminate thrombocytopenia of unknown cause in routine hematologic practice.     

Immune amegakaryocytic thrombocytopenia of the newborn: association with anti-HLA-A2.

Three newborn babies with congenital immune amegakaryocytic thrombocytopenia are described. Two were siblings. The mothers' sera showed antibody against both pooled random platelets and lymphocytes of type HLA-A2. Two babies had transient thrombocytopenia and leucopenia and made a full haematological recovery. One baby died aged four hours having had no haematological investigations during life. Histological examination of the bone marrow in all three babies (two by trephine biopsy; one at necropsy) confirmed a deficiency of megakaryocytes. It is suggested that anti-HLA-A2 may cause neonatal thrombocytopenia by depressing megakaryocytes instead of, or in addition to, any direct effect on platelets.     

Acquired pseudo-pseudo Bernard-Soulier syndrome complicating Gaucher's disease.

AIMS--To investigate the abnormality in platelet function in two patients with type I Gaucher's disease causing a chronic bleeding tendency despite normalisation of the platelet count after spleen removal. METHODS--Routine laboratory methods were used to assess baseline coagulation. Platelet aggregometry was used to assess platelet responses to a range of agonists, and abnormalities were further assessed in mixing experiments using washed platelets and patients' plasma. RESULTS--Platelets from both patients with Gaucher's disease failed to agglutinate to ristocetin, despite normal platelet surface glycoprotein (GP) Ib and plasma von Willebrand factor activity. The agglutination of normal washed platelets was abolished by incubation in patient plasma. The inhibitory activity did not lie in the IgG fraction of patient plasma, and was found to be loosely associated with the patient platelet surface. CONCLUSIONS--The inhibition of ristocetin induced platelet agglutination in patients with Gaucher's disease causes a prolonged skin bleeding time. This could be due to the accumulated glucocerebroside in the plasma coating the platelet membrane. It is suggested that the term pseudo-pseudo Bernard-Soulier syndrome would be appropriate, as on initial screening, the abnormality has the features of Bernard-Soulier syndrome, but further investigation shows normal plasma von Willebrand activity and platelet surface GP Ib concentrations. The inhibitory activity is not due to a platelet specific antibody as is the case in pseudo-Bernard Soulier syndrome.     

A monoclonal antibody against rat platelets. I. Tissue distribution in vitro and in vivo.

In this study we describe a new monoclonal antibody (MoAb PL.1) against rat platelets. Immunohistology of various rat tissues showed staining of platelets, especially in the spleen, and staining of megakaryocytes in bone marrow and spleen red pulp. In the liver small platelet aggregates and endothelial cells were stained. After in-vivo administration of MoAb PL.1 an acute severe thrombocytopenia was observed. In general the distribution of the antibody and/or antibody-coated platelet aggregates showed the same pattern as after in-vitro incubation, i.e. staining of rat platelets and platelet aggregates in spleen red pulp, and staining of megakaryocytes in spleen and bone marrow. Platelet aggregates were observed in the liver and electron microscopy indicated that they were associated with Kupffer cells. Furthermore, liver endothelial cells were positively stained. Comparison of the molecular weight of the antigens recognized by this MoAb and by human anti-platelet MoAbs, as well as comparison of staining patterns of megakaryocytes indicated that MoAb PL.1 is probably directed to a GPIIb/IIIa complex analogue. Since MoAb PL.1 is of the non-complement-binding mouse IgG1 isotype, it can be used for studying clearance of platelet aggregates by Fc-receptors of the MPS. It also promises to be a useful tool in the study of platelet involvement in rats with experimental nephritis.     

Differentiation between Bernard-Soulier syndrome and immune thrombocytopenia by immunostaining of peripheral blood.

Peripheral blood smears from seven patients with Bernard-Soulier syndrome were examined by an immunocytochemical staining procedure using a monoclonal antibody specific for platelet glycoprotein Ib. No platelet staining was observed except for very slight staining of the large sized platelets from one of the patients. Application of the assay to blood smears from 12 patients with immune thrombocytopenia showed that their peripheral platelets stained normally, so the assay can be used to differentiate between immune thrombocytopenia and Bernard-Soulier syndrome and to confirm a diagnosis of the syndrome.     

Antiplatelet antibody‐induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia

Immune thrombocytopenia (ITP ) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody‐mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell‐mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB /c mice were administered various anti‐αII b, anti‐β3 or anti‐GPI b antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse‐anti‐mouse β3 sera and an anti‐ αII b monoclonal antibody (MWR eg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.     

Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes

Platelets are specialized cells produced by megakaryocytes in the bone marrow that represent the first defense against hemorrhage, yet they also play a pathological role in thrombosis, inflammation, and cancer. Millions of platelet transfusions are conducted each year, and the supply of this blood component is limited. There are many diseases where platelet production or function is impaired with severe consequences for patients. With such clinical need, new insight into the formation of platelets would have a major impact on patients and healthcare. We developed an innovative 3D system to study platelet production that represents the first spatial reconstruction of the bone marrow environment. In this system human megakaryocytes were able to migrate toward the vascular niche, extend proplatelets, and release functional platelets into vascular tubes. The combination of different bone marrow components and the compliance of silk-based vascular tubes makes this model a unique tool for the study of platelet formation and production for use in healthcare needs.     

Shear Stress-Induced Binding of Large and Unusually Large von Willebrand Factor to Human Platelet Glycoprotein Ibα

Platelet membrane glycoprotein (GP) Ib(alpha), a component of the GP Ib-IX-V complex, is a receptor for von Willebrand factor (VWF). A small quantity of large VWF multimers binds to platelets under high shear stress, and induces aggregation. We studied the shear-induced attachment of large and unusually large VWF multimers to the GPIb(alpha) extracellular domain (glycocalicin), human platelets, and GPIb(alpha) gxpressing Chinese hamster ovary (CHO) cells. Compared to binding in the presence of botrocetin and ristocetin, shear stress only induced low-level NVWF (normal plasma VWF multimers) binding. This shear stress induced interaction is also dependent on VWF multimeric size. Elevated binding levels of endothelial cell VWF (enriched in unusually large VWF multimers) to glycocalicin-coated beads were observed under low shear conditions, which did not result in the attachment of normal plasma VWF.     

Human/BALB radiation chimera engrafted with splenocytes from patients with idiopathic thrombocytopenic purpura produce human platelet antibodies.

We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post-transplant and declined after 6 weeks, while human anti-human platelet antibodies were detected 2-8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross-reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP.     

Effect of platelet growth factors on proliferation of guinea pig bone marrow and splenic stromal precursor cells and proliferation of cultural descendants from bone marrow precursor cells

Elimination of platelets from guinea pig splenocyte suspension (laking megakaryocytes) with EDTA considerable reduces the efficiency cloning of splenic stromal precursor cells. It means that platelet-derived growth factors are necessary for stromal precursor cells from different organs (bone marrow, thymus, and spleen). The dependence on the platelet growth factors can vary within a wide range in descendants from cultured bone marrow precursor cells (passaged bone marrow fibroblasts at different stages of differentiation.