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A method and approach are described to differentiate classic autoimmune thrombocytopenia (ATP) from immune complex-associated thrombocytopenia in systemic lupus (SLE), hepatitis/chronic liver disease (LIV-ITP) and HIV-1 related thrombocytopenia (HIV-1-ITP). The platelet immunologic profile of IgG, C3C4 and IgM was measured with a solid-phase ELISA, employing 125I-staphylococcal protein A to detect indicator antibody binding. Polyethylene glycol was employed to precipitate immune complexes (PEG-IC). Platelet-associated IgG (PAIgG) was 2.8-, 5.6- and 5.8-fold higher in SLE, LIV-ITP and HIV-1-ITP patients respectively compared to ATP patients: platelet C3C4 was 3.2-, 4.8- and 4.5-fold higher respectively; platelet IgM was 2.2-, 3.7- and 3.8-fold higher respectively; serum PEG-IC levels were 4.2-, 4.8- and 2.1-fold higher respectively. With all parameters measured, there was no overlap between the 75th percentile for ATP patients and the 25th percentile for all three cohorts. The likelihood of having a platelet C3C4 level higher than the highest ATP level was 69% for SLE, 90% for LIV-ITP and 94% for HIV-1-ITP respectively; with PEG-IC measurements the likelihood was 83%, 100% and 100% respectively. Serum IgG, C3, C4, IgM and PEG-IC were examined for a possible relationship with platelet measurements. Except for a positive correlation between serum and platelet IgM in ATP, r = 0.5, P < 0.04, there was no positive correlation with any of the parameters measured. An inverse correlation was noted between PEG-IC level and platelet C3C4 in SLE, r = 0.7, P < 0.04. Thus platelet immunologic profile and serum PEG-IC level measurements differentiated classic ATP from immune complex-associated thrombocytopenias (SLE, LIV-ITP, HIV-1-ITP). Except for IgM measurements in ATP, platelet measurements could not be attributed to their respective serum concentration.

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+ CD41+) were isolated from normal human bone marrow or induced in culture from CD34+ CD41- bone marrow cells by treatment with thrombopoietin (TPO) or IL-3. We found that although this population included the majority of cells that can form CFU-MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+ CD41+-induced cells was greater than that of isolated CD34+ CD41+ cells in that the isolated cells only formed CFU-MK and BFU-e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+ CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+ CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU-e. A major portion of the cells that expressed CD34+ CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c-mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+ CD38- cells), expressed c-mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c-mpl was predictive of commitment to the MK lineage.

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41+ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41+ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.

CD4 expression is not limited to T cells and monocytes. In both mouse and man the antigen has been detected on some early haemopoietic progenitors and we have shown that some mature megakaryocytes (MK) express CD4, the surface molecule that serves as the high-affinity receptor for human immunodeficiency virus type-1 (HIV-1). Using a serum-free culture system in which sorted CD34+ haemopoietic progenitors are cultured with thrombopoietin (TPO), IL-3, IL-6 and SCF, we now show that CD4 expression is induced in virtually all developing haemopoietic cells. This phenomenon was particularly striking in the MK lineage, where CD4 expression began whilst the cells were still CD34+ but after they expressed CD41 (GPIIb/IIIa). CD4 expression and endomitotic polyploidization occur at the same time in MK development. In culture, maximum CD4 expression occurred 4-6 d after CD41 expression and lasted for a few days. Expression of CD4 declined gradually thereafter and most MK were CD4- by the end of the culture period. The amount of CD4 on the surface of some MK, as measured by intensity of fluorescence staining, exceeded that of normal monocytes and approached the brightness of T cells. Appearance of the surface antigen correlated with the presence of mRNA for CD4, as measured by RT-PCR.

Thrombin-treated tumour cells enhance their adhesion to platelets, fibronectin and von Willebrand factor in vitro, and enhanced their pulmonary metastasis in mice in vivo. A unique seven transmembrane spanning thrombin receptor has recently been cloned which is activated following thrombin proteolysis of the N-terminal end of the receptor with exposure of a tethered ligand. An N-terminal 14-mer (SFLLRNPNKYEPF) or 6-mer (SFLLRN) of the tethered ligand can serve as a thrombin receptor activation peptide (TRAP) by mimicking the action of thrombin on platelets, endothelial cells and smooth muscle cells. We have examined six human tumour cell lines for their response to TRAP, for the presence of this thrombin receptor mRNA by RT-PCR, protein by immunoblot and for their in vitro and in vivo response to TRAP. All six cell lines contain the receptor mRNA, and when treated with 100 microM 6-mer TRAP or 1 u/ml thrombin increase their adhesion to platelets 2-3-fold. Four of the six cell lines undergo tyrosine phosphorylation within 30 s to 1 min after exposure to 6-mer TRAP or thrombin. Thus tumour cells respond to thrombin via activation of their seven transmembrane spanning thrombin receptor.

A monoclonal antibody, LK-4, has been developed which distinguishes platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes on platelet glycoprotein GPIIIa of Triton-solubilized platelet extracts. An ELISA assay has been developed which traps GPIIIa with Concanavalin A, enriching the platelet extract for the PLA antigens. A second monoclonal antibody, DEK-10, which reacts equally with GPIIIa of PLA1/PLA1 and PLA2/PLA2 platelet extracts is employed as an internal standard to correct for individual differences in GPIIIa content, GPIIIa extracted by Triton X-100 and GPIIIa trapped with Concanavalin A. This ELISA assay clearly differentiated 11 different PLA1/PLA1 subjects from eight PLA2/PLA2 women with a history of neonatal alloimmune thrombocytopenia as well as six unrelated obligate heterozygotes and should be useful in evaluating the PLA genotype of pregnant women and their families.

Ig secretion of peripheral blood mononuclear cells (PBMC) was measured in Epstein Barr virus (EBV) seropositive autoimmune thrombocytopenic purpura (ATP) patients and controls following in vitro infection with EBV. EBV infected PBMC from patients with ATP secreted Ig for a longer period of time than EBV infected control cells and had higher peak Ig production. Removal of T cells or CD8 cells from control PBMC increased the duration and level of Ig production to that achieved by ATP PBMC. Total T or CD8 cell depletion of ATP PBMC had no effect on the enhanced level or duration of Ig secretion. Depletion of control CD4 lymphocytes decreased Ig production. Treatment of EBV infected control PBMC with either ATP sera or purified IgG (plus complement) increased the duration and level of production of Ig to that observed in ATP PBMC, control PBMC depleted of all T cells or control PBMC depleted of CD8 cells. This antibody effect could be reversed by reconstitution with control T cells. Thus, patients with ATP produce an antibody which leads to suppressor cell dysfunction. Defective function of these cells may lead to a disorder of immune regulation in which autoantibodies are produced against platelets.

Platelet volume distribution curves were obtained in 20 control subjects and in 21 patients with idiopathic/autoimmune thrombocytopenic purpura. A striking increase in microthrombocytes as well as megathrombocytes was noted in 86% of patients on one or more occasions, particularly in the prsence of severe thrombocytopenia. The entire spectrum of platelet volume distribution curves noted in patients could be reproduced experimentally in rabbits following intravenous injection of anti-platelet antibody. Differential centrifugation studies with control subjects revealed that microthrombocytes were light platelets and megathrombocytes were heavy platelets. Electron microscopy in patients with thrombocytopenia revealed that microthrombocytes were composed of intact small platelets as well as platelet fragments. It is concluded that severe peripheral destruction of platelets is associated with an increase in microthrombocytes as well as megathrombocytes.

Rabbits were either splenectomized or subjected to splenic blockade with phenylhydrazine in order to determine the splenic platelet and megathrombocyte (large-platelet) pools. The average splenic platelet pool calculated from both methods was 35% of total platelets. The average splenic megathrombocyte pool was 54% of total megathrombocytes. Adrenaline injection into rabbits or dogs revealed a rapid increase in both platelet count and megathrombocyte number which peaked at 2-6 min and returned toward normal in 5-10 min. The platelet volume distribution curve was shifted to the right, indicating the release of large platelets (megathrombocytes) into the circulation. The peak rise in megathrombocyte number was significantly greater than the peak rise in platelet count. Rabbits subjected to splenic massage shifted their platelet volume distribution curve to the right in the absence of a rise in platelet count. It is concluded that the spleen preferentially sequesters megathrombocytes.