Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) ...VIIa (rFVIIa; NovoSeven®) and 2·5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC‐1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of > 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet–leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet‐rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.
The adhesion receptors known as integrins perform key functions for hematopoietic cells. The platelet integrin alphaIIbbeta3 is critical in hemostasis, and the beta1 and beta2 integrins on leukocytes ...have many roles in cell-mediated immunity. Mutations in the beta2 subunit lead to integrin nonexpression and to an immune deficiency, leukocyte adhesion deficiency-1. Mutations in either the alpha or beta subunit of alphaIIbbeta3 usually lead to integrin nonexpression and a bleeding tendency termed Glanzmann thrombasthenia. Here we describe a unique patient with clinical features of both Glanzmann thrombasthenia and leukocyte adhesion deficiency-1. The patient has normal expression of beta1, beta2, and beta3 integrins, but all are dysfunctional. The key findings are that "inside-out" signaling pathways leading to integrin activation are defective and that this is associated with abnormal integrin clustering. The integrins themselves are intact and capable of function following extracellular stimulation. T cell motility is normal, as are the expression levels and electrophoretic characteristics of all cytoskeletal and signaling proteins tested, except PKC-alpha, which has enhanced expression in the patient's cells. To our knowledge, this is the first description of a dysfunction affecting three classes of integrins. We propose that it is caused by a lesion in an intracellular factor or signaling pathway essential for integrin activation in hematopoietic cells and results in lack of regulation of clustering, an essential component of integrin-mediated adhesion.
We report a woman with an obstetric history ofa stillbirth at 28 weeks, associated with hypertension and severe intrauterine growth restriction and a miscarriage at 9 weeks. She was persistently ...positive for immunolgobulin G (IgG) anticardiolipin antibodies and IgG anti‐Beta‐2‐glycoprotein I (anti‐Beta2GPI) antibodies. She has delivered three healthy babies when managed antenatally with aspirin and low‐molecular‐weight heparin prophylaxis. Genotyping revealed that she was homozygous for the 316 Trp to Ser Beta2GPI polymorphism. Studies examining the binding of her plasma Beta2GPI to purified cardiolipin showed markedly reduced binding in comparison with Beta2GPI in pooled normal plasma.
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