Studies of alpha-granule proteins in cultured human megakaryocytes.

alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34(+) cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-granule protein in megakaryocyte cultures was contained within the cells (averages: 41-71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage.

HIV antibody screening remains indispensable for ensuring viral safety of blood components despite NAT implementation.

BACKGROUND: The main objective of the implementation of NAT for the screening of blood-borne viruses was to compensate for the failure of serologic assays during the window period. Because this new screening procedure theoretically covers the entire period of infectivity, the necessity for maintaining serologic assays in blood screening strategy could become questionable. STUDY DESIGN AND METHODS: To investigate this issue, a panel of 35 samples has been studied by NAT. These samples had been collected from HIV-1 antibody-positive individuals presenting a persistently low viral RNA load (<400 copies/mL) in the absence of antiviral therapy. All samples were analyzed with the minipool (x8) NAT routinely used in blood bank setting (HIV-1 and HCV assay based on transcription-mediated amplification) and with single-donation testing. RESULTS: The minipool NAT failed to detect the presence of HIV RNA in 15 of the 35 samples (11 remained negative when retested). Single-donation testing gave negative results in 4 samples (3 remained negative when retested). Fourteen of the 18 samples with a viral load greater than 50 copies per mL were positive by minipool NAT versus 6 of the 17 samples with fewer than 50 copies per mL (p = 0.02). CONCLUSION: The results clearly demonstrate that anti-HIV screening should not be withdrawn from biologic qualification procedures of blood donations, even when single NAT is performed.

Pathogenic effects of anti-glycoprotein Ib antibodies on megakaryocytes and platelets.

Antibodies directed against the glycoprotein (GP) Ib have been identified as the potential cause of various platelet disorders: Immune thrombocytopenic purpura (ITP) may be caused by such autoantibodies; Anti-thrombotic drugs targeting GPIb also induce thrombocytopenia. In order to elucidate the potential mechanism(s) of the anti-GPIb effects, we have examined by electron microscopy (EM) the effect of several antibodies directed against GPIb and GPIIb-IIIa on human culture megakaryocytes (MK). Virtually all antibodies to GPIb enhanced the interaction of newly formed platelets with MK when compared to other antibodies. These effects were retrieved when antibodies were tested on platelets. We conclude that antibodies to GPIb can potentially inhibit platelet release by MK, and can also induce homotypic platelet adhesion. These results may have implications in the pathophysiology of thrombocytopenia and platelet recovery in ITP, and shed light on the pathological effect of anti-GPIb antibodies used as antithrombotic drugs.