ALBERT

Description: Activation of platelet 12-lipoxygenase (12-LO) generates large quantities of 12(S)- HETE, an oxidized metabolite of arachidonic acid. For decades, the function of this lipid was unknown. Recently, we demonstrated that platelets undergo oxidative fragmentation when exposed to nanomolar concentrations of 12(S)-HETE (JCI43:973, 2004). 12-LO activation occurs upon binding of human or rabbit anti-platelet GPIIIa to a specific platelet epitope GPIIIa49-66 (Cell106:551, 2001) which is regulated by a specific IgG conformation (JBC283: 3224, 2008). Fragmentation requires activation of the NADPH oxidase pathway downstream of the release of 12(S)-HETE since NADPH oxidase deficient (gp91 phox −/−) platelets fail to respond to exogenous 12(S)-HETE, whereas 12-LO−/− platelets are fully reactive. Since several lipid mediator receptors have been identified, including the leukotriene BLT1 and BLT2 receptors, we reasoned that the stereospecific action of the 12(S)-HETE was also likely mediated by a G-protein coupled receptor (GPCR). Recent data published in an abstract has suggested that some tumor cells might respond to 12(S)- HETE through the orphan GPCR, GPR31 (Guo, et al., IMPaCT, 2007). We searched for evidence of this receptor in platelets and in HUVEC. GPR31 mRNA was found in highly purified platelet and HUVEC preparations by RT-PCR. Further, incubation of gel filtered platelets with [3H]12(S)-HETE demonstrated saturation binding that was 30- fold above background. Binding of the radioligand was inhibited by a 100-fold molar excess of non-radioactive 12(S)-HETE, but was not effected by addition of 12(R)-HETE, a stereoisomer of the 12-LO product (n=3). In two experiments the Kd for 12(S)-HETE binding to platelets was ~20 nM with a Bmax of ~16 fmol/106 platelets. Oxidative platelet fragmentation induced by anti-GPIIIa49-66 Ab is a 12(S)-HETE-dependent process. We now show that this can be inhibited by 13(S)-HODE with an IC50 of ~400 nM (n=3). 13- HODE has been suggested to be a competitive inhibitor of 12(S)-HETE (Pidgeon, et al., Cancer Metastasis Rev26:503, 2007). CHO cells which do not express an endogenous 12-(S)-HETE receptor were transfected with a GPR31-bearing plasmid. [3H]12(S)-HETE binding to GPR31-CHO cells was dose-dependent and saturable while the radioligand did not bind to mock transfected cells. Radioligand binding to the GPR31-CHO cells was inhibited by both 13(S)-HODE as well as cold 12(S)-HETE with an IC50 of

Description: HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell106: 551, 2001; JCI113: 973, 2004). Since HCV-ITP, like HIV-ITP, is associated with circulating immune complexes, we asked whether the complexes could contain platelet fragments induced by oxidative platelet fragmentation and whether HCV-ITP could be induced by molecular mimickry with an HCV peptide. The incidence of Hepatitis-C related ITP varies from 10–40% increasing with severity of liver disease. HIV-ITP is more frequent in drug abusers compared to non-drug abusers (37% vs 16%); and more severe in HIV-drug abusers than non-drug abusers (platelets

Description: Abstract 4802 Endomitosis, the uncoupling of DNA replication with cytokinesis, occurs during the maturation of megakaryocytes (MK) and leads to the MK polyploidization (4N to 128N DNA content). The mechanism that controls the ploidy in MKs is not well understood. We investigated the correlation of cell proliferation rate with MK ploidy in several differentiating megakaryoblastic cell lines. Meg-01, CHRF-288, and K562 cells were treated with a Src kinase inhibitor (3 μm Su6656) for 4 days and the ploidy was assessed by propidium iodide. Proliferation rate was determined by the doubling time of synchronized cells cultured in 10% serum. We found a significant correlation between ploidy and proliferation rate in these cell lines, with the K562 line having both the highest proliferation rate and the percentage of ploidy cells. We then differentiated the megakaryocyte cell line L8057 cells by thrombopoietin using various concentrations of serum (5%, 10%, and 20%) to control proliferation. The proliferation rate of L8057 cells varied with serum concentration and was also highly correlated the percentage of ploidy cells (23%, 32.8%, and 40.9% respectively). Thus, our data suggest that factors control the cell proliferation rate may contribute to the polyploidization of megakaryocyte. Disclosures: No relevant conflicts of interest to declare.

Description: Patients with HIV related thrombocytopenia (HIV-ITP) have a unique Ab against GPIIIa49-66 (Ab) which induces complement-independent, Fc-independent, peroxide-induced lysis of platelets (Cell, 106:551, 2001). This Ab correlates inversely with platelet count and induces oxidative-fragmentation of human and mouse platelets, and thrombocytopenia in mice. Oxidation is induced by H2O2 generated from platelet NADPH oxidase activated with the platelet 12-LO product, 12(S)-HETE (JCI 113: 973, 2004). The substrate for 12-LO is arachidonic acid (AA), produced by PLA2. Similar oxidative fragmentation is also induced by the Ca++ionophore A23187 as well as phorbol myristate acetate (PMA) (Blood 102:126A, 2003). Most patients with classic autoimmune thrombocytopenia (AITP) as well as HIV-ITP respond dramatically to treatment with steroids. The effect of Dex in AITP requires opsonized platelets interacting with Fc macrophage receptors, while Ab-induced oxidative fragmentation of platelets does not. We therefore investigated possible mechanisms of Dex inhibition of oxidative platelet fragmentation in gel-filtered human platelets (measured by flow cytometry and DCFH oxidation) induced by Ab, A23187 or PMA over 4 hr at 37 C. Dex inhibited both oxidation and platelet fragmentation ( IC 50 0.8uM, range 3.2uM to 0.2uM), n=5. Regardless of the stimulus, 12(S)-HETE production was completely inhibited during the first 60 min of Dex incubation with 2–3 fold inhibition at 60–240 minutes. Similar Dex inhibition was noted when exogenous AA was added confirming a reduction of 12-LO activity, n=4, p

Description: Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the β3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti–GPIIIa49-66–induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated.

Description: Patients with HIV-1 immune-related thrombocytopenia (HIV-1–ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1–seropositive drug abusers are more prone to develop immune thrombocytopenia than non–drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non–drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti–GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r2 = 0.7, P 〈 .01). Ab raised against peptide PHC09 in GPIIIa−/− mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti–GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P 〈 .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.

Description: Anti-platelet GPIII49–66 Ab obtained from HIV-1-ITP patients (or raised in rabbits) induces platelet oxidation, fragmentation and death by activating platelet 12-lipoxygenase (generating 12(S)-HETE) and NADPH-oxidase with exposure of membrane fragment phosphatidyl serine and thrombin-generating capacity (Cell106:551, 2001; JCI113: 973, 2004). Our recent studies reveal that activation of oxidative platelet death requires classic Ca++ flux (fura-2, AM) (completely inhibited by 100uM EGTA or 10uM BAPTA), and is associated with mild GPIIIa activation (PAC Ab binding) and prominent P-selectin release (n=4). However, robust activation of oxidative fragmentation/death is induced in the presence of 1uM PGE1, 10uM dibutyrl cyclic AMP (n=6) and occurs in Gαq KO mouse platelets (all conditions which inhibit ADP, collagen or thrombin-induced platelet activation). We have also observed that platelet oxidation/fragmentation can be induced independently of anti-GPIIIa49–66 by 10mM A23187, a Ca++ ionophore or 0.4uM PMA, a PKC activator. Both A23187 and PMA induce oxidation of platelets loaded with the oxidative fluorochrome, DCFH. Their reactivity is inhibited by the oxidation scavengers catalase (H2O2) and DPI (inhibitor of NADPH oxidase) and is absent in NADPH oxidase gp91phox(−/−) KO as well as 12-LO(−/−) KO mouse platelets (n=4). Thus, anti-GPIIIa49–66 could be inducing the intracellular effects of ionophore and PMA. We next looked for a possible physiologic mechanism. Platelet GPIIIa49–66 was panned with a phage-peptide display library. Twenty 7-mer peptide clones were found which reacted with GPIIIa49–66. One of these peptides (VHCVQLY) had 70% homology with ADAMTS-18, a disintegrin and metalloprotease with thrombospondin (TSP)-like motifs, constitutively secreted by endothelial cells. An 18-mer peptide of ADAMTS-18 was therefore synthesized from the C-terminal TSP motif and conjugated to biotin, Bio-VQTRSVHCVQQGRPSSSC-OH. The peptide alone had no effect on platelet oxidation/fragmentation. However, an anti-biotin Ab employed to cluster the peptide did induce oxidation/fragmentation (n=6). Recombinant ADAMTS 18 was then made with the expression vector pBudCE4.1 in 293T cells. It induced platelet 12(S)-HETE and oxidation/fragmentation in an identical kinetic fashion as anti-GPIIIa49–66 Ab. Both expressed rADAMTS-18 and HUVEC conditioned media ADAMTS-18 could be activated by thrombin (0.5 u/ml and then neutralized with hirudin) with optimum effect at 1 hr (n=4). HUVEC ADAMTS-18 was inactive in the absence of thrombin. ADAMTS-18 induced oxidation/fragmentation could be inhibited ~50% by an scFV Ab raised against the ADAMTS-18 (18-mer) peptide as well as GPIIIa49–66 peptide, as well as RGDS (GPIIIa ligand binding site) (n=7). Both peptides GPIII49–66 and RGDS were synergistic (~75% inhibited) when combined at optimum individualized concentration, suggesting 2 binding sites on platelet GPIIIa. Thus a mechanism is proposed for platelet thrombus clearance, induced by platelet membrane oxidative fragmentation leading to thrombin generation and activation of constitutively secreted endothelial cell ADAMTS-18.

Description: Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by a clonal population of hematopoietic stem cells with an acquired somatic mutation in the PIG-A gene, giving rise to populations of circulating mature cells that are unable to synthesize glycosylphosphatidylinositol (GPI). The disease is most readily diagnosed by flow cytometry analysis of red blood cells, using antibodies specific for the GPI-linked protein CD59, or analysis of granulocytes, using antibodies specific for the GPI-linked protein CD24, along with the FLAER reagent, a fluorescent protein that binds to the GPI structure and which is detected only on the surface of GPI (+) cells. However, other mature blood lineages can be derived from the PNH clone. Notably, thrombosis is a major life threatening complication of PNH and may be triggered by complement activation on platelets that belong to the GPI-negative stem cell clone. The PNH clone size generally predicts thrombosis, but sometimes the proportion of PNH red cells and granulocytes are highly discordant, in which case there might be a role for the determination of the proportion of PNH platelets. Historically, flow cytometry analysis of platelets in patients with PNH has been technically difficult. Here is described a method to do this that avoids technical challenges by using aspirin and sepharose gel filtration of platelets to prevent their activation as well as simultaneous determination of CD59 expression and uptake of the FLAER reagent. Red cells were analyzed based on CD59 expression and granulocytes based on CD24 and FLAER. We analyzed blood samples from 48 patients with PNH and or AA/PNH who provided informed consent, 16 of whom had a prior history of thrombosis. To separate platelet rich plasma (PRP), whole blood collected in EDTA tubes was centrifuged at 200g for 7 minutes at room temperature with the brake turned off. After this step, there was no further centrifugation or vortexing of the platelets. A solution of aspirin was made up immediately prior to use and was added to the PRP at a final concentration of 0.5mMolar. Aspirinated PRP was then loaded on top of a sepharose-2B column using Tyrode's buffer. The platelet-rich turbid drops were collected, to isolate platelets from red cells and coagulation proteins. 50 ul of platelet rich buffer was then incubated with FLAER-Alexa-488 (Pinewood, 1:20 dilution) and CD59-PE (Serotec, 1:10 dilution) in the dark for 30' at room temperature. To prevent doublet events from confounding the analysis, the platelet suspension was diluted 1:200 in Hanks with 0.1% BSA. The sample was passed through a 35 uM Falcon cell strainer, and platelets were identified by forward/side scatter acquired on a log-log scale on a BD Facscan. The median proportion of PNH red cells, granulocytes and platelets was 24%, 86%, 76% respectively in the group without a history of thrombosis and 23% ,82%, and 65% in the group with a history of thrombosis. The proportion of PNH platelets was highly correlated with the proportion of PNH granulocytes (r=0.84). In two patients with almost undetectable PNH red cells and over 90% PNH granulocytes, the proportion of PNH platelets was over 90%; both were on prophylaxis and neither had thrombosis. It is predicted that this technique may be useful for determining thrombosis risk, particularly when the results from the analysis of rbc's and granulocytes are discordant. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2255 Background: Several studies have demonstrated a significant association between platelet plasma markers (soluble CD40 ligand [sCD40L] and soluble p selectin [sPselectin]) and in-vivo platelet activation and cardiovascular events. However, the reliability and pre-analytical variation of these assays remain uncertain. We therefore sought to investigate the reproducibility of these assays, type of collection (plasma or sera), correlation between sCD40L and sPselectin, and the effect of aspirin. Methods: Following an overnight fast, 40 healthy volunteers had early morning phlebotomy during 4 consecutive weeks. Subjects took aspirin 81mg daily x7 days between weeks 3 and 4. Platelet poor plasma was obtained via centrifugation at 2500 × g for ten minutes within 15 minutes of phlebotomy while serum samples were similarly processed at 30 minutes after phlebotomy. Samples were aliquoted and stored at −80°C no more than five minutes after completion of centrifugation. Concentrations of sPselectin and sCD40L were determined in plasma and serum by quantitative enzyme linked immunosorbent assay (Bender MedSystems: BMS219 & BMS293, respectively). Reproducibility over time of levels of sPselectin and sCD40L was assessed by coefficient of variation (CV). Correlation was assessed using Pearson r statistic. The difference between levels pre and post aspirin was measured with Wilcoxon Signed- Rank test. Data is presented as median [interquartile range]. Results: Soluble CD40L measurements were reproducible over time in plasma (Week 1: 0.72 ng/mL [0.35–1.63], Week 2: 0.72 [0.36–1.69], Week 3: 0.66 [0.34– 1.7]; CV: 9.6 %) and serum (Week 1: 2.43 [2.12– 2.99], Week 2: 2.26 [1.8– 2.98], Week 3: 2.44 [1.75– 3.05]; CV 8%). Soluble P-selectin measurements were also reproducible over time in plasma (Week 1: 58.9 ng/mL [46.2–70.8], Week 2: 55.5 [45.4–68.6], Week 3: 52.6 [43.8–62.8]; CV: 9.4%) and serum (Week 1: 116 ng/mL [94.6–145.5], Week 2: 113.6 [90.7–149.6], Week 3: 111.9 [94.1–148.8]; CV: 6%). Measurement of sCD40L in plasma correlated with levels in serum before aspirin (r=0.88, p〈 0.0001) and after aspirin (r=0.87, p〈 0.0001) as did levels of sPselectin in plasma correlate with levels in serum before aspirin (r=0.66, p〈 0.0001) and after aspirin (r=0.54, p= 0.0004). There was no significant correlation between sCD40L and sPselectin before (r=-0.06, p=0.704) or after aspirin (r=-0.0956, p=0.573) in plasma or in sera (before aspirin, (r= 0.03, p=0.853) or after aspirin (r=0.11, p=0.482)). After 1-week of aspirin 81mg/day, there was a reduction in sCD40L in serum (2.43 ng/mL vs. 2.07; p

Description: Abstract 1434 We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures: No relevant conflicts of interest to declare.