Impact of Platelet Count on Bleeding in the Setting of Anti-Platelet Therapy

Anti-platelet therapy (APT) is used for secondary prevention of thrombosis. The most commonly prescribed anti-platelet drugs are aspirin and P2Y12 inhibitors, including clopidogrel, prasugrel and ticagrelor. Dual anti-platelet therapy (DAPT) consisting of aspirin and a P2Y12 inhibitor is often used in the first 1-12 months after an initial thrombotic event and has a greater anti-thrombotic effect than single agents, but is also associated with a higher risk of bleeding. Due to this risk of hemorrhage, the appropriate use of DAPT in patients requiring percutaneous coronary intervention (PCI) with baseline or periprocedural thrombocytopenia remains unclear. To study the impact of thrombocytopenia on bleeding with APT, we used intravital imaging in a murine hemostasis model and adoptive platelet transfer to generate mice with specific platelet counts with or without platelet inhibition. To generate experimental mice, we used transgenic mice in which platelets express a chimeric GPIb receptor with the extracellular domain replaced with a domain of the human IL-4R (hIL-4R/GPIb-Tg). Endogenous platelets were depleted by injection of anti-hIL-4R antibody, and the recipient mice were then transfused with wild-type (WT) platelets from donor mice treated, or not, with single or dual APT (aspirin 20 mg/kg; clopidogrel 25 mg/kg) to achieve specific platelet counts ranging from 50,000 to 400,000 platelets/μL. We also compared these mice with WT mice (with normal platelet counts, ~1,200,000 platelets/μL) treated with APT. Platelet inhibition was confirmed prior to performing in vivo experiments. Hemostasis was determined by intravital imaging in our saphenous vein laser injury model, in which a 50 μm injury was induced by laser ablation. Real-time top-down epifluorescence imaging was used to determine time to initial hemostasis, rebleeding events, and platelet and fibrin accumulation. In each mouse, 3-5 injuries were induced at different sites and each injury was visualized for 10 minutes. Following real-time imaging, spinning disk confocal Z-stacks of platelet plugs were obtained for 3D reconstruction to compare platelet plug volume. In untreated WT mice, hemostasis was achieved in ~20 seconds. In WT mice treated with DAPT, initial hemostasis was often rapidly achieved but this was followed by significant rebleeding events. Paradoxically, platelet accumulation was increased in WT + DAPT mice due to extravascular accumulation of platelets which occurred during bleeding. However, in plugs that stabilized, plug volume was reduced in WT + DAPT mice. In hIL-4R/GPIb-Tg mice with reduced platelet counts, untreated platelets were able to form a stable hemostatic plug even at 50,000/μL, although time to hemostasis was slightly prolonged. However, as platelet counts decreased in mice with DAPT-treated platelets, initial hemostasis became more prolonged and many injuries never achieved initial hemostasis. These results suggest that DAPT may not be safe in the setting of severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Ultrasound and Transcriptomics Identify a Differential Impact of Cisplatin and Histone Deacetylation on Tumor Structure and Microenvironment in a Patient-Derived In Vivo Model of Gastric Cancer

Pharmaceutics ◽

10.3390/pharmaceutics13091485 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1485

Author(s):

Aina Venkatasamy ◽

Eric Guerin ◽

Anais Blanchet ◽

Christophe Orvain ◽

Véronique Devignot ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Microenvironment ◽

Real Time ◽

Histone Deacetylase Inhibitors ◽

Structural Changes ◽

Histone Deacetylation ◽

In Vivo Model ◽

Specific Cell ◽

The Impact

The reasons behind the poor efficacy of transition metal-based chemotherapies (e.g., cisplatin) or targeted therapies (e.g., histone deacetylase inhibitors, HDACi) on gastric cancer (GC) remain elusive and recent studies suggested that the tumor microenvironment could contribute to the resistance. Hence, our objective was to gain information on the impact of cisplatin and the pan-HDACi SAHA (suberanilohydroxamic acid) on the tumor substructure and microenvironment of GC, by establishing patient-derived xenografts of GC and a combination of ultrasound, immunohistochemistry, and transcriptomics to analyze. The tumors responded partially to SAHA and cisplatin. An ultrasound gave more accurate tumor measures than a caliper. Importantly, an ultrasound allowed a noninvasive real-time access to the tumor substructure, showing differences between cisplatin and SAHA. These differences were confirmed by immunohistochemistry and transcriptomic analyses of the tumor microenvironment, identifying specific cell type signatures and transcription factor activation. For instance, cisplatin induced an “epithelial cell like” signature while SAHA favored a “mesenchymal cell like” one. Altogether, an ultrasound allowed a precise follow-up of the tumor progression while enabling a noninvasive real-time access to the tumor substructure. Combined with transcriptomics, our results underline the different intra-tumoral structural changes caused by both drugs that impact differently on the tumor microenvironment.

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Differential Effects of HIV Protease Inhibitors on Release and Activation of Platelet-Derived TGF-β1: Potential Association with HIV-Linked CVD

Blood ◽

10.1182/blood-2019-126225 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2341-2341

Author(s):

Kouzbari Karim ◽

Gostynska Sandra ◽

Sonia Elhadad ◽

Dube Pratibha ◽

Jeffrey Laurence ◽

...

Keyword(s):

Protease Inhibitors ◽

Low Dose ◽

Cardiac Fibrosis ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Minor Effect ◽

The Impact ◽

Tgf Β1 ◽

In Vivo Assessment

Combination antiretroviral therapies (cART) have markedly reduced mortality in HIV infection. However, cardiovascular disease (CVD), including heart failure linked to fibrosis, remains a major cause of morbidity and mortality in HIV/cART patients. The magnitude of this risk increases with use of certain protease inhibitors (PI), but the underlying mechanism remains unclear. We showed that the PI ritonavir leads to increased plasma levels of the pro-fibrotic cytokine TGF-β1, cardiac dysfunction, and pathologic cardiac fibrosis in wild-type (wt) C57BL/6 mice. Mice with targeted depletion of platelet TGF-β1 had reduced cardiac fibrosis and partially preserved cardiac function following ritonavir exposure (Laurence, et al. PLoS One 2017;12:e0187185). Several groups have examined the effects of a variety of cART agents on agonist-induced platelet aggregation, but correlations with clinical CVD are weak. Since platelets are a rich source of TGF-β1, we hypothesized that ritonavir and other PIs linked clinically to an increased CVD risk directly activate platelets to release TGF-β1 and activate latent (L)TGF-β1 to initiate signaling for organ fibrosis. We examined the impact of clinically relevant doses of ritonavir, alone and in combination with two other contemporary PIs, atazanavir and darunavir, which are currently used along with low dose ritonavir in so-called PI-boosted cART regimens. We incubated human platelet-rich plasma and washed platelets with PIs alone or in combinations at various doses for 10 min at 37°C in a platelet aggregometer (BioData. Corp). Total and active TGF-β1 levels were measured by ELISA. For in vivo assessment, we treated wt mice with a low dose of ritonavir, as used in PI-boosted cART, and measured the levels of plasma TGF-β1 by ELISA, and TGF-β1 signaling in tissues by immunofluorescence imaging for pSmad2. We found that ritonavir dose-dependently increased total TGF-β1 release from freshly-isolated platelet-rich plasma and washed human platelets. This release was blocked by ceefurin-1 and MK517, potent inhibitors of the ATP binding cassette transporter ABCC4. Darunavir alone did not cause release of TGF-β1, and did not alter significantly ritonavir-induced TGF-β1 release (Figure-1A). Atazanavir alone did induce release of TGF-β1 from platelets and did not affect the extent of such release induced by ritonavir (Figure-1A). Since total TGF-β1 released from platelets must be activated in order to signal, we tested whether these PIs could activate LTGF-β1. Ritonavir alone, in low dose, activated TGF-β1 by 4-5-fold (Fig-1B). Darunavir alone did not activate LTGF-β1, and had only a minor effect on ritonavir-induced TGF-β1 activation (Fig-1B). In marked contrast, while atazanavir also did not activate LTGF-β1, it significantly inhibited ritonavir-induced LTGF-β1 activation (Fig-1B). For in vivo assessment, wt mice were injected daily for 8 weeks with ritonavir, which dose-dependently increased plasma TGF-β1 levels (mean levels with vehicle 2.1 ng/ml; 6.4 ng/ml with 5 mg/kg ritonavir; 8.5 ng/ml with 10 mg/kg ritonavir). Increased TGF-β1 levels correlated with development of pathologic fibrosis and increased phosphorylated Smad signaling in hearts of ritonavir-treated vs. vehicle-treated mice. Clinical correlations with these in vitro and in vivo mouse studies are important. The fact that ritonavir effected both release and activation of platelet TGF-β1 is consistent with its ability to induce cardiac fibrosis and dysfunction in mice, and its association with accelerated CVD in HIV-infected individuals. Our findings that low dose ritonavir in combination with darunavir induced release and activation of platelet TGF-β1, whereas atazanavir blocked TGF-β1 activation, are consistent with the strong association of ritonavir-boosted darunavir, but not ritonavir-boosted atazanavir, with CVD in the setting of HIV (Ryom, et al. Lancet-HIV 2018;5:e291-e300). Future work will examine the effects of other contemporary cART agents, including cobicistat, which is currently replacing ritonavir in many PI-boosted therapies and some integrase-boosted regimens, on TGF-β1 release and activation, for which correlations with clinical CVD are not yet available. Identification of the mechanism of pathologic fibrosis in the heart, and potentially other organs affected by certain cART regimens, such as the kidney, may suggest specific therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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Thrombocytopenia in septicemia: the role of disseminated intravascular coagulation

Blood ◽

10.1182/blood.v56.1.88.88 ◽

1980 ◽

Vol 56 (1) ◽

pp. 88-92 ◽

Cited By ~ 86

Author(s):

PB Neame ◽

JG Kelton ◽

IR Walker ◽

IO Stewart ◽

HL Nossel ◽

...

Keyword(s):

Disseminated Intravascular Coagulation ◽

Gel Chromatography ◽

Severe Thrombocytopenia ◽

Intravascular Coagulation ◽

Platelet Counts ◽

Normal Platelet ◽

Platelet Survival ◽

Possible Cause

Abstract The mechanism of isolated thrombocytopenia in septicemia is unknown, but compensated disseminated intravascular coagulation (DIC) has been suggested as a possible cause. To investigate this possibility, platelet counts and sensitive assays for in vivo thrombin and plasmin generation, including fibrinogen gel chromatography and fibrinopeptide A (FPA) assays, were obtained on 31 septicemic patients. Fifteen of 17 patients with gram-negative septicemia and 8 of 14 patients with gram- positive septicemia had thrombocytopenia. Platelet survival studied demonstrated a decreased platelet survival. In 11 of 12 patients with severe thrombocytopenia (platelet count less than 50,000mul), there was laboratory evidence of intravascular coagulation. In contrast, there was little evidence of intravascular coagulation in 8 of 11 patients with moderate thrombocytopenia (platelet counts 50,000 to less than 150,000/mul) or in 7 of 8 patients with normal platelet counts. This report indicates that while DIC accompanies thrombocytopenia in many patients with severe thrombocytopenia, there is frequently little evidence for intravascular coagulation in patients with moderate thrombocytopenia. It is apparent that factors other than intravascular thrombin must play a role in producing the thrombocytopenia of septicemia.

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Effect of bodyweight on VerifyNow Aspirin platelet function test: a retrospective review

Journal of NeuroInterventional Surgery ◽

10.1136/neurintsurg-2020-016842 ◽

2020 ◽

pp. neurintsurg-2020-016842

Author(s):

Melissa Sandler ◽

Cuong Hoang ◽

Hannah Y Mak ◽

Michael R Levitt

Keyword(s):

Stent Thrombosis ◽

Retrospective Review ◽

Platelet Reactivity ◽

Thrombotic Event ◽

Optimal Dose ◽

Platelet Inhibition ◽

Platelet Function Test ◽

Assay Result ◽

The Mean ◽

The Impact

BackgroundAntiplatelet therapy is used to prevent stent thrombosis in intracranial stents, but the optimal dose of aspirin is unknown. This study sought to determine whether the degree of platelet inhibition with aspirin is affected by bodyweight as observed through a platelet reactivity assay.MethodsThis is a retrospective review of patients who underwent neurovascular stent placement and had a VerifyNow Aspirin assay result. The primary outcome was the correlation between the VerifyNow Aspirin result, bodyweight, and the initial dose of aspirin. Secondary outcomes included the impact of the VerifyNow P2Y12 result and of weight on the incidence of bleeding or a thrombotic event.ResultsOf the 142 included patients, 62.7% weighed ≥70 kg and 88.7% were initiated on aspirin 300–325 mg daily. 83.8% achieved a therapeutic VerifyNow Aspirin result. There was minimal correlation between the VerifyNow Aspirin result, bodyweight, and aspirin dose (R2=0.02). Between patients who weighed <70 kg versus ≥70 kg, there was no difference in the mean aspirin reaction units (ARU) (449 vs 435, p=0.32) or in the incidence of bleeding (28% vs 17.1%, p=0.14) or a thrombotic event (4% vs 5.3%, p=0.59). No patient experienced stent thrombosis and eight patients experienced in-stent stenosis. In a multivariate analysis, only the VerifyNow P2Y12 result predicted the development of either bleeding or a thrombotic event (p<0.01).ConclusionsBodyweight did not influence the likelihood of obtaining a therapeutic VerifyNow Aspirin result. The clinical utility of obtaining VerifyNow Aspirin assays for this patient population is unknown.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Gvhd Severity Is Regulated by the Adoptive Transfer Low Numbers of NKT Cells by a Mechanism Distinct From CD4+CD25+ Regulatory T Cells.

Blood ◽

10.1182/blood.v114.22.229.229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 229-229

Author(s):

Dennis Leveson-Gower ◽

Janelle Olson ◽

Emanuela I Sega ◽

Jeanette Baker ◽

Robert Zeiser ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Adoptive Transfer ◽

Conflicts Of Interest ◽

Serum Levels ◽

Nkt Cells ◽

Donor Type ◽

Ifn Γ ◽

The Impact

Abstract Abstract 229 NKT cells, a subset of which are CD1d reactive, play an important immunoregulatory role in suppressing dysfunctional immune reactions, including graft-versus-host disease (GVHD). To explore the biological activity and mechanism of donor-type NKT in suppression of GVHD, we utilized highly purified (>95%) populations of donor (C57Bl6; H-2b) NKT (DX5+TCR+CD4+) cells adoptively transferred into lethally irradiated recipient (Balb/c; H-2d) animals with T cell depleted bone marrow (TCD-BM). Highly purified (>95%) NKT cells (5.5×105) from luciferase positive (luc+) C57BL/6 mice were infused into lethally irradiated Balb/c recipients with TCD-BM(5×106) from wild-type (WT) C57BL/6 mice, and the animals were monitored by bioluminescence imaging (BLI). By day 4 after transfer, an NKT derived signal was observed in spleen and lymph node (LN) sites, and between days 7 and 10, NKT had also migrated to the skin. Total photons emitted peaked near day 25 after transplantation, followed by a steady decline. To assess the impact of donor-type NKT cells on GVHD induction by conventional CD4+ and CD8+ T cells (Tcon), we co-transferred various doses of highly purified WT NKT at day 0 with TCD-BM, followed by 5×105 luc+Tcon/animal on day 2. As few as 2.5×104 NKT cells significantly improved survival of mice receiving 5×105 Tcon. Animal survival with Tcon only was 20% and for Tcon with NKT cells was 74%(p=0.0023). In contrast to what is observed with CD4+CD25+FoxP3+ regulatory T cells (Treg), the NKT cells did not suppress Tcon proliferation assayed by both in vivo BLI and in a mixed-leukocyte reaction. Analysis of serum cytokines with or without 2.5×104 NKT, following HCT with TCD-BM and Tcon, indicated the addition of NKT cells resulted in elevated levels of INF-γ, IL-5, and IL-6 in serum; significant differences were not observed in serum levels of IL-2, IL-4, IL-10, IL-17, or TNF-α. Intracellular levels of cytokines in Tcon were analyzed from the same groups. At 8 days after HCT, mice receiving NKT had fewer TNFα-positive cells in LNs (CD4: 45% to 27%; CD8 36% to 24%); by day 11, however, TNFαa levels between groups were equivalent. IFN-γ levels, which were high in both NKT treated and untreated groups at day 8 (85%-95%), decreased significantly in NKT treated mice by day 11 (CD4: 40%; CD8: 43%), but were abundant in Tcon only mice (CD4: 78%; CD8: 80%) (p=.0001). No significant changes were found in the intracellular levels of IL-2, IL-4, IL-5, IL-10, or IL-17 of Tcon in the presence or absence of NKT cells. NKT from both IL-4 -/- and IFN-γ -/- mice were less effective at suppressing GVHD than WT NKT, implicating these cytokines in the suppressive mechanism. Finally, we found that NKT do not have a major impact on the graft-versus-tumor effect of Tcon against a luc+ BCL-1 tumor. These studies indicate that NKT persist in vivo upon adoptive transfer and suppress GVHD, even at extremely low cell numbers, which is important given the relative paucity of this cell population. The mechanisms of GVHD suppression appear to be distinct to those of Treg and involve the production of IL-4 and IFN-γ by NKT resulting in a decrease in Tcon, which produce pro-inflamatory cytokines. Disclosures: No relevant conflicts of interest to declare.

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Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Blood ◽

10.1182/blood.v114.22.3622.3622 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3622-3622

Author(s):

Frederick Karl Racke ◽

Maureen E Baird ◽

Rolf Barth ◽

Tianyao Huo ◽

Weilian Yang ◽

...

Keyword(s):

Conflicts Of Interest ◽

Leukemic Cell ◽

The Novel ◽

Drug Induced ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Counts ◽

Pkc Isoforms

Abstract Abstract 3622 Poster Board III-558 Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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A Case of Clopidogrel Induced TTP Recurring After Four Years

Blood ◽

10.1182/blood.v116.21.4678.4678 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4678-4678

Author(s):

Nanda K. Methuku ◽

Abhinav B. Chandra ◽

Anuradha Belur ◽

Lech Dabrowski

Keyword(s):

Platelet Count ◽

Renal Insufficiency ◽

Peripheral Blood ◽

Blood Smear ◽

Reticulocyte Count ◽

Conflicts Of Interest ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Multiple Organs

Abstract Abstract 4678 Case description - A 61 year old woman was started on clopidogrel after having PTCA with stent placement in February 2006. Four weeks after starting clopidogrel she developed thrombocytopenia with platelet nadir of 17,000. Her LDH was 700 IU/L and she was anemic with hemoglobin of 7.4 gm/dl with elevated reticulocyte count. Peripheral blood smear showed schistocytes and diagnosis of TTP secondary to clopidogrel was made. She did not have renal insufficiency. Clopidogrel was discontinued and patient was started on plasmapheresis with recovery of platelet counts. Early attempts in weaning plasmapheresis resulted in drop in platelet count and Rituximab was given to the patient weekly for four weeks. Subsequently, patient was weaned off plasmapheresis. For four years patient was followed periodically with CBC showing platelet counts greater than 250,000. In May 2010, four years after initial event patient was admitted to hospital for abdominal pain and found to have splenic infarcts. Subsequently, she also developed bilateral cerebral infarcts. Platelet count had decreased to less than 100,000. Her LDH was elevated at 419 IU/L. Reticulocyte count was 2.3%. Peripheral blood smear revealed significant number of schistocytes. There was no renal insufficiency or fever. Trans-esophageal echocardiogram (TEE) was done that did not reveal any vegetations. Patient was diagnosed as having recurrence of TTP and started on plasmapheresis with recovery in platelet counts. Pt was also treated with Rituximab. Discussion- We describe a case of TTP initially occurring within weeks of starting clopidogrel. Patient was treated with plasmapheresis and Rituximab and clopidogrel was discontinued. Patient had recurrence after four years as manifested by infarcts in multiple organs, with mild thrombocytopenia, elevated LDH and significant number of schistocytes on peripheral blood smear. It is very uncommon for clopidogrel associated TTP to recur after such a prolonged period of 4 years. Most cases of clopidogrel associated TTP have mild thrombocytopenia. This patient had severe thrombocytopenia on first presentation of TTP but had mild thrombocytopenia on recurrence. This case illustrates the importance of extended follow up and high index of suspicion for TTP as delays in initiation of plasmapheresis has a poor clinical outcome. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Is An Essential Regulator of Late Stage Megakaryocyte Maturation and Platelet Production

Blood ◽

10.1182/blood.v118.21.2358.2358 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2358-2358

Author(s):

Lothar Vassen ◽

Tarik Moroy

Keyword(s):

Bone Marrow ◽

Late Stage ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Pcr Analysis ◽

Severe Thrombocytopenia ◽

Facs Analysis ◽

Platelet Counts ◽

Platelet Release ◽

Deficient Mice

Abstract Abstract 2358 Absence of Gfi1b in mice is embryonically lethal and causes failure to produce functional megakaryocytes and platelets. Thrombopoiesis, the production of platelets by megakaryocytes, is an essential process in hemostasis that needs to be well controlled. Too many platelets can cause thrombosis, too few cause excessive bleeding. How terminal megakaryocyte maturation and platelet release works is incompletely understood but requires many factors such as Fli1, Gata1, MyH9, p45-NFE2, or c-Myc. Expression array analysis of hematopoietic cells from conditionally Gfi1b deficient mice (Gfi1bfl/fl Mx-Cre) revealed an up-regulation of many factors important for megakaryocyte function like Itga2b, Itgb3, CD9, CD41, CD61, PF4 and Ppbp. Gfi1b ablation in adult Gfi1bfl/fl Mx-Cre mice leads to a severe drop in platelet counts to less than 20% of wt mice with an increase in mean platelet volume (MPV) by 40%. However, megakaryocyte numbers rise up to 100 fold over normal levels when Gfi1b is absent. FACS analysis of bone marrow cells of Gfi1b deficient mice showed a higher number of MEPs, a higher proportion of smaller megakaryocytes and an aberrant population of cKithiCD41hiCD9veryhi cells, which are not present in wt animals. Gfi1b−/− megakaryocytes can reach wt size and normal ploidy as shown by FACS analysis and immunofluorescence microscopy. Transmission electron microscopy (TEM) of pIpC induced Gfi1bfl/fl Mx-Cre megakaryocytes revealed an excess number and larger size of so called “demarcation membranes” in unusual parallel layers and a strongly reduced number of dense granula. Strikingly, both intact and fragmented megakaryocytes were frequently found within bone marrow blood vessels in Gfi1b−/− mice. In addition, a high percentage of megakaryocytes were found in a state of disintegration, without signs of proper platelet release. These features are rarely seen in wt mice. It is known that Gfi1b is required for erythropoiesis and Gfi1bfl/fl Mx-Cre mice show signs of anemia and stress erythropoiesis, which might explain high MEP numbers, which could explain the high numbers of megakaryocytes. To better define the function of Gfi1b in late stage megakaryocyte development, we decided to abrogate Gfi1b expression more specifically by using mice that express Cre recombinase under the megakaryocyte specific promoter of the PF4 gene (PF4-Cre). We observed that Gfi1bfl/fl PF4-Cre mice develop a very severe thrombocytopenia reaching only 2% of wt platelet counts in peripheral blood accompanied by an increase of MPV by 80% over wt levels. Most of Gfi1bfl/fl PF4-Cre mice died at 6–8 weeks of age from severe internal bleedings. Megakaryocyte numbers increase in these mice by 5 to 10 fold and they also reach high ploidy, but their morphology is highly disturbed. Gfi1bfl/fl PF4-Cre megakaryocytes contain less cytoplasm, few dense granula organized in a small patch and a lobulated, ring-shaped nucleus localized close to the cell membrane giving the cells an almost “inside-out” appearance and indicating a disturbed cytoskeleton organization. Gfi1bfl/flPF4-Cre mice show a stress induced splenic erythropoiesis and an increase in MEP numbers, probably a consequence of their substantial hemorrhaging owing to the low platelet counts. The high MEPs number might explain the increase in megakaryocytes in these mice compared to wt controls. Immunofluorescence analysis of Gfi1bfl/fl PF4-Cre megakaryocytes compared to wt counterparts showed less expression of van Willebrand factor (vWF), an important regulator of thrombopoiesis. Q-PCR analysis on mRNA from sorted Gfi1bfl/fl PF4-Cre wt megakaryocytes revealed a lower expression of vWF, but higher PF4, very high Mpl and high CCNE1 expression. Our data show that Gfi1b controls the production of platelets from megakaryocytes, but does not affect the maturation of megakaryocytes as such. However, Gfi1b is required to maintain the cellular organelle structure in megakaryocytes and, more specifically is required to control the formation of dense granula and demarcation membrane formation. Gfi1b ablation in megakaryocytes results in a phenotype with high similarities to Gata1-low mice and syndromes involving mutations in the Gata1 target Gpib, the receptor for vWF, causing Bernhard-Soulier Syndrome (BBS). It is thus possible that Gfi1b is another candidate gene involved in megakaryocyte related diseases. Disclosures: No relevant conflicts of interest to declare.

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Release of Procoagulant and Proinflammatory Cell-Derived Microparticles (MP) During Blood Storage: Effects of Storage Time, Leukoreduction, and Residual Platelets,

Blood ◽

10.1182/blood.v118.21.3373.3373 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3373-3373

Author(s):

Wenche Jy ◽

Sherry Shariatmadar ◽

Marco Ricci ◽

Orlando Gomez-Marin ◽

Carlos Bidot ◽

...

Keyword(s):

Inflammatory Markers ◽

Storage Time ◽

Time Course ◽

Conflicts Of Interest ◽

Annexin V ◽

Bioactive Substances ◽

Platelet Counts ◽

The Impact ◽

Stored Blood ◽

Over Time

Abstract Abstract 3373 Introduction Several studies have indicated that transfusion with older blood carries more risk of adverse reactions than transfusion with younger blood, but this remains controversial. It is not clear why older blood may carry increased risks, or what the “safe age” of stored blood is. It is known that multiple bioactive substances are generated from blood during storage, and one or more of these substances may be involved in transfusion-related complications. Among them, MP are a recognized marker of the storage lesion, and their involvement in transfusion-related complications has been postulated. However, questions such as MP species, quantity, biological activity, and factors affecting their release are not well elucidated. The purpose of this study was to quantify MP species and their activity in stored RBC as a function of storage time, and to evaluate the impact of leukoreduction and residual platelets on MP release. Methods (I) MP generation and functional activity Thirty-four bags of packed RBC (16 non-leukoreduced, 18 leukoreduced) of known blood types (A+, B+, AB+, O+) were obtained from the blood bank within 2–4 days of drawing, and then stored at 4°C. Time of receipt was defined as day 0. At days 0, 10, 20, 30, and 40, 40 mL samples were centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for: (1) subtypes of MP by flow cytometry comprising (a) red cell MP (RMP) assessed by CD235a, (b) leukocyte MP (LMP) by CD45, (c) platelet MP (PMP) by CD41, (d) endothelial MP (EMP) by CD144, and (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV); (2) MP-mediated thrombin generation assay (TGA); (3) MP-mediated inflammatory activity by CD 11b expression in neutrophils following incubation with MP. (II) Reconstitution of increasing platelet counts in leukoreduced RBC. To investigate the effect of residual platelets on RMP generation, we mixed a constant amount of RBC with increasing amounts of type-matched platelets (0 to 250,000/μL f.c.) in standard storage bags and measured time-dependent MP release. Results (A) Time-course of MP generation (i) Non-leukoreduced. RMP, PMP and LMP all increased with time, but with different patterns. RMP increased little to day 10 but then rose exponentially, and by day 40 they were 4–6 fold higher than at day 0. PMP counts rose steadily from day 0 and peaked at day 20, being 2–3 fold higher than at day 0. LMP showed no significant change until day 20 when they started to increase, and then increased sharply after day 30, and by day 40 were 1.5–2 fold higher than at day 0. Levels of PMP (days 0 to 20) and RMP (days 20 to 40) correlated with increasing MP-mediated procoagulant and inflammatory markers. (ii) Leukoreduced. Pre-storage leukoreduction decreased RMP generation by 20–40%, completely suppressed PMP and LMP generation, and reduced total MP-mediated procoagulant and inflammatory markers by 40–60%. CBC showed that leukoreduction not only removed >99% WBC but also reduced residual platelets by >95% (from 90 ±30 ×103/μL to 3.5 ±1.3 ×103/μL). This suggests that residual leukocytes and platelets potentiate RMP generation. (B) Effects of residual platelets on RMP generation. To further study the effects of platelets on RMP generation, we mixed known counts of platelets with leukoreduced RBC, and then evaluated RMP generation over time. We found that RMP levels released were proportional to the platelet counts, as were the procoagulant and inflammatory markers. These results show that platelets in stored RBC play a key role in RMP generation. Conclusion Multiple MP types (PMP, LMP, RMP) are released during storage, and their levels increase over time but their patterns of change are different. Procoagulant and inflammatory markers increase in parallel with PMP and RMP. Our data support the hypothesis that age of stored blood could be important in transfusion-related complications, via MP production. Leukoreduction sharply reduces MP generation and procoagulant and inflammatory markers, suggesting that known benefits of leukoreduction may be attributable to reduced MP production. The finding that residual platelets in stored RBC can potentiate RMP generation suggests that minimizing platelets in non-leukoreduced packed cells could reduce the risk of transfusion-related complications. (Supported by NIH grant 1R01HL098031). Disclosures: No relevant conflicts of interest to declare.

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