ALBERT

Description: Key Points Normal plasma VWF multimers act as a cofactor in the factor I–mediated cleavage of C3b to iC3b and inhibit complement activation. Large VWF multimers, including ultra-large VWF multimers, do not have factor I cofactor activity and permit complement activation.

Description: Key Points Reduction in ADAMTS13 function and complement dysregulation coexist in a significant number of patients with aHUS. Variations in the ADAMTS13 gene (polymorphisms and rare variants) are partly responsible for the reduced ADAMTS13 function in aHUS.

Description: Abstract 308 Graft-vs-host disease (GVHD) is an alloimmune response after allogeneic hematopoietic stem transplantation (HSCT) mediated by donor T cells against antigen presented by recipient dendritic cells. Our studies with a murine model of GVHD indicate that the complement system regulates the alloimmune response leading to acute GVHD. We used the disparity in MHC class I and II antigens between BALB/c (H-2d) as donors and either wild-type (WT) or complement deficient C57BL/6 (H-2b) as recipients. We found that mice deficient in the central component of the complement system (C3−/−) had significantly lower GVHD-related mortality and morbidity compared to WT recipient mice. Within 8 weeks after BMT, 80% of WT recipient mice and only 25% of C3−/− mice died (p=0.0008, n=20 in each group). While WT mice showed a moderate to severe GVHD in the skin, intestine, liver, lung and kidney, C3−/− mice had mild changes in these organs, reflected in a significantly lower GVHD scores compared to WT mice. Donor T cells proliferation is a critical step in development of GVHD. Therefore, we analyzed donor-derived T cells in C3−/− mice 7 days post-transplant, and found a significantly lower number of CD4+ and CD8+ T cells in spleen, lymph node and Peyer's patches compared to those in WT mice. Complement deficiency not only affected the number of T cells but also their polarization. In the spleen and lymph nodes of C3−/−mice, we found a significantly lower number of IFNg-producing T cells and Th17+IFNg+ cells compared to WT recipients, consistent with a reduced Th1 and Th17+ differentiation of donor T cells in C3−/− recipient mice. Since the interaction between recipient-derived dendritic cells (DCs) and donor T cells is one of the initial events in the pathogenesis of the alloimmune response in GVHD, we analyzed both lymphoid and nonlymphoid DCs in the C3−/− recipient mice. Murine lymphoid DCs are divided into CD8a+ and CD8a− subsets that stimulate Th1 and Th2 cells, respectively. We detected a significantly lower number of CD8a+ (Th1-driven) and higher number of CD8a− (Th2-driven) DCs in the spleen of C3−/− recipient compared to WT mice. Additionally, the number of CD8a+ DCs was significant decreased in lymph node of C3−/− mice. In the nonlymphoid tissues, CD103+ DCs are developmentally and functionally related to the CD8a+ DCs. We studied DCs in the lung, liver, intestine and skin of recipient mice and found a significant reduction in the number of CD103+ DCs in the lung of C3−/− compared to WT recipient mice. Thus, C3 deficiency is associated with a decrease in Th1-driven DCs in both lymphoid (CD8a+) and nonlymphoid organs (CD103+) resulting in a reduced donor Th1 differentiation in C3−/− recipient mice. In the present study, we show for the first time that complement plays a role in GVHD. Our results are consistent with the findings of previous studies showing that C3 production by DCs in allograft is essential for maturation of DCs, effective antigen presentation to alloreactive T cells, and the development of Th1 response. We demonstrated that a similar effect of complement after BMT might be important in regulating Th1-driven DC activation and donor Th1/Th17differentiation, and in determining the severity of GVHD. Our study improves the understanding of the molecular mechanisms of GVHD and provides a rationale for using complement inhibitors as a novel potential therapeutic tool in GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: A significant number of patients with ovarian cancer develop venous thromboembolism that is associated with a worse prognosis. The etiology of an increased frequency of venous thrombosis in cancer patients is not clear, and various hypotheses, including the presence of hyperreactive platelets, have been postulated. Hyperreactive platelets have a lower threshold for aggregation, and hence there is a higher number of degranulated platelets in circulation and a higher concentration of platelet granular contents in plasma. We compared ADP- and collagen-induced platelet aggregation in patients with ovarian cancer to those in patients with benign ovarian tumors. To reduce the effect of confounding factors such as surgery or chemotherapy, all blood samples were collected prior to any surgical interventions or chemotherapy. To detect hyperreactivity of platelets, we used both low and high doses of platelet agonists. To evaluate platelet preactivation at baseline, we measured the plasma concentration of b-thromboglobulin (b-TG) and platelet factor-4 (PF-4) as markers of platelet α granule secretion. All studies were approved by the Institutional Review Boards of the University of Texas, M.D. Anderson Cancer Center. Whole blood samples were collected from 34 patients with ovarian cancer and 19 patients with benign ovarian tumors into sodium citrate anticoagulant and processed within 2 hours after collection. Platelet rich plasma (PRP) was prepared by 15 min of 850 rpm centrifugation at room temperature. Aggregation studies were conducted in a light transmission aggregometer (Bio/Data Corporation), using ADP (at 2mM and 20 mM) and collagen (at 19 mg/ml and 190 mg/ml). Platelet poor plasma (PPP) was prepared by centrifuging PRP samples at 2500 rpm for 20 min at room temperature. All PPP samples were stored at -80ºC and the quantity of b-TG and PF-4 was determined by ELISA. We found that platelets isolated from ovarian cancer patients showed aggregation responses similar to platelets from patients with benign ovarian tumors (Figure 1). There was a statistically significant difference in the high-dose collagen-induced platelet aggregation between cancer and benign tumor samples, with increased aggregation of platelets from patients with benign tumors (79 ± 4.9%) in comparison to aggregation of platelets from cancer platelets (69 ± 6.3%). To investigate preactivation of platelets in cancer patients, we measured b-TG and PF-4 in PPP samples. We did not detect a higher concentration of b-TG and PF-4 in cancer PPP samples (Figure 2). In fact, PPP from cancer patients had a lower concentration of both α granule constituents. In the case of PF-4, the difference was statistically significant (9.8 ± 1.5 ng/ml for cancer patients versus 11.7 ± 1.7 ng/ml for patients with non-malignant tumors). We conclude that platelets from ovarian cancer patients are not hyperreactive and are not degranulated or preactivated. Links between ovarian cancer, venous thromboembolism and platelets may be absent or may involve non-hemostatic platelet functions. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: In resting platelets, the cytoplasmic domains of glycoprotein (Gp) Ibα and β3 integrin link the GpIb-IX-V and αIIbβ3 complex with adapter proteins, cytoskeletal elements and lipid and tyrosine kinases. Biochemical and pharmacological analyses of intact platelets and genetic analyses of recombinant GpIb-IX-V and αIIbβ3 in CHO cells point towards the hypothesis that a series of cytoskeletal proteins form a functional tether connecting the cytoplasmic domains of GpIbα and β3 integrin. To test the hypothesis that dynamic contractility of this tether modulates VWF-induced signaling between GpIb-IX-V and αIIbβ3, we examined how pathological shear stress affects connections between tethering elements and how altering the contractile function of the tether affects VWF-induced platelet adherence and aggregation. We observed in resting platelets that there is no co-immunoprecipitation of GpIbα and αIIbβ3. The large dimeric actin-binding protein filamin A co-immunoprecipitates with both GpIbα and β3 integrin, but its association with αIIbβ3 is inhibited by DNaseI, indicating that it binds indirectly to αIIbβ3 through cytoskeletal connections. These connections were investigated in resting platelets by examining a series of immunoprecipitates (IP) for co-precipitating proteins using immunoblotting (IB). We observed the following IP/IB pairs [(+) designates that the co-precipitation is DNaseI-sensitive]: β3/talin (+); talin/α-actinin (+); talin/vinculin (+); and talin/filamin A (−). We also observed that myosin heavy chain (MHC) and the tyrosine kinase Syk co-immunoprecipitate with αIIbβ3 in resting platelets. When washed platelets in buffer containing calcium (1 mM) and VWF (5 μg/ml) were sheared at 120 dynes/cm2 for 2 minutes, the cytoskeletal linkage separates: there was enhanced filamin A, actin and α-actinin binding to GpIbα, enhanced vinculin binding to α-actinin, enhanced talin binding to αIIbβ3, and both myosin and the tyrosine kinase Syk disassociated from the β3 tail. Inhibiting myosin contractility with the myosin light chain kinase (MLCK) inhibitor ML-9 (1 mM) inhibited the shear-induced association between talin and β3, as well as platelet aggregation in response to 120 dynes/cm2 in the cone plate viscometer and platelet-dependent thrombosis from whole blood onto type I collagen in a parallel plate flow chamber with a shear rate of 500 sec−1 (shear stress of ~ 20 dynes/cm2). The data presented support a model of shear-induced platelet aggregation in which a series of cytoskeletal proteins serve as a mechanotransducing scaffolding linking the cytoplasmic domains of GpIbα and β3 integrin. When GpIb-IX-V engages ligand under shearing forces, the force is transduced from GpIbα to filamin to actin/α-actinin to vinculin to talin to β3, and contractility driven by the activation of αIIbβ3-associated myosin enhances talin binding to β3, thereby effecting a conformation change that creates a ligand-receptive αIIbβ3. Such interactions may be regulated by cytosolic ionized calcium (which effects MLCK activation) and tyrosine kinases (αIIbβ3-associated Syk disassociates from β3 following shear). These results provide evidence that the structural scaffolding connecting GpIb-IX-V with αIIbβ3 localizes signaling elements to functionally important compartments that modulate αIIbβ3 activation and shear-induced platelet aggregation.

Description: The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation. The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above. The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation. RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent. These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.

Description: Background Hematologic toxicity is a common treatment complication of chronic hepatitis C virus (HCV) infection, especially when interferon (IFN) and ribavirin are used. The side effects of treatment are often augmented in cancer patients due to baseline cytopenias. These adverse events often lead to dose reduction or discontinuation of antivirals. Hematopoietic growth factors (GF) and blood transfusions are used to counteract toxicities allowing patients to complete treatment. We aimed to evaluate the incidence and management of hematological toxicity associated with different types of HCV treatment in cancer patients. Methods Medical records of cancer patients treated for HCV infection at MD Anderson Cancer Center between 2009 and 2014 were reviewed. Those seen from 8/2009 to 10/2012 were analyzed retrospectively, whereas those seen from 11/2012 to 7/2014 were prospectively studied. Patients who received combination treatment with peg-IFN and ribavirin (PR), telaprevir or boceprevir plus PR (TBPR), sofosbuvir plus PR (SPR), sofosbuvir with simeprevir (SS) and sofosbuvir with ribavirin (SR) were included in the study. Data regarding treatment interventions (dose reductions and/or discontinuation of antivirals), use of GFs or blood transfusions in the management hematological side effects were analyzed. Categorical variables were analyzed using the χ2 or Fischer's exact test. Results Sixty-five patients were identified (Table). The need for treatment interventions, GFs or blood transfusions was comparable between patients with hematologic malignancies and solid tumors. Seventeen (81%) of the PR group, 13 (93%) of the TBPR group, 6 (67%) of the SPR group, 9 (64%) of the SR group and 0 of the SS group required treatment interventions (p

Description: Platelets have been called ancient granulocytes because they are capable of recognizing and destroying microbial invaders through their release of a variety of anti-microbial proteins. Experimental evidence indicates that this may involve direct platelet-microbial interactions or occur indirectly through platelet-leukocyte interactions, neutrophil histone-platelet interactions and/or platelet-directed neutrophil extracellular trap formation. To test the hypothesis that platelets contribute to innate lung immunity, we examined the clinical course, lung histopathology and pathogen burden of mice -/+ antibody-mediated platelet depletion who were subjected to aerosolized bacteria (Streptococcus pneumoniae or Pseudomonas aeruginosa). Mice (C57BL/6, 10 weeks) were injected by tail vein with a purified rat monoclonal antibody directed against mouse GpIbα (or control antibody) resulting in platelet depletion to ~ 20% at 3 hours that persisted for 48 hours. 30 minutes after GpIbα antibody injection mice were placed into a closed system for 60 minutes where they were exposed to 10 ml nebulized S. pneumoniae (2-6 x 1011 CFU/ml) or P. aeruginosa (1-4 x 1010 CFU/ml). All platelet depleted animals infected with S. pneumoniae died by day 4 after infection, whereas all platelet replete mice survived the infection. Infection with P. aeruginosa resulted in death of all animals, although the platelet replete mice lived longer than the platelet depleted mice. No platelet depleted sham-infected animals died. The lungs of animals were harvested, fixed and prepared for histopathology with hematoxylin and eosin staining; or harvested, homogenized and plated for bacterial CFU measurements. Whole lungs of platelet-depleted and infected mice were congested. Histopathology showed increased red cells in the venules of platelet-depleted S. pneumoniae-infected mice but no difference in air-space hemorrhage in comparison with platelet-depleted sham-infected lungs and platelet-replete S. pneumoniae-infected lungs. Platelet depletion resulted in ~ 5-fold increase in lung S. pneumoniae CFUs and ~ 10-fold increase in P. aeruginosa CFUs. These results indicate that platelets provide defense against pneumonia. The mechanism of this effect is not known. Experiments are ongoing to determine if it is a consequence of platelet-dependent hemostasis, platelet-leukocyte interactions and/or direct platelet-microbial interactions leading to the release of bactericidal compounds. Disclosures Kroll: Boerhinger-Ingelheim: Membership on an entity's Board of Directors or advisory committees; Aplagon Therapeutics: Membership on an entity's Board of Directors or advisory committees.