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Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) degrades the purines ATP and ADP that are key regulators of inflammation and clotting. We hypothesized that NTPDase1 polymorphisms exist and that they regulate this pathway. We sequenced the ENTPD1 gene (encoding NTPDase1) in 216 subjects then assessed genotypes in 2 cohorts comprising 2213 humans to identify ENTPD1 polymorphisms associated with venous thromboembolism (VTE). The G allele of the intron 1 polymorphism rs3176891 was more common in VTE vs. controls (odds ratio 1.26–1.9); it did not affect RNA splicing, but it was in strong linkage disequilibrium with the G allele of the promoter polymorphism rs3814159, which increased transcriptional activity by 8-fold. Oligonucleotides containing the G allele of this promoter region bound nuclear extracts more avidly. Carriers of rs3176891 G had endothelial cells with increased NTPDase1 activity and protein expression, and had platelets with enhanced aggregation. Thus, the G allele of rs3176891 marks a haplotype associated with increased clotting and platelet aggregation attributable to a promoter variant associated with increased transcription, expression, and activity of NTPDase1. We term this gain-of-function phenotype observed with rs3814159 G “CD39 Denver.”—Maloney, J. P., Branchford, B. R., Brodsky, G. L., Cosmic, M. S., Calabrese, D. W., Aquilante, C. L., Maloney, K. W., Gonzalez, J. R., Zhang, W., Moreau, K. L., Wiggins, K. L., Smith, N. L., Broeckel, U., Di Paola, J. The ENTPD1 promoter polymorphism −860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated “human only” (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6 months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available. Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry. Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or “heart failure” gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n=21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.

Hereditary spastic paraplegia (HSP) type 2 is a proteolipid protein (PLP1)-related genetic disorder that is characterized by dysmyelination of the central nervous system resulting primarily in limb spasticity, cognitive impairment, nystagmus, and spastic urinary bladder of varying severity. Previously reported PLP1 mutations include duplications, point mutations, or whole gene deletions with a continuum of phenotypes ranging from severe Pelizaeus–Merzbacher disease (PMD) to uncomplicated HSP type 2. In this manuscript we report a novel PLP1 missense mutation (c.88G>C) in a family from Argentina. This mutation is in a highly conserved transmembrane domain of PLP1 and the mutant protein was found to be retained in the endoplasmic reticulum when expressed in vitro. Due to the variable expressivity that characterizes these disorders our report contributes to the knowledge of genotype–phenotype correlations of PLP1-related disorders.

Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine. Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays. Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment. Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. Disclosures Kastelowitz: University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

Cystathionine beta-synthase (CBS) deficient homocystinuria (HCU) is an inherited metabolic defect that if untreated, typically results in cognitive impairment, connective tissue disturbances, atherosclerosis and thromboembolic disease. In recent years, chronic inappropriate expression of the inflammatory response has emerged as a major driving force of both thrombosis and atherosclerotic lesion development. We report here a characterization of the abnormalities in cytokine expression induced in both a mouse model of HCU and human subjects with the disease in the presence and absence of homocysteine lowering therapy. HCU mice exhibited highly significant induction of the pro-inflammatory cytokines Il-1alpha, Il-1beta and TNF-alpha. Similarly, in untreated/poorly compliant human subjects with HCU we observed constitutive induction of multiple pro-inflammatory cytokines (IL-1alpha, IL-6, TNF-alpha, Il-17 and IL-12(p70)) and chemotactic chemokines (fractalkine, MIP-1alpha and MIP-1beta) compared to normal controls. These HCU patients also exhibited significant induction of IL-9, TGF-alpha and G-CSF. The expression levels of anti-inflammatory cytokines were unaffected in both HCU mice and human subjects with the disease. In the human subjects, homocysteine lowering therapy was associated with either normalization or significant reduction of all of the pro-inflammatory cytokines and chemokines investigated. We conclude that HCU is a disease of chronic inflammation and that aberrant cytokine expression has the potential to contribute to multiple aspects of pathogenesis. Our findings indicate that anti-inflammatory strategies could serve as a useful adjuvant therapy for this disease.

Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells.

Prelamin A processing is unique amongst mammalian proteins and results in the production of a farnesylated and carboxymethylated peptide. We examined the effect of pathogenic LMNA mutations on prelamin A processing, and of the covalently modified peptide on cardiac and skeletal myoblast differentiation. Here we report a mutation associated with dilated cardiomyopathy prevents prelamin A peptide production. In addition, topical application of the covalently modified C-terminal peptide to proliferating skeletal and cardiac myoblasts induced myotube and striated tissue formation, respectively. Western blot analysis revealed that skeletal and cardiac myoblasts are the first cell lines examined to contain unprocessed prelamin A, and immunostaining of peptide-treated cells revealed a previously unidentified role for prelamin A in cytoskeleton formation and intercellular organization. These results demonstrate a direct role for prelamin A in myoblast differentiation and indicate the prelamin A peptide may have therapeutic potential.

Objectives We examined the prevalence, genotype-phenotype correlation, and natural history of lamin A/C gene (LMNA) mutations in subjects with dilated cardiomyopathy (DCM). Background Mutations in LMNAhave been found in patients with DCM with familial conduction defects and muscular dystrophy, but the clinical spectrum, prognosis, and clinical relevance of laminopathiesin DCM are unknown. Methods A cohort of 49 nuclear families, 40 with familial DCM and 9 with sporadic DCM (269 subjects, 105 affected), was screened for mutations in LMNAusing denaturing high-performance liquid chromatography and sequence analysis. Bivariate analysis of clinical predictors of LMNAmutation carrier status and Kaplan-Meier survival analysis were performed. Results Mutations in LMNAwere detected in four families (8%), three with familial (R89L, 959delT, R377H) and one with sporadic DCM (S573L). There was significant phenotypic variability, but the presence of skeletal muscle involvement (p < 0.001), supraventricular arrhythmia (p = 0.003), conduction defects (p = 0.01), and “mildly” DCM (p = 0.006) were predictors of LMNAmutations. The LMNAmutation carriers had a significantly poorer cumulative survival compared with non-carrier DCM patients: event-free survival at the age of 45 years was 31% versus 75% in non-carriers. Conclusions Mutations in LMNAcause a severe and progressive DCM in a relevant proportion of patients. Mutation screening should be considered in patients with DCM, in particular when clinical predictors of LMNAmutation are present, regardless of family history.

Background Growth Arrest Specific gene 6 (Gas6) is a ligand for the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases found on the surface of platelets. Previous studies have shown that stimulation of these receptors results in amplification of platelet activation and thrombus stabilization via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, leading to phosphorylation of the β3 integrin. Previous work (from our lab and others) demonstrated that inhibition of the Gas6/TAM pathway results in impaired platelet aggregation, reduced aggregate stability, and decreased platelet spreading. Additionally, knockout mice deficient in the receptor or ligand are protected from venous and arterial thrombosis, but retain normal tail bleeding times. Here, we describe development and characterization of novel Mer-selective small molecule inhibitors (SMIs) for thrombosis applications. Objectives To determine if Mer-selective SMIs can inhibit platelet aggregation and protect mice from thrombosis using in vitro and in vivo models Methods We used aggregometry and in vivo murine models of arterial and venous thrombosis to compare two Mer-selective SMIs (UNC Mer TKI1 and UNC Mer TKI2) and determine the most effective inhibitor of platelet aggregation and thrombus formation. The inhibitory effect of two doses (1µM and 5 µM) of the compounds were determined using standard light-transmission aggregometry after a 30 minute incubation with washed human platelets at 37 ¢ªC and compared to platelets treated with vehicle control or with a TKI control (UNC TKI Null), a SMI with similar structure but minimal anti-TAM activity. Both collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury models were used to determine effects on thrombosis mediated by UNC TKIs. Wild type C57Bl/6 mice were treated with one of the two inhibitors and compared to mice treated with vehicle control. Mean values +/- SEM are shown and statistical significance (p Results UNC Mer TKI1 exhibited more potent inhibition of platelet aggregation in vitro relative to UNC Mer TKI2, although both compounds mediated dose-dependent effects. At a concentration of 1uM, the maximum percent aggregation in UNC Mer TKI1-treated samples (n=7) was significantly greater than samples treated with UNC TKI Null (n=7), 20% DMSO vehicle (n=7), or UNC TKI2 (n=7), with mean values of 69 +/- 2.2%, 76.7 +/-1.8% (p UNC Mer TKIs also mediated protection from thrombus formation in mice. Following FeCl3 injury to the carotid artery, vehicle-treated mice (n=11) developed stable vessel occlusions with a mean time of 6.77 +/- 0.25 min. In contrast, stable occlusion occurred at a mean time of 46.6 +/- 7.72 min (n=9, p=0.001) for UNC Mer TKI1-treated mice. Survival times following venous injection of collagen and epinephrine were also significantly increased in mice treated with either UNC Mer TKI relative to the UNC TKI Null or vehicle controls. Mice pre-treated with UNC Mer TKI1 (n=9, p=0.04 compared to vehicle alone) or UNC Mer TKI2 (n=9, p=0.03 compared to vehicle alone) survived for 19.84 +/- 4.4 and 21.25 +/- 4.65 minutes, respectively. In contrast, mice given UNC TKI Null (n=3) or vehicle (n=21), only survived for 3.21 +/- 2.4 min and 3.09 +/- 0.22 minutes, respectively. Conclusion UNC Mer TKIs mediate dose-dependent inhibition of platelet aggregation and protect mice from arterial and venous thrombosis. Their pronounced activity compared to an inactive scaffold protein with minimal anti-TAM activity suggest that Gas6/TAM pathway inhibition is the mechanism of action for these novel compounds. UNC Mer TKI1 has more potent anti-thrombotic properties than UNC Mer TKI2. Disclosures: Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Liu:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Wang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.

Gray platelet syndrome (GPS) is a rare autosomal recessive bleeding disorder characterized by thrombocytopenia, large platelets, and deficient alpha granules in platelets and megakaryocytes. The genetic cause of GPS was recently elucidated by our lab and others, and deleterious mutations were found in the gene NBEAL2 in several affected individuals. NBEAL2 stands for Neurobeachin-like 2 and is a large (30kb, 54 exon) gene on human chromosome 3p21.31. NBEAL2 is a member of a family of proteins that contain a BEACH (Beige and Chediak Higashi) domain which is a highly conserved domain that has been associated with protein functions such as vesicular trafficking, membrane dynamics, and receptor signaling. Very little is known about NBEAL2 other than its involvement with GPS. The relative expression of the NBEAL2 transcript, both in tissues and in subcellular compartments, is not known. It has previously been published that NBEAL2 protein coding isoforms are present in a variety of human tissues, but the abundance of NBEAL2 transcript in each tissue is not known. Furthermore, there is currently no available antibody that recognizes the 302 kDa NBEAL2 protein to validate that mRNA presence translates to protein expression. We have examined the relative transcript abundance of NBEAL2 in a human cDNA library and have validated a novel NBEAL2 antibody in a human megakaryocytic cell line (Dami) and human platelets. We obtained a human mRNA tissue library from Invitrogen which was reverse transcribed to cDNA. Tissues analyzed include bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, bone marrow, peripheral leukocytes, CD33+, and CD36+. To investigate relative transcript abundance, we used a Taqman qPCR probe designed to identify all six protein coding isoforms, as determined by Ensembl (http://uswest.ensembl.org). Highest NBEAL2 expression was seen in CD33+ cells, which was 54.3 fold higher than the tissue with lowest expression (skeletal muscle). Relatively high expression was also seen in the peripheral leukocytes, bone marrow, lung, esophagus, and cervix. Alternatively, NBEAL2 expression was low in the brain, despite the homology to the Neurobeachin (NBEA) brain specific protein. While high transcript abundance may infer function, relative protein expression is necessary to validate these findings. Therefore, we designed a novel NBEAL2 antibody against a 14 amino acid peptide (SLEPRRPEEAGAEVC) encoded by exon 1 of NBEAL2 that is 100% conserved between human and mouse. Western blot characterization of this antibody showed the expected approximately 300 kDa band in both soluble and insoluble Dami lysates and human platelet rich plasma lysates. Furthermore, the NBEAL2 antibody was also used for immunofluorescence of Dami cells to determine the approximate subcellular localization of the protein. Preliminary results suggest that NBEAL2 is localized to the cytoplasm, a finding that is consistent with a subcellular localization prediction program (Euk-mPLoc 2.0). In conclusion, we have determined the relative abundance of NBEAL2 transcript in several human tissues, and have begun to characterize a novel antibody against NBEAL2 using the human megakaryocytic Dami cell line and human platelets. Ongoing studies with this novel tool in the Nbeal2 knockout mouse model will likely provide new information about this elusive protein. Disclosures: Di Paola: CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.

Background ADP activates platelets through P2Y1 and P2Y12 receptors. Various ADP/P2Y inhibitors are used clinically for arterial thrombosis prophylaxis. However, these agents exhibit inter-individual response variability and bleeding complications. A more recently described platelet activation signaling pathway is activated by Growth Arrest Specific gene 6 (Gas6), a ligand for the Tyro3/Axl/Mer (TAM) family of platelet surface receptor tyrosine kinases. Previous have shown that inhibiting this pathway decreases platelet activation responses and β3 integrin-mediated thrombus stabilization, and protects mice from arterial and venous thrombosis, without evidence of significant bleeding side effects. Here, we investigate the effects of a novel Mer-selective small molecule inhibitor (SMI) on platelet aggregation and murine models of induced thrombosis relative to ADP/P2Y pathway antagonists. Objectives We hypothesized that inhibition of the Gas6/TAM pathway with a novel SMI would decrease platelet aggregation and thrombosis, comparable to that seen with known ADP inhibitors. Additionally, we examined the effect of the combination of these 2 inhibitor types on platelet aggregation. Methods We compared the inhibitory effect of 2 known ADP inhibitors (1uM MRS2179 administered concurrently with 1uM 2-MeSAMP) to 1uM of a novel Mer-selective SMI (UNC Mer TKI), using standard light-transmission aggregometry with washed human platelets (30-minute incubation at 37 ¢ªC) and two murine thrombosis models (collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury). Thrombosis studies were performed using WT C57BL/6 mice treated with either UNC Mer TKI or ADP/P2Y inhibitors compared to mice treated with vehicle only. Mean values +/- SEM are shown and statistical significance (p Results ADP/P2Y antagonists and Gas6/TAM inhibitor both mediate protection from thrombosis in mice relative to vehicle-treated controls. Following FeCl3 –induced carotid artery injury, control mice (n=11) experienced stable vessel occlusion at a mean time of 6.77 +/- 0.25 minutes. In contrast, stable occlusion occurred at 46.6 +/- 7.72 minutes (n=9, p=0.001) in mice treated with UNC Mer TKI, and 18.74 +/- 4.35 minutes (n=3, p Survival times following venous injection of collagen and epinephrine significantly differed between mice treated with ADP/P2Y antagonists or UNC Mer TKI, compared to vehicle control. Mice pre-treated with UNC Mer TKI (n=9, p=0.04) or ADP/P2Y inhibitors (n=5, p Both the ADP/P2Y antagonists and UNC Mer TKI also inhibit platelet aggregation. At 1uM doses, the maximum percent aggregation in UNC Mer TKI-treated samples (n=7) differed significantly from samples treated with vehicle alone, with mean values of 69 +/- 2.2% (p=0.04), 77 +/- 1.8% (n=7), and 76.9 +/- 2.1% (n=7), respectively. 1uM ADP/P2Y inhibitor-treated samples (n=7) exhibited a mean maximum aggregation of 62 +/- 5.2% (p=0.008), and the combination of ADP/P2Y inhibitors and UNC TAM TKI had a maximum percent aggregation of 31.3 +/- 7.7% (n=7, p=0.001). The Chou-Talalay Combination Index (a quantitative estimation of the effect of combined inhibitors) was 0.78, indicating a synergistic, rather than additive, effect. Similarly, using the Bliss additivity equation, 38.1 +/- 7.9% inhibition of aggregation was predicted for an additive interaction, but the actual % observed in samples treated with UNC Mer TKI combined with ADP/P2Y inhibitors was 64.3 +/- 8.9%, a statistically significant difference (p = 0.02), suggesting a synergistic effect of the combination therapy. Conclusion A novel Mer-selective SMI mediated inhibition of platelet aggregation and protection from arterial and venous thrombosis in a manner comparable to that seen with known ADP/P2Y inhibitors. Additionally, the two drugs mediate synergistic inhibition of platelet aggregation when used in combination. This observation suggests that combination therapies consisting of a Mer-inhibitor and an ADP/P2Y inhibitor could allow for dose reduction of one or both agents, thereby decreasing off-target effects and/or bleeding complications. Disclosures: Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.

Purines are critical for energy metabolism, cell signalling and cell reproduction. Nevertheless, little is known about the regulation of this essential biochemical pathway during mammalian development. In humans, the second, third and fifth steps of de novo purine biosynthesis are catalyzed by a trifunctional protein with glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART) enzymatic activities. The gene encoding this trifunctional protein is located on chromosome 21. The enzyme catalyzing the intervening fourth step of de novo purine biosynthesis, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17. To investigate the regulation of these proteins, we have generated monoclonal and/or polyclonal antibodies specific to each of these enzymatic domains. Using these antibodies on western blots of Chinese hamster ovary (CHO) cells transfected with the human GARS-AIRS-GART gene, we show that this gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa. This carboxy-truncated human GARS protein is produced by alternative splicing resulting in the use of a polyadenylation site in the intron between the terminal GARS and the first AIRS exons. The expression of both the GARS and GARS-AIRS-GART proteins are regulated during development of the human cerebellum, while the expression of FGARAT appears to be constitutive. All three proteins are expressed at high levels during normal prenatal cerebellum development while the GARS and GARS-AIRS-GART proteins become undetectable in this tissue shortly after birth. In contrast, the GARS and GARS-AIRS-GART proteins continue to be expressed during the postnatal development of the cerebellum in individuals with Down syndrome.

Abstract 1179 Background Previous studies by our laboratory have demonstrated changes in apheresis collected, PLT concentrates during storage that are enhanced by Mirasol®PRT treatment. These include increased expression of P-selectin on PLTs and increased formation of neutrophil (PMN)/PLT aggregates. To explore the effect of these changes in a microvascular environment, we measured PLT adherence in a microfluidic chamber coated with collagen. Methods Three, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was treated with Mirasol PRT and both were stored under standard conditions. Aliquots were sterilely removed from products during storage. To 1000 μl of fresh, ABO type specific, citrate anticoagulated whole blood was added 60 μl of apheresis platelet concentrate with or without PRT treatment on day 1 or 7 of storage and incubated for 5 min at 37C. The sample was recalcified with 20 mM CaCl2 and then pulled through the inlet of a microfluidic chamber at a flow sheer rate of 100 s−1. The flow chamber was composed of two parts, a glass slide patterned with collagen and a series of four polydimethylsiloxane microfluidic chambers per inlet. Stored PLTs were labeled with fluorescently labeled anti-CD41 to distinguish from PLTs of the whole blood. The chamber was mounted on an inverted fluorescence microscope, and video microscopic images sampled on observation field every 7 sec over 7 min. The number of fluorescent cells (stored PLTs) per field and the total area covered by all PLTs were determined at 7 min. Results The area, expressed as % of total field, covered by all of the PLTs in the sample was not different for whole blood alone (29 ± 3); Day 1 untreated (23 ± 9); Day 1 PRT treated (26 ± 8); Day 7 untreated (21 ± 5); and Day 7 treated (25 ± 9) PLTs. In contrast, there was an increase in the number of treated apheresis PLTs at Day 1 compared to untreated apheresis platelets binding to the chamber (2.26 ± 0.61 untreated, vs. 5.45 ± 1.45 treated, numbers are mean ± SEM for numbers of cells adhered normalized to the PLT count in the product, significant, p=0.0229). There was also an increase in binding of treated compared to untreated PLTs at Day 7 (2.43 ± 0.47 untreated, vs. 4.42 ± 1.35 treated). However, the results for untreated PLTs on Day 1 and 7 and treated PLTs on study days were not different. The increased binding of Mirasol PRT treated PLTs parallels the small decrease in recovery seen in clinical studies of patients receiving treated PLT concentrates compared to untreated products. Conclusion PRT treatment increases adherence of PLTs early in storage but does not appear to progress further. The changes may have implications for clinical responses to platelet transfusions. Evaluation of adherence in the microfluidic chamber provide a model which simulate the microvascular environment. Disclosures: Ambruso: Terumo BCT: Research Funding. Seewald:Terumo BCT: Research Funding. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment.

Abstract 3303 Background: Growth Arrest Specific gene 6 (Gas6) signals through platelet-surface Mer receptors, leading to platelet activation and thrombus stabilization via activation of PI3K and Akt, and β3 integrin phosphorylation. This amplifies outside-in signaling via αIIb β3, a necessary step for stable platelet aggregation. iMer is a truncated form of the Mer receptor tyrosine kinase's extracellular domain, produced by alternative splicing, that inhibits Gas6 signaling. A selective UNC Mer small molecule inhibitor (UNC Mer TKI) inhibits signaling by inhibiting Mer tyrosine phosphorylation. Objectives: We hypothesized that inhibiting the Gas6/Mer pathway with UNC Mer TKI would decrease platelet activation responses and thrombus formation to a greater degree than iMer. So, we comparatively evaluated iMer's and UNC Mer TKI's inhibition of Gas6/Mer signaling in vitro and in vivo. Methods: We measured the effect on platelet inhibiton of iMer and UNC Mer TKI using lab assays of human platelet function and murine thrombosis models. In vitro studies included standard aggregometry and aggregate formation on collagen surfaces in a microfluidic flow chamber. In vivo studies included a FeCl3-induced model of carotid artery injury and a collagen/epinephrine-induced pulmonary embolism (PE) model to compare thrombosis protection between littermate C57BL/6 mice treated with inhibitors or vehicle control. A paired t-test was used to compare samples in aggregation, microfluidic flow surface area coverage, as well as elapsed time to initial and stable occlusions in the FeCl3model and survival time in the PE model. Results/Discussion: Inhibitor-treated platelets exhibited significantly decreased aggregation. Collagen-stimulated samples treated with 1.2 μM iMer had mean aggregation of 45 +/− 13% (Stand Dev), compared to 72 +/− 8% in controls (n=6, p=0.02). 1.2 μM UNC Mer TKI-treated samples had mean aggregation of 56 +/−15%, compared to 74 +/−10% in controls (n=5, p Adhesion of platelets to collagen under physiologic flow conditions resulted in 1.9% (+/− 1%) coverage in 1.2 μM iMer-treated samples compared to 8.7 +/− 2% mean surface area coverage in controls (n=7, p10 platelets/aggregate). Following 6% FeCl3-induced carotid artery injury for analysis of iMer's thrombosis protection, vehicle-treated control mice (n=6) had a hazard ratio for stable artery occlusion of 7.9 (95%CI 1.7–36.3) compared to 30mg/kg iMer-treated mice (n=8, p In the PE model, 1.2 μM iMer-treated mice (n=5) had longer mean survival (14.1 +/− 14.5 min) following collagen/epinephrine injection than controls (n=5, 1.78 +/12.4 min, p=NS), with 2 iMer–treated mice and no controls surviving for 30 min. A much greater effect was seen with the 3 mg/kg UNC Mer TKI-treated mice (n=5) who had a mean survival time of 37.24 +/−31.2 min, with 3 surviving for a full 60 min, compared to 1.63 +/− 0.6 min (p=0.03) for controls (n=5), of which only 1 survived for 60 min. Conclusions: iMer and UNC Mer TKI are Gas6/Mer inhibitors that decrease platelet activation and protect mice from thrombosis, and may have translational applications as novel anti-platelet agents. UNC Mer TKI has similar efficacy, and higher potency, especially in the animal models where similar (FeCl3 model) or superior (PE model) results were seen with a 10-fold lower concentration. UNC Mer TKI also has the advantage of possible oral preparation, which may increase its potential therapeutic appeal. Disclosures: Branchford: University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Sather:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Brodsky:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

Patients with Down syndrome are known to be hyperuricemic and this condition is thought to result from the over-production of purines (Pant et al., 1968). Only one purine metabolism gene is located on human chromosome 21 in the critical region associated with Down syndrome. This gene encodes for the trifunctional protein composed of phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide transformylase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). Increased levels of this protein, known collectively as GART, have also been observed in the cultured fibroblasts of Down Syndrome patients (Scoggin et al., 1980) It is therefore highly likely that the increased levels of purines described in these patients is directly due to their over-expression of the GART trifunctional protein.

Abstract 189 Background: Growth Arrest Specific gene 6 (Gas6) signaling through platelet-surface Tyro3/Axl/Mer (TAM) receptors leads to platelet activation and thrombus stabilization. This occurs via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, stimulating tyrosine phosphorylation of the β3 integrin. This process amplifies outside-in signaling via αIIbβ3, which is necessary for stable aggregate formation. iMer is a truncated form of the extracellular domain of the Mer receptor tyrosine kinase, produced by alternative splicing, that inhibits Gas6/TAM signaling, likely by acting as a decoy receptor for Gas6. Objectives: We hypothesized that inhibiting the Gas6/TAM pathway with a novel Gas6-sequestering protein would decrease platelet activation responses. We therefore evaluated iMer's inhibition of Gas6 signaling in human and murine platelets in vitro and in vivo. Methods: We measured the inhibitory effect of iMer on platelet activation using laboratory evaluations of platelet function and a murine carotid artery thrombosis model. In vitro studies included aggregometry, adhesion to collagen in a flow chamber, and platelet spreading. These platelet activation responses were tested in human platelets in the presence or absence of the inhibitor and also in wild type (WT) and Gas6/TAM −/− murine platelets. A ferric-chloride model of carotid artery injury was used to compare susceptibility to thrombosis between littermate C57BL/6 mice treated with either iMer or vehicle. Platelet aggregation data was evaluated by the Wilcoxon Signed Rank Test, and times to occlusion following carotid artery injury were compared using the Mann-Whitney Rank Sum Test. Results/Discussion: Western blot analysis demonstrated decreased β3 integrin phosphorylation in iMer-treated human platelets after addition of human Gas6 when compared to controls, consistent with decreased Mer signaling in the presence of iMer. iMer-treated human platelets exhibited significant decreases in ADP- and collagen-induced platelet aggregation. ADP-stimulated samples treated ex vivo with iMer showed an aggregation mean of 74% (SD= +/− 3%), compared to 86% aggregation (+/− 3%) in controls (p=0.016). Collagen-stimulated samples treated ex vivo with iMer exhibited a mean of 70% aggregation (+/− 8%), compared to 88% aggregation (+/−2%) in controls (p=0.004). Electron micrographs of adhered human platelets revealed that iMer delayed, but did not permanently abrogate, platelet spreading on fibrillar collagen (100 μg/mL). Flow cytometric analysis of human platelets showed reduced expression of platelet-surface activation markers (P-selectin and PAC-1) despite stimulation with fibrillar collagen (1 μg/mL). Microfluidic flow assay demonstrated that adhesion of untreated human platelets to collagen at a wall shear rate of 100s−1 resulted in 21.3% (SD=+/− 8%) mean surface area coverage, while ex vivo iMer-treated samples showed only 1.1% (+/− 0.9%) coverage. These results are consistent with those of WT mice compared to that of Gas6/TAM −/−mice in preliminary studies using the same system. Following ferric chloride injury to the carotid artery, 71% of vehicle-treated control mice (n=7) had initial occlusions that remained stable, and only 14% remained patent. In contrast, only 25% of the iMer-treated mice (n=8) formed initial occlusions that remained stable, while 50% remained patent. The iMer treated mice also had a significant decrease (p=0.02) in the duration of first occlusion time (i.e. length of time the initial occlusion lasted), suggesting decreased thrombus stability. Conclusions: iMer is a novel inhibitor of the Gas6/TAM pathway that decreases platelet activation responses and protects mice from arterial thrombosis by decreasing phosphorylation of β3 integrin, which has been shown to be necessary for thrombus stabilization. This compound may, therefore, have translational applications as a novel anti-platelet agent. Disclosures: No relevant conflicts of interest to declare.

Abstract 856 Fibrinogen plays a central role in maintaining hemostasis. Disruption of its normal function may lead to hemorrhagic or thrombotic events. Fibrinogen is encoded by three genes, FGA, FGB, and FGG, clustered on chromosome 4q28-q31. Hereditary defects of fibrinogen, although uncommon, can affect the quantity (hypofibrinogenemia and afibrinogenemia) or the quality (dysfibrinogenemia) of the circulating protein. We report a family with two affected individuals (father and daughter) that presented with a mild bleeding predisposition, but also exhibited thrombotic events, i.e. unprovoked deep vein thrombosis in a father and an in utero middle cerebral artery stroke in a daughter. The Clauss fibrinogen levels for the father and daughter were 105 and 98 mg/dL (normal 150–400 mg/dL) and fibrinogen antigen levels were 138 and 152 mg/dL (normal 170–400 mg/dL), respectively. Thrombin times were prolonged in both patients. All other coagulation factors were within normal range and the patients did not carry either the prothrombin G20210A or factor V Leiden mutation. All exons and intron-exon junctions of the three fibrinogen genes were sequenced. The father and daughter were found to be heterozygous for a novel missense mutation in the gamma chain (Ala315Asp) while this mutation was not observed in an unaffected family member (mother of the daughter). Transient transfection experiments using CHO-K1 cells showed that gamma chain with p.Ala315Asp mutation was detected in both cell lysates and supernatant, although in reduced amount as compared with cells transfected with wild-type (WT) FGG cDNA. Structurally, there was no significant difference in fibrin fiber diameter, as measured manually by scanning electron microscopy, between the affected (father and daughter), and the daughter's unaffected mother. Similarly, there was no difference in thrombus height between the three individuals. As expected, hydraulic permeability of platelet rich plasma clots correlated with the fibrinogen levels in each person. Clotting kinetics in plasma of affected family members was consistent with hypofibrinogenemia, but did not suggest abnormal fibrin polymerization. Clot structure and fibrin deposition was further characterized with a microfluidics flow assay, an experimental technique in which platelet and fibrin accumulation is observed in real-time using fluorescent probes in whole blood under shear stress on a collagen/tissue factor micropatterned surface. Interestingly, fibrin density and platelet accumulation, as measured by platelet and fibrin surface area coverage, was highest in the father and daughter, the two individuals with hypofibrinogenemia and thrombotic phenotype. In order to evaluate the possible changes resulting from the Ala315Asp mutation we examined predicted alterations in protein structure, energy, and protein-protein interaction between two fibrinogen molecules via in silico molecular modeling using published protein crystal structure. The molecular modeling showed that alanine for aspartic acid substitution predicted to result in substantial changes in overall conformation of the interaction interface (distal D-domain) between the two fibrinogen molecules due to introduction of a bulkier side chain. Potential energy (energy of minimization) for Ala315Asp mutant homodimer, and for WT-mutant heterodimer were lower than the WT homodimer, predicting a more stable structure from an energetic standpoint. Overall interaction energy (measurement of the energy of association between the two molecules) was lower for Ala315Asp mutant homodimer as compared with WT homodimer or WT-mutant heterodimer, suggesting more favorable interaction for Ala315Asp mutant dimers. Overall, the in silico modeling studies predicted that Ala315Asp mutation may lead to more rapid or stronger interaction between fibrinogen molecules. These data suggest that a novel FGG mutation, c.944C>A, that predicts pAla315Asp change, if expressed and incorporated into fibrinogen molecule in vivo, may result in a more stable or more resistant to lysis clot. Characterizing the association between a specific genetic defect and its phenotypic expression will advance the current understanding of the molecular and biochemical mechanisms of the inherited disorders of fibrinogen, and may contribute to the development of safer therapeutic interventions for the patients with fibrinogen disorders. Disclosures: No relevant conflicts of interest to declare.

STCH is a member of the stress70 chaperone family, which plays a major role in the processing of cytosolic and secretory proteins. Members of the stress70 protein chaperone family participate in protein processing events by binding denatured or misfolded peptide sequences and then releasing these polypeptide chains by an ATP-dependent mechanism. Members of this protein family possess an ATPase activity located in the highly conserved amino-terminal domain and a less well-conserved carboxy-terminal domain necessary for peptide binding. The Stch gene encodes a 60-kDa peptide that is constitutively expressed in all human cell types and shares a high degree of amino acid identity with HSP70 and BiP. The protein differs from previously identified stress70 gene products by the presence of a unique hydrophobic signal sequence and the absence of a carboxy-terminal peptide-binding domain. This results in the protein being highly enriched in the lumen of a crude cellular microsome fraction and possessing an ATPase activity that, unlike other HSP70-like proteins, is not peptide inducible. Analysis of the rat Stch cDNA demonstrates that the unique properties of this C-terminal truncated HSP70-like molecule are conserved through mammalian evolution. As with the human Stch gene, the rat cDNA encodes a hydrophobic leader sequence, andmore » the putative translation product lacks a C-terminal peptide-binding domain. 7 refs., 2 figs.« less

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.