bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Venous Outflow Obstruction and Venous Valvular Incompetence as Mechanisms for Venous Stasis Syndrome Following Deep Vein Thrombosis: A Population-Based Cohort Study.
Authors: Ashrani Aneel A. , Heit John A. , Lahr Brian D. , Petterson Tanya M. , Bailey Kent R.
Published: October 2019

Background: Venous stasis syndrome (VSS) is a relatively common long-term sequelae of deep vein thrombosis (DVT), although it frequently is noted in individuals with no prior history of DVT.

Objective: To evaluate whether: (1) venous stasis syndrome (VSS) is associated with a prior history of DVT; (2a) venous outflow obstruction (VOO) and/or (2b) venous valvular incompetence (VVI) are associated with DVT; and (3) VSS is associated with VVI and/or VOO.

Design: Case-control study nested within a population-based inception cohort study.

Population: 230 residents of Olmstead County, MN (OCM) with a first lifetime VTE over the 25-year period, 1966 – 1990 (cases), and 135 age, gender and year of incident VTE-matched OCM residents without prior history of VTE (controls).

Measurements: Physical examination and patient questionnaire for symptoms or signs of VSS, and strain gauge outflow plethysmography, continuous wave venous Doppler ultrasound, and passive venous drainage and refill testing for VOO and VVI performed between 1996 – 1998.

Results: Of the 365 study participants, 43 (12%) had VOO, 136 (37%) had VVI, and 265 (73%) had VSS. In multivariate logistic regression analyses: (1) age at the follow-up visit [OR Δper 10 years: 1.70 (1.41, 2.04)], prior DVT in the affected limb [OR: 4.03 (2.32, 7.01)], and presence of prior varicose veins [OR: 4.36 (1.84, 10.31)] were significantly associated with VSS; (2a) age at the follow-up visit [OR Δper 10 years (95% CI): 1.84 (1.39, 2.44)] and prior DVT in the affected limb [OR: 5.01 (2.61, 9.63)] were significantly associated with VOO; (2b) prior DVT in the affected limb (OR: 3.91 (2.56, 5.97)], presence of prior varicose veins [OR: 2.19 (1.32, 3.63)] and symptoms of VSS prior to incident DVT [OR: 3.42 (1.46, 8.00)] significantly increased the odds for VVI; and (3) VOO (p=0.004) and VVI (p<0.0001) were highly associated with VSS. Having a DVT in the left leg was associated with a greater odds of developing VOO, VVI or VSS in that leg when compared to their association with right leg DVT (OR: 6.69 vs. 3.65; 4.82 vs. 3.09; 4.71 vs. 3.97, respectively). Interestingly, prior DVT in the opposite leg was associated with an increased odds of subsequent VVI [OR: 2.00 (1.28, 3.10) and VSS [OR: 2.20 (1.31, 3.70)], but not VOO, in the test leg.

Conclusions: Prior DVT imparts an increased risk for subsequent VSS, likely due to VOO and/or VVI. The odds of VOO or VSS increases with age. Presence of varicose veins increases the odds for VVI and VSS. We speculate that the increased odds of left sided VOO, VVI and VSS in patients with prior DVT may be secondary to May-Thurner syndrome. The increased odds of VVI and VSS in the limb opposite to the one affected by prior DVT could reflect occult DVT in the test limb, inferior vena cava thrombosis, or other mechanisms leading for VVI and VSS.
The Interaction of Prothrombin and Its Activation Products with Platelets.
Authors: Samis John A. , Manuel Reginald P. , Nesheim Michael E.
Published: October 2019

The interaction of human 125I-prothrombin and its activation products with unactivated and thrombin-stimulated human platelets was studied. 125I-prothrombin binding to unactivated platelets was found to be a reversible and calcium-dependent process (n=48,000 sites/platelet, Kd=3.0μM). Thrombin stimulation of platelets resulted in increased prothrombin binding both in the presence and absence of calcium (n=84,000 sites/platelet, Kd=6.0μM and n=53,000 sites/platelet, Kd=8.0μM, respectively). Thrombin stimulation of platelets also increased the calcium-independent binding of prethrombin-1 compared with unstimulated cells. Using thrombin-stimulated platelets as a membrane surface for prothrombinase, 125I-prothrombin was converted exclusively to 125I-Fragment 1.2 and 125I-thrombin when the reactions were carried out in the presence of the reversible thrombin inhibitor, 5-dimethylaminonapthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). 125I-thrombin was the major prothrombin activation product that remained bound to the thrombin-stimulated platelets (50,000 molecules/platelet). Platelet-bound 125I-thrombin either added directly or generated from 125I-prothrombin by the action of prothrombinase was found to be inacessible to human antithrombin (AT)/heparin-induced inactivation. Utilization of phosphatidylcholine/phosphatidylserine (PCPS) vesicles as a membrane surface for prothrombinase in the presence of DAPA demonstrated that 125I-prothrombin activation proceeded initially through a meizothrombin intermediate that was replaced at later times by Fragment 1.2 and the A and B chains of α-thrombin. 125I-Fragment 1.2 was the main prothrombin activation product that remained bound to the PCPS vesicles after Sephadex G-150 gel filtration chromatography. The above results indicate that human prothrombin binds specifically to unstimulated human platelets via its Fragment 1 domain only in the presence of calcium and thrombin-stimulation results in the exposure of additional calcium-dependent and -independent prothrombin binding sites. In addition, thrombin was the major prothrombin activation product that remains associated with the thrombin-stimulated human platelets and this platelet-bound thrombin is protected from AT/heparin- induced inactivation. During prothrombin activation on platelets, large amounts of thrombin remain bound and Fragment 1.2 is released from the platelet surface. In contrast, Fragment 1.2 was the major prothrombin activation product that binds PCPS vesicles. Therefore, although both thrombin-stimulated human platelets and PCPS vesicles may each serve as an effective membrane component of prothrombinase, they appear to differ both in their prothrombin activation pathway as well as their ability to bind the products of prothrombin activation.
Signalling Pathways Have Different Expression Profiles in Human Platelets Isolated from Men and Women.
Authors: Rothwell Stephen W. , Eidelman Ofer , Jozwik Catherine , Mueller Gregory , Poth Merrily , Zeitlin Pamela L. , Guggino William , Jacobowitz David M. , Srivastava Meera , Pollard Harvey B.
Published: October 2019

Background: Cardiovascular diseases (CVD) are the leading causes of death for men and women in the U.S. Platelets play a central role in the inflammation and coagulation mechanisms responsible for these disorders, and anti-platelet drugs are therefore the current mainstays in prevention and therapy. Understanding the signaling pathways involved in normal and abnormal platelet function using proteomic techniques offers a novel strategic approach to the discovery of potential new therapeutic targets.

Hypothesis: The incidence and outcomes of cardiovascular disease are profoundly dependent on gender, and it is possible that differences in platelet signaling mechanisms may contribute to these disparities. We have therefore hypothesized that the platelet signaling proteome is gender-specific.

Methods: Platelets were isolated and purified from four normal adult male and four normal adult female donors under an IRB-approved protocol. Conventional 2D-gel electrophoresis (2DGE) and mass spectrometry were used to profile the high abundance platelet proteome. Antibody microarrays, bearing 507 monoclonal antibodies against low abundance proteins involved in signaling pathways and related functions, were used to interrogate the lower abundance platelet. Western blots were used to validate a subset of the data.

Results: Discovery studies with 2DGE revealed at least nineteen lower abundance proteins which could be significantly distinguished on the basis of gender (P<0.05). Two of these proteins, a thrombopoeitin-activated G-protein component (#1101) and a megakaryocyte-relevant co-factor for DNA synthesis (#1583), were selectively elevated in male platelets. Concurrent studies on the sensitive antibody microarray platform revealed that more than 420 proteins were significantly expressed in a gender-specific manner (P<0.05). Of these, 62 remained significant by the stringent criterion of a False Discovery Rate (FDR) of <10% (SAM Algorithm, 2000 permutations). By rank-ordering the proteins by expression level, we found that platelets from normal male and female donors were significantly and uniquely enriched in different signaling proteins. These gender-specific signaling proteins included IL-1beta, and specific isoforms of iNOS (inducible nitric oxide synthase), PLC (phospholipase C), and PKC (protein kinase C). Western blot analysis validated a subset of the differentially expressed platelet proteome.

Conclusions: These data support the hypothesis that the platelet signaling proteome is gender-specific. We interpret these data to indicate that gender-specific differences in the platelet signaling proteome may contribute to the gender-specific disparity in outcomes for cardiovascular diseases in men and women.
Nitrite Is Reduced to Nitric Oxide by Xanthine Oxidoreductase and Nitric Oxide Synthase in the Erythrocyte Membrane in Hypoxemia.
Authors: Rathod Krishnaraj S. , Webb Andrew J. , Lovell Matthew J. , Lecomte Florence , Ahluwalia Amrita
Published: October 2019

Evidence is emerging that inorganic nitrite, previously thought to be an inactive metabolite of nitric oxide (NO.), provides an alternative source of functionally active NO. under ischemic conditions when normal NO. synthesis, via nitric oxide synthase (NOS), is suppressed. Our previous studies have demonstrated that the reduction of nitrite to NO. in the ischemic heart is cardioprotective and dependent on xanthine oxidoreductase (XOR) activity (Webb et al., PNAS, 2004; 101:13683–13688). Recent evidence in erythrocytes suggests that nitrite is converted to NO. by a different mechanism involving deoxyhemoglobin (Cosby et al., Nat Med, 2003; 9:1498–1505). We hypothesized that erythrocytes may also contain XOR with nitrite reductase activity, and we investigated this possibility using suspensions of washed erythrocytes prepared from blood taken from healthy volunteers. Whole erythrocytes were incubated with nitrite (100 μM) in anaerobic conditions at pH 7.4 and at pH 6.8, mimicking normal physiology and severe acidosis respectively, and NO. production measured in parts per billion (ppb) using ozone chemiluminescence. Addition of erythrocytes to nitrite caused an elevation of NO. generation at pH 7.4 (0.28±0.06; p<0.05; n=6) and pH 6.8 (0.50±0.09; p<0.05; n=6) significantly above that produced by simple acidification (0.07±0.02 at pH 7.4, n=6; 0.17±0.06 at pH 6.8). NO. generation at pH 6.8 was enhanced by ~43% (P<0.01, n=6) in the presence of XOR substrate xanthine (10 μM) and attenuated by ~65% (P<0.05, n=6) by the XOR inhibitor allopurinol (100 μM). In contrast, xanthine and allopurinol did not alter nitrite-derived NO. generation at pH 7.4 (n=6). Treatment of erythrocytes with the NOS inhibitor L-NAME (300 μM) abolished nitrite-derived NO. generation at pH 7.4 but inhibited the response by only ~57% at pH 6.8 (n=6, P<0.05). Western blot analysis of erythrocyte membranes confirmed the presence of endothelial NOS (eNOS), as has been demonstrated recently (Kleinbongard et al., Blood, 2006; 107(7):2943–2951). Furthermore the presence of XOR in whole purified erythrocytes was confirmed both immunohistochemically and using immunoflorescence. In summary we have demonstrated that erythrocytes reduce nitrite to NO. through the enzymatic actions of XOR and eNOS. These mechanisms of NO. production complement that of deoxyhemoglobin and are likely to contribute to the protective effects of nitrite attributed to NO. generation in hypoxemic and ischaemic conditions.
Nitroglycerin as a Modulator of Hemoglobin Function.
Authors: Hrinczenko Borys
Published: October 2019

Recent evidence suggests that oxygen acts as an allosteric modulator of nitric oxide (NO) binding to hemoglobin. This may have important physiological implications. Those effects, thought by some to be mediated by nitrosylation of the conserved cysteine β93 residues (S-nitrosohemoglobin; SNO-Hb), may also have therapeutic relevance in sickle cell disease and potentially be a useful strategy in controlling vasoactive effects of hemoglobin-based blood substitutes. Nitroglycerin (NTG), the classic vasodilator drug, acts in part through metabolism to NO. Normal (AA) and sickle (SS) blood suspensions were treated with nitroglycerin from 0.03 to 300 μM for periods up to 3.25 hours. Such treatment significantly lowered the oxygen affinity (increases P50, PO2 at which hemoglobin is half-saturated with O2) of both AA and SS red cells. The decrease in oxygen affinity is greater when oxygenated cells are treated than deoxygenated cells, with changes in P50 as great as 7.4 mm Hg in the AA cells and 9.9 mm Hg in the SS cells. Those shifts occur within 15 minutes, before significant increases in methemoglobin levels. This is opposite to what has been seen with other NO donors (2-(N,N-diethylamino)-diazenolate-2-oxide, S-nitrosocysteine, and sodium trioxodinitrate), where increased oxygen affinity occurs. Other classic NO donors also yielded significant methemoglobin formation within 15 minutes. In vivo effects of NTG infusion up to 1 μg/kg/min fail to demonstrate any change in P50 or methemoglobin in study subjects with normal AA erythrocytes. (Shigihara A. Masui1994;43:222) In addition, NTG has been shown to enhance oxygen release from erythrocytes without significantly increasing tissue blood flow in the hepatic microcirculation in a rat model. (Kosaka H, et al. Nat Med1997;3:456) Furthermore, a study in a dog model demonstrated NTG-induced enhanced oxygen unloading in the ischemic microcirculation. (Bin JP, et al. Circulation2006;113:2502) Elevated tandem erythrocyte S-nitrosothiol content, reflecting mainly SNO-Hb, was also noted. However, SNO-Hb increases oxygen affinity (Bonaventura C, et al. J Biol Chem1999;274:24742) and cannot explain the increased oxygen unloading. The effect of NTG on erythrocytes in our in vitro study model mimics in vivo results in the microcirculation (as opposed to the macrovascular circulation) and demonstrates that the reported enhanced oxygen unloading is due to a decrease in oxygen affinity of hemoglobin. Other biotransformation products of NTG may account for these effects as opposed to SNO-Hb. This study demonstrates that selective NO donors (i.e., NTG) may target erythrocytes for enhanced R (oxyhemoglobin) to T (deoxyhemoglobin) transformations specifically in the microcirculation and thereby improve tissue oxygenation.
An Erythoid-Specific Component of the Rhesus Stage Selector Protein, rNF-E4, Modulates γ-Globin Gene Expression Specifically.
Authors: Wu Ruiqiong , Desgardin Aurelie , Jane Stephen M. , Cunningham John M.
Published: October 2019

Understanding the molecular mechanisms that regulate γ-globin gene expression is essential for development of new therapeutic strategies for individuals with sickle cell disease and β-thalassemia. We have previously identified a tissue- and developmentally- specific multiprotein transacting factor complex, the human stage selector protein (SSP), which facilitates the interaction of the g-globin gene promoters with the upstream locus control region enhancer in fetal erythoid cells. This complex interacts with the stage selector element (SSE) in the proximal g-globin promoter, a regulatory motif phylogenetically conserved in primate species with a distinct fetal stage of β-globin like gene expression. Given these observations, we hypothesized that a similar complex modulates γ-globin in the rhesus macaque, a non-human primate model that has been utilized to study β-globin like gene expression. We focused our efforts on NF-E4, given that a human isoform of this factor confers erythroid and fetal specificity to the SSP complex. Fetal liver erythroblasts were obtained from rhesus embryos and analyzed by reverse transcriptase(RT)-PCR analysis for NF-E4 expression. NF-E4 like transcripts were identified in day 60, 80 and 120 embryonic erythroblasts, but not other rhesus tissues, demonstrating an erythroid-specific pattern of expression. Utilizing 5′ RACE, we cloned a full length NF-E4 transcript, identifying an open reading frame encoding a 131 amino acid polypeptide. This 20kD polypeptide shares a high degree of homology with human NF-E4, especially in its carboxy-terminal domain. Like human NF-E4, GST pulldown chromatography confirmed the ability of the rhesus factor to interact directly with CP2 and ALY, the other core components of the SSP. To evaluate rNF-E4 function in vivo, we utilized retrovirally mediated gene transfer to enforce expression of this factor in K562 cells, a model of human fetal erythropoiesis. Initial co-immunoprecipitation studies confirmed the in vivo interaction of rNF-E4 with other components of the SSP. Interestingly, we observed a specific 3-fold induction of γ-globin gene expression in rNF-E4 expressing cells when compared to controls. Moreover, we demonstrated that, like enforced expression of human NF-E4, rNF-E4 induced a significant increase in ε-globin gene expression. Taken together, our results suggest a conservation of NF-E4 expression and function in species with a fetal stage of globin gene expression. Moreover, the identification of rNF-E4 provides a platform for the pre-clinical development of therapeutic agents that induce high levels of NF-E4 in adult erythroblasts.
Conditional Disruption of the Murine Prothrombin Gene.
Authors: Mullins Eric S. , Flick Matthew J. , Kombrinck Keith W. , Degen Sandra J. , Sun William , Degen Jay L.
Published: October 2019

Thrombin is the core protease in the hemostatic system. Thrombin directs thrombus formation through the proteolytic conversion of fibrinogen to fibrin and the local activation of protease-activated receptors on platelets and other cells. In addition, thrombin controls the coagulation system through the activation of fXI, protein C and other key hemostatic factors. Interestingly, the biological role of thrombin is seemingly not limited to the maintenance of vascular integrity. There is appreciable evidence that thrombin-mediated proteolysis plays an important role in development, the inflammatory response, tissue repair, tumor cell metastasis and other physiological and pathological processes. Unfortunately, the embryonic and perinatal lethal phenotype previously described in prothrombin null (fII−/−) mice limited the utility of those knockout animals in better defining the larger role of fII in vivo. In order to develop the means to explore the importance of thrombin in disease processes within adult animals, a mouse line was generated carrying a conditional (“floxed”) fII knockout allele (fIIfx mice). Homozygous fIIfx/fx mice and compound heterozygous mice carrying one fII floxed allele and one fII null allele (fIIfx/− mice) developed to term, were present in offspring in the expected Mendelian frequencies, survived to adulthood and retained normal reproductive success. In the absence of Cre-mediated recombination, fIIfx/− mice maintained circulating fII levels that were low (approximately 10% of normal), but spontaneous bleeding events were never encountered in these animals. Studies of fIIfx/− mice carrying a Cre recombinase transgene known to be constitutively expressed in the liver showed that prothrombin levels can be reduced to levels incompatible with post-natal survival. More sophisticated studies using the polyI:C-inducible Mx-Cre system revealed that unchallenged Mx-Cre+/fIIfx/− mice consistently survived to adulthood. However, induction of Cre under conditions that result in near-complete recombination of target floxed alleles within the liver resulted in the development of spontaneous bleeding events and death within 7 days. Multiple sites of hemorrhage were evident in these challenged adults, including lower gastrointestinal and intracranial sites of bleeding. Immunological analysis of plasma collected from these animals revealed that they carried levels of fII below current detection limits (< 1% of normal). Studies are underway to establish both the lowest level of plasma fII compatible with long-term survival and define the effects of extremely low fII levels on disease processes in vivo.
Missense Mutations in the Proline-Rich Region of Coagulation Factor XII in Hereditary and Idiopathic Angioedema.
Authors: Dewald Georg , Bork Konrad
Published: October 2019

Hereditary angioedema (HAE) is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic HAE types (I and II) are caused by mutations in the complement C1 inhibitor gene, resulting in a functional deficiency of C1 inhibitor. Recently, a novel type of hereditary angioedema has been described, these patients showing normal C1 inhibitor concentration and activity in plasma (HAE with normal C1 inhibitor, HAE type III). With few exceptions, all reported patients have been women, and exposition to estrogens appears to be an important precipitating factor. Screening of twenty unrelated female patients with this new type of HAE for mutations in the coagulation factor XII gene revealed two different missense mutations, both located in exactly the same position within exon 9, namely in the second position of the codon (ACG) encoding Thr309 of the mature protein. Five patients showed a C→A transversion (1032C→A), predicting a threonine-to-lysine substitution (Thr309Lys); one additional patient showed a C→G transversion (1032C→G), resulting in a threonine-to-arginine substitution (Thr309Arg). Thus, in 6 of 20 unrelated patients the wild-type threonine residue is substituted by a basic amino acid residue. The mutations are located in the proline-rich region of factor XII which may play some role in the binding of factor XII to negatively charged surfaces, and, thus, may eventually influence mechanisms of contact activation. The predicted structural and functional impact of the mutations, their absence in healthy control individuals (n=145), and their co-segregation with the phenotype in five families with altogether 20 affected women provide strong support that they cause disease (Biochem. Biophys. Res. Commun.343: 1286–1289, 2006). We speculated if these mutations may also be present in singular angioedema cases, i.e., patients with no affected family members. We therefore studied 58 such patients with idiopathic recurrent angioedema (23 men, 35 women) by sequencing of exon 9 of the F12 gene. In two patients, both female, the 1032C→A (Thr309Lys) mutation was observed. Thus, this mutation may also play a role in a subgroup of patients with idiopathic angioedema, in particular in female patients. The known estrogen-dependent regulation of factor XII expression may well be seen in association with the disease manifestation mainly in women.
hFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.
Authors: Kikushige Yoshikane , Yoshimoto Goichi , Miyamoto Toshihiro , Ishikawa Fumihiko , Iwasaki Hiromi , Harada Mine , Akashi Koichi
Published: October 2019

Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.
SLAM-Family Expression on Human Mobilized Peripheral Blood Progenitors.
Authors: Savona Michael R. , Kiel Mark J. , Leavitt Andrew D. , Morrison Sean J.
Published: October 2019

Background and significance: A simple but precise method to identify hematopoietic stem cells within mobilized peripheral blood would be useful for transplantation. Our lab has recently identified a family of surface markers whose differential expression distinguishes mouse hematopoietic stem cells from other hematopoietic progenitors. The founding member of the signaling lymphocyte attractant molecule (SLAM) family, CD150, was expressed on all hematopoietic stem cells (HSCs) but not on other hematopoietic progenitors. Other SLAM-family members, including CD244 and CD48, were expressed by non-self-renewing multipotent progenitors and most colony-forming restricted progenitors, respectively. As a result, mouse stem cells can be highly purified as CD150+CD48− cells, dramatically simplifying and improving the purification of mouse HSCs. To begin to test whether SLAM family markers can facilitate the identification and purification of human hematopoietic stem cells, we have assessed the frequency of CD150+CD48− cells in mobilized peripheral blood and compared their distribution to that of CD34+CD38− cells, which are known to be highly enriched for human hematopoietic stem cells.

Methods: Mobilized human peripheral blood samples were stained with anti-CD150 (conjugated to the FITC), anti-CD48 (PE), anti-CD41 (PE), anti-CD34 (APC), and anti-CD38 (PE-Cy5) antibodies. Samples were analyzed by flow-cytometry.

Results: We have identified a population of CD150+CD48−CD41− cells within human mobilized peripheral blood that is present at a similar frequency as the same population in mobilized mouse peripheral blood (mean 0.039±0.11%). The CD34+CD38− population was similarly infrequent. Interestingly, 16.3±19.5% of CD150+CD48−CD41− cells were also CD34+ whereas only 1.13±3.45% of the CD34+CD38− population was CD150+CD48−CD41− raising the possibility that SLAM-family members may substantially improve the purity of human hematopoietic stem cells.

Conclusion: Murine and human hematopoietic tissues have a similar frequency of CD150+CD48−CD41− cells. It is possible that the use of SLAM-family markers might enhance the identification and purification of human hematopoietic stem cells beyond what is possible using CD34 and CD38. We are currently performing reconstitution assays to test this functionally.

Peripheral Blood Mononuclear Cells Peripheral Blood Mononuclear Cells
Characterization of Placenta Derived Adherent Cells (PDAC): A Novel Type of Stem Cells Isolated from Human Placenta.
Authors: Edinger James , Ye Qian , Paludan Casper , Wang Jia-Lun , Hofgartner Wolfgang , Hariri Robert
Published: October 2019

The placenta is a rich source of ethically non-controversial stem cells. In this work, we describe the development of procedures to isolate and culture adherent stem cells from a full term, postpartum human placenta (Placenta Derived Adherent Cells, PDACs). PDACs are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, cord, placental cotyledons, or any combination thereof. PDACs were established in a medium containing low concentrations of fetal calf serum and limited growth factors. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. We found that PDACs differentiate down the adipocyte, chondrocyte and osteocyte lineages. In an induction medium containing IBMX, insulin, dexamethasone and indomethacin, PDACs turned into fat laden adipocytes in 3 to 5 weeks. Under osteogenic induction culture conditions, PDACs were found to form bone nodules and have calcium depositions in their extracellular matrix. Chondrogenic differentiation of PDACs was performed in micropellets and was confirmed by formation of glycosaminoglycan in the tissue aggregates. In addition, we showed that PDACs suppress an MLR consisting of suitable effectors (T cells or NK cells) and target populations (allogeneic irradiated PBMCs or mature DCs). The range of suppression of T cell proliferation was 50 to 95 %. The observed suppression of the MLR is likely cell-to-cell contact dependent in the range of 20%. In summary, the PDACs described herein exhibit in vitro properties of pluripotent stem cells. The placenta is an excellent raw material for the isolation of stem cells.
Marrow Makes Thyroid but Not Adrenal Tissues in Humans.
Authors: Davenport Jake , Jorgensen Marda L. , Scott Edward W. , Cogle Christopher R.
Published: October 2019

Human marrow has shown potential to contribute to organ various organs. We previously demonstrated marrow contribution to the brain in humans, without evidence for fusion. Given the observations of marrow contributing to the other end-organs, we questioned whether marrow also repair endocrine glands like the thyroid and adrenal glands. To address this question, we identified thyroid (n=3) and adrenal tissues (n=3) obtained from women who received hematopoietic cell transplantation from male donors. Thyroid tissues were stained for thyroid peroxidase (TPO), CD45, and XY FISH. Adrenal tissues were stained for synaptophysin, CD45, and XY FISH. Analysis of 450 thyroid follicles (150 per patient) found 2% to 5% of follicles containing a male/marrow-derived thyroid follicle demonstrating follicular orientation, TPO expression, lacking CD45, and containing a Y chromosome. The median number of male/marrow-derived cells in follicles was 1. None of the male/marrow-derived cells demonstrated a fusion karyotype (XXY or XXXY). In contrast, adrenal tissues analyzed in the women demonstrated no male cells throughout all layers. In conclusion, our results show that human marrow can contribute to thyroid follicles and express thyroid proteins, however at a modest level. Furthermore, adrenal tissues in these women demonstrated no evidence of marrow contribution. These results expose a potential strategy of thyroid repair by illuminating the potential of marrow contribution to thyroid epithelia.
Substrate Preferences for Antiplasmin Cleaving Enzyme Hydrolysis of Peptides Derived from the α2-Antiplasmin Sequence.
Authors: Jackson Kenneth W. , Christiansen Victoria J. , Lee Kyung N. , Mason Christina F. , McKee Patrick A.
Published: October 2019

Antiplasmin cleaving enzyme (APCE) is a proteinase that specifically, but slowly cleaves the Pro-Asn bond in long-form α2-antiplasmin (Met-α2AP) in human plasma. This slow cleavage produces a steady-state plasma mixture of Met-α2AP and an N-terminally shortened form of antiplasmin, Asn-α2AP. The Asn-α2AP form crosslinks to fibrin ~13-fold faster than Met-α2AP. A faster crosslink rate causes a greater number of antiplasmin molecules to become bound during fibrin formation, thereby enhancing resistance to fibrinolysis. Inhibition of plasma APCE may decrease the number of antiplasmin molecules crosslinked and result in clots that are more easily removed during fibrinolysis. Therefore, an inhibitor specific for APCE could potentially be used to regulate fibrinolysis. Human Met-α2AP exists in two polymorphic forms at position six in the mature sequence, with arginine predominant, and tryptophan accounting for a lesser percentage. We have determined the relative cleavage rates of synthetic peptides from a peptide library that span the cleavage site. The peptides contained all common amino acids except cysteine in the polymorphic position (P7 position). Arg was the optimal amino acid in this position with a relative cleavage rate ~5–10-fold faster than other amino acids except Lys, which was ~70% of the Arg rate. The P7 position Arg enhancement was also observed when Arg was in the P6 or P5 position, but no enhancement was observed when Arg was moved to positions P8, P4, P3 or P2. It was also determined that APCE is preferentially an endoproteinase rather than an aminodipeptidase, with a 10-fold greater rate of hydrolysis of the internal Pro-Asn bond in the Met-α2AP 1–17 peptide sequence MEPLGRQLTSGP-NQEQV over the Pro-Asn bond penultimate to the amino-terminal bond in the Met-α2AP 11–27 peptide sequence GP-NQEQVSPLTLLKLGN in peptide hydrolysis experiments. We conclude that APCE inhibitors designed with a positive charge placed upstream of the Pro-X scissile bond equivalent to five to seven amino acid residues may prove to be highly potent and specific. In addition, such inhibitors should also prove useful for blocking the activity of the closely related enzyme fibroblast activation protein. This work was supported by NIH grant HL072995.
Enhanced Synthesis of Functional Recombinant Factors IX and VII by BHK Cells Engineered to Overexpress VKORC1 Combined with siRNA Silencing of the γ-Carboxylation Inhibitor Calumenin.
Authors: Wallin Reidar , Wajih Nadeem , Owen John
Published: October 2019

Because of the importance and demand for recombinant factors IX, VII and protein C in prophylactic medicine, our laboratory is focused on engineering eukaryotic cells with enhanced capacity to produce fully γ-carboxylated functional forms of these recombinant vitamin K-dependent proteins. Here we report our work on recombinant human factor IX (r-hFIX) and recombinant human factor VII (r-hFVII). Successful engineering was accomplished by overexpressing the vitamin K cofactor producing enzyme VKORC1 of the vitamin K cycle in BHK cells stably overexpressing r-hFIX and r-hFVII respectively. VKORC1 provides the γ-carboxylase with reduced vitamin K1 (vit.K1H2) cofactor and is the rate limiting step in vitamin K-dependent post translational γ-carboxylation of proteins. We have identified the ER chaperone protein calumenin as an inhibitor of vitamin K-dependent γ-carboxylation. Here we show that siRNA silencing of calumenin in BHK cells engineered to overexpress VKORC1 and r-hFIX and r-hFVII respectively significantly increases production of both coagulation proteins as functional fully γ-carboxylated proteins. Engineering of the γ-carboxylation system and silencing of calumenin do not affect total synthesis of r-hFIX and r-hFVII by BHK cells. However the fraction of functional r-hFIX and r-hFVII isolated from the total pool of each recombinant coagulation factor pool was found to be 80 and 85% compared to 12 and 14 % from the pools isolated from non engineered BHK cells stably overexpressing r-hFIX and r-hFVII respectively. This new recombinant technology enforced on the vitamin K-dependent γ-carboxylation system demonstrates that r-hFIX and r-hFVII used as pharmaceuticals can be produced at a much higher yield than currently is accomplished from r-hFIX and r-hFVII overexpressing cells not modified by the new technology.
Plasmin-Mediated Coagulation Factor Xa Derivative, Xa33/13, Enhances Plasminogen Activation and Fibrin Clot Lysis.
Authors: Pryzdial Ed L.G. , Talbot Kimberley , Meixner Scott C.
Published: October 2019

Previous reports showed that coagulation factor Xa (FXa) treated with the fibrinolysis protease, plasmin, loses procoagulant activity and can accelerate tissue plasminogen activator (tPA). The resulting FXa derivative, Xa33/13, acquires binding sites for plasminogen and was consequently presumed to be the form of FXa involved in generating plasmin. By evaluating the function of purified Xa33/13, the current work addresses the hypothesis that conversion of FXa to Xa33/13 is the basis of enhanced plasmin generation by tPA and that this newly acquired FXa function can also contribute to clot lysis. Using purified proteins in a chromogenic assay, both Xa33/13 and FXa were found to increase the generation of plasmin activity by tPA at least 10-fold. To explain the involvement of FXa reported previously, Western blots demonstrated a correlation between the disappearance of FXa, appearance of Xa33 antigen and enhanced plasmin production. The Western blots also revealed that detection of Xa33 antigen is lost at later time points, which explains the observation that plasmin generation is eventually shut down during the chromogenic assay. Despite fibrin being recognized as the principal accelerator of tPA, at concentrations as low as 10 nM, either Xa33/13 or FXa reduced the lysis time of thrombin-mediated fibrin clot in a turbidity assay. The Xa33/13 or FXa could be added during clot formation before the tPA or after clot formation with the tPA to facilitate a 50–70% reduction in the time required to achieve half maximal lysis at 0.1 μM. The time-dependent formation of Xa33 antigen in plasma that was induced to clot with tissue factor, anionic phospholipid vesicles and Ca2+, demonstrated that the plasmin-mediated fragmentation pattern of purified FXa represents the complex physiological mixture. However, the use of specific inhibitors and immuno-depleted plasma indicated that plasmin is not required for production of Xa33 antigen in plasma. Cumulatively the data presented here support a role for coagulation FXa in fibrinolysis. These diametrically opposing functions derived from the same molecule, may contribute to the balance between coagulation and fibrinolysis, thereby facilitating tolerance for a wide normal physiological range of FX and possibly other coagulation proteins.
Morphological and Functional Studies of Ultra Long Von Willebrand Factor (ULVWF) Anchored on Endothelial Surface.
Authors: Huang Jing , McEver Rodger P. , Heuser John , Sadler J. Evan
Published: October 2019

Von Willebrand factor multimers that are tethered on endothelial cells are ultra large, hyperactive and susceptible to ADAMTS13 cleavage. However, the morphology of acutely secreted ULVWF that are deposited on endothelial surface and the mechanism by which they persist under fluid shear stress remain elusive. In this study, we use immunofluorescence and electron microscopy to characterized HUVEC surface associated VWF. By conducting perfusion assays in a parallel plate flow chamber in the presence of an agonist and a fluorescent anti-VWF antibody, we directly visualized VWF secretion and dynamics in real time. Upon histamine or forskolin stimulation, VWF multimers secreted by confluent HUVEC formed extended strings parallel to flow direction. While strings formed independent of platelet adhesion, they bound platelets without requiring platelet activation. Quick-freeze, deep-etch electron microscopy of immuno-gold labeled VWF showed that multiple single strings can merge and form parallel or twisted bundles, suggesting lateral association between individual VWF multimers. These “strings” or “bundles of strings” were tethered to the plasma membrane by a limited number of anchoring points, remained stable for over 30 minutes of continuous laminar flow at the shear stress of 2.5dyn/cm2, and about 50% of these strings were disrupted (bent or washed away) by a reversal of flow direction. Static adhesion assays indicated that both “RGDS” binding integrin and P-selectin were involved in binding of CHO-P cells to ULVWF containing HUVEC supernatant. However, “RGDS” peptide, but not soluble P-selectin or a polyclonal P-selectin antibody by itself, could interfere with the formation of fluorescent VWF strings under flow. These observations suggest that “RGDS” binding integrins are implicated in the presentation of ultra-long VWF strings on endothelial lumen, and that multiple protein-protein interactions could contribute to this process.
Altered Von Willebrand Factor Subunit Proteolysis and Multimer Processing Associated with the Cys1599(2362)Phe Mutation in the B2 Domain.
Authors: De Marco Luigi , Casonato Alessandra , Gallinaro Lisa , Sztukowska Marita , Mazzucato Mario , Battiston Monica , Pagnan Antonio , Ruggeri Zaverio M.
Published: October 2019

Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband’s plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband’s plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband’s plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.
Role of Tissue Factor in Platelet Adhesion: Real-Time Perfusion Studies.
Authors: Tonda Raul , Galan Ana M. , Navalon Fulgencio , Munday Adam D. , Chen Junmei , Lopez-Vilchez Irene , Lopez Jose A. , Escolar Gines
Published: October 2019

Exposure of Tissue Factor (TF) to circulating blood and excessive thrombin generation are major contributors to acute thrombosis. Platelets also play a central role in the pathogenesis of occlusive events. Despite the importance of both components, convincing evidence of direct adherence of platelets onto TF immobilized on a surface is lacking. Here, we studied the interactions of platelets with different TF-rich substrata. Real-time perfusion techniques were applied to visualize thrombus formation and to evaluate the time necessary for platelets to both attach to the surface and to form thrombi. We used three different sources of TF as substrates:Thromborel S®, which is TF purified from human placenta (pTF) (at 0.4 ng/cm2);Innovin®, recombinant TF that has been relipidated (rTF) (at 0.4 ng/cm2); andTF expressed on the membranes of TF-transfected CHO cells (CHO-TF).

As negative controls, we used equivalent surfaces sprayed with 0.5% bovine serum albumin or cultured sham-transfected CHO cells (CHO-C-), respectively. Platelet-rich plasma (PRP) from blood anticoagulated with low-molecular-weight heparin was perfused over these substrata at a shear rate of 300s−1 for 5 min. Images were captured with a digital camera at high resolution and streamed to a computer. Continuous real-time photographs were taken during the full 5 min. Single frames were captured at predetermined times and the mean area of the platelet aggregates at these times was measured. Every experiment was replicated at least 4 times. Results are expressed as mean ± S.E.M. Under these conditions, platelets bound and formed thrombi on all substrata containing TF, although the time to aggregate formation and aggregate area varied depending on the substratum. Platelet deposition was minimal on the BSA-coated surfaces or on CHO-C- cells.

Initial platelet deposits on pTF were observed after 120 sec of perfusion. After 5 min, thrombi reached an area of 122±41 μm2. Relipidated rTF was less effective in supporting platelet deposition, requiring 135 seconds on average for the initiation of platelet aggregates. After 5 min, the average area of platelet aggregates was 34.3±9.6 μm2. Formation of platelet aggregates on CHO-TF cells was noticeable after 80 seconds. Aggregates formed at 5 min reached an area of 213±58 μm2. In conclusion, our studies demonstrate that platelets adhere to and aggregate on different TF substrata. The presence of other adhesive molecules in cell-derived TF preparations (pTF, CHO-TF) appears to accelerate the interaction of platelets with TF. These observations suggest that in addition to its role in blood coagulation, TF also has role in platelet adhesion by providing an adhesive surface at sites of vessel injury.

SAF2003-05780, SGR2005-00952, FIS PI040887, FIS CP04-0011, NHLBI 5R01HL064796.