bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of the Non-Human Sialic Acid Neu5Gc into Human Leukemic Cells and Targeting by Natural Human Anti-Neu5Gc Antibodies.
Authors: Nguyen Dzung H. , Tangvoranuntakul Pam , Varki Ajit
Published: October 2019

Humans are incapable of synthesizing the common mammalian cell surface sialic acid N-glycolylneuraminic acid (Neu5Gc), due to an irreversible genetic mutation that occurred after our last common ancestor with great apes. Despite this, we found trace levels of Neu5Gc in certain normal human tissues, and higher levels in fetal and malignant tissues, apparently due to incorporation from dietary sources. Circulating anti-Neu5Gc antibodies also occur in most normal humans, with marked individual variations in levels. We now show that while normal human blood cells metabolically incorporate very little free Neu5Gc from the culture medium, human leukemic cell lines do so efficiently, displaying it on their cell surfaces, as detected by flow cytometry using a monospecific polyclonal chicken antibody, and confirmed chemically by HPLC. Specific deposition of IgG from Neu5Gc-reactive human sera onto Neu5Gc-expressing leukemic cells could be demonstrated. This was associated with lytic cell killing, apoptosis, and antibody-dependent cell-mediated cytotoxicity. Human sera with low levels of anti-Neu5Gc antibodies did not mediate such effects. These data show for the first time that the “natural” anti-Neu5Gc antibodies found in normal humans can be functionally active. The selective incorporation of Neu5Gc into leukemic cells could provide an approach for targeting leukemic cells in vivo, via naturally occurring Neu5Gc-specific antibodies. However, in contrast to unstimulated blood cells, activated human T-lymphocytes also incorporated some Neu5Gc, albeit to a lesser degree than leukemic cells, allowing IgG binding and complement deposition. Thus, exposure of rapidly dividing activated T cells to Neu5Gc could potentially affect ongoing immune responses. Since incorporation into leukemic cells was most efficient, it may still be possible to define a therapeutic window to selectively target malignant cells.
Hemostatic Effects of Fibrinogen-γ Chain Dodecapeptide-Conjugated Polymerized Albumin Particles In Vitro and in Vivo.
Authors: Okamura Yosuke , Watanabe Naohide , Takeoka Shinji , Suzuki Hidenori , Murata Mitsuru , Ikeda Yasuo , Handa Makoto
Published: October 2019

We have studied a prototype of platelet substitutes focusing on a dodecapeptide, HHLGGAKQAGDV (H12)1), which is specific for fibrinogen γ-chain carboxy-terminal sequence (γ 400–411). In this study, we conjugated H12 to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable carriers to produce particles having hemostatic ability, and evaluated their in vitro and in vivo effects.

H12 (H12; 9.6 x 103 /particles) containing cysteine to the N-terminal was conjugated to polyAlb (260 ± 60 nm), and the effect of H12-polyAlb on platelet thrombus formation was evaluated in vitro with thrombocytopenic whole blood ([platelet] = 2.0 x 104 /μL, anticoagulated with PPACK) under flow conditions (shear rate; 150 s−1). Thrombocytopenic rats were made by busulphan injection at a dose of 20 mg/kg, and a 2.5 mm length x 1.0 mm depth template-guided incision (QuikheelTM, Becton-Dickinson, San Jose, CA) was made 1 cm from the tip of tail. The tail was immersed in a 50 mL cylinder of saline and the time taken to stop bleeding was measured.

When thrombocytopenic blood in the presence of H12-polyAlb ([rHSA]=0.14 mg/mL) was flowed on collagen-plate, the surface coverage of DiOC6-labeled platelets evaluated by fluorescence microscopy was increased to 3.9 ± 1.1 % (n=3) from 2.1 ± 0.4 % (n=3) obtained in the absence of H12-polyAlb. In the same experiments, rhodamine-labeled H12-polyAlb was found to be involved in platelet-platelet interactions by binding to activated platelet surface, thus enhancing platelet thrombus formation. Next, in vivo hemostatic effect was tested by measuring tail bleeding time of thrombocytopenic rats 5 minutes after the intravenous administration of H12-polyAlb. The bleeding times of normal ([platelet] = 8.1 ±0.9 x 105 /μL) and thrombocytopenic rats ([platelet] = 2.0 ±0.3 x 105 /μL) were 187 ± 51 s and 609 ± 153 s (n=6), respectively. H12-polyAlb administration at a dose of 4 mg/kg significantly shortened the bleeding time to 342 ± 73 s (n=10), whereas polyAlb was without effects (553 ± 104 s, n=6). These results indicate that H12-polyAlb can be a suitable candidate for an alternative to human platelet concentrates infused into thrombocytopenic patients.
Long Distance Trans DNA Hypomethylation in Cyclin D1 Deregulated B Cell Malignancies.
Authors: Liu Hui , Wang Jin , Huang Jing , Epner Elliot M.
Published: October 2019

Cyclin D1 is a common target gene in B cell malignancies. Cyclin D1 is deregulated by translocation in most patients with mantle cell lymphoma (MCL) and 15–20% of patients with multiple myeloma (MM). Cyclin D1 is not expressed in normal lymphocytes. Gene targeting experiments in cyclin D1 overexpressing MCL and MM were carried out and genetic variants isolated which had lost the translocated or inserted 11:14 chromosomes. These clones no longer expressed cyclin D1 but expressed high levels of cyclin D3. Analysis of DNA methylation patterns in these clones demonstrated that the translocated chromosome exerts a long distance trans DNA hypomethylating effect on the normal chromosome (transvection) at the cyclin D1 locus and at least 100 kilobases upstream. Thus, in the absence of the translocated chromosome, the normal chromosome is densely DNA methylated. This long distance trans hypomethylating effect was also demonstrated in a MM cell line (U266) which contains an insertion rather than a translocation of IgH sequences. Combined FISH/ immunofluorescent antibody labelling studies have shown the presence of both the normal and translocated chromosome 11’s at the outer nucleolar membrane, where they are tethered by the proteins CTCF and nucleophosmin, as demonstrated using chromatin immunoprecipitation assays. Tethering of the translocated and nontranslocated chromosomes by CTCF/nucleophosmin provides a mechanism for pairing and long distance DNA transhypomethylation.
Correlation between Real-Time Microvascular Abnormalities and Whole Blood Viscosity (WBV) in Type-2 Diabetes Mellitus (T2DM) Patients.
Authors: Cheung Anthony T. , Ramanujam Sahana , Barbosa Michelle A. , Asfour Violet K. , Medina Maria V. , Miller Joshua W.
Published: October 2019

Novel technologies were used to study the microvascular abnormalities in Type-2 diabetes mellitus (T2DM) patients and to correlate these real-time pathological complications with whole blood viscosity (WBV) over a wide range of shear rates (1–1,000s−1). Computer-assisted intravital microscopy (CAIM) was used to non-invasively and objectively quantify the microvascular abnormalities in the conjunctival microcirculation in T2DM patients (n=12; age range = 45 to 68) and healthy non-diabetic control subjects, with the medical history of each subject blinded to the investigators. Fifteen recognizable microvascular abnormalities existed in T2DM patients and not in the control subjects, though not all the abnormalities were found in each patient. A severity index (SI) -- the arithmetic sum of microvascular abnormalities in each patient quantified via CAIM -- was computed to give a score to correlate with medical history, WBV and shear rates. T2DM SI (6.9±1.7) differed significantly (P<0.01) from control SI (0.6±0.7) and correlated significantly with disease severity, but not with the duration of the disease since diagnosis. The results, together with results from a previous study, lend support to the hypothesis that diabetic complications may have occurred in the pre-diabetic period; indicative of the existence of a “time window” before the onset of clinically detectable hyperglycemia. To adequately assess WBV to correlate with T2DM SI, a computer-assisted scanning capillary viscometer, the Rheolog™, was used to generate a viscosity profile for each patient or control subject over a range of shear rates (1 to 1,000s−1) using one 3-ml citrated venous blood sample from each subject. Based on the viscosity profiles generated, WBV of T2DM patients at various shear rates differed significantly from control values. For example, averaged WBV of T2DM patients at a shear rate of 300s−1 (4.01±0.33cp) differed significantly (P<0.02) from averaged WBV of control subjects (3.34±0.05cp). WBV correlated with SI of T2DM patients, disease severity and medical records. This correlation may have unique clinical implications - it can be used to study shear-stress induced pathogenesis on endothelial dysfunction in various vascular diseases and in early detection of T2DM in carriers (e.g. siblings of T2DM patients) before clinical diagnosis of T2DM during the pre-diabetic “time window” of early detection. Follow-up studies on the correlation between real-time microvascular abnormalities (SI), disease severity, WBV and shear rates in Alzheimer’s Disease patients and in early detection of T2DM are in progress.
Platelet-Derived Microparticles Induce Angiogenesis and Stimulate Post-Ischemic Revascularization.
Authors: Dashevsky Olga , Brill Alexander , Rivo Julia , Varon David
Published: October 2019

Platelet attachment to the subcellular matrix at injured sites of the vasculature is followed by their activation and release of microparticles. Platelet-derived microparticles (PMP) have been shown to be involved in the regulation of hemostasis. However, little is known about the role of PMP in the regulation of angiogenesis and related clinical conditions. We have recently demonstrated that platelets as a cellular system induce angiogenic responses both in vitro and in vivo. In the present study, we investigated the potential role of PMP in angiogenesis. A strong dose-dependent pro-angiogenic effect of PMP in the rat aortic ring model (5.3±2.1 mm2 surface covered with sprouting vessels versus 0.24±0.2 mm2 in the control, p<0.001) was observed. This effect was reversed by selective inhibition of VEGF, bFGF and PDGF (surface covered with vessels 0.7±0.5 mm2, 1.7±1.5 mm2, and 2.4±1.2 mm2, respectively, p<0.02 versus control), but not by inhibition of heparanase (5.1±0.8 mm2, p>0.5 versus control). PMP exert their stimulatory effect via PI3-kinase, Src kinase and ERK, whereas protein kinase C seems not to be involved, as judged by the aortic ring sprouting model. Using confocal and electron microscopy, we also demonstrate that PMP bind to non-activated endothelial cells. In addition, PMP markedly increased invasion of human endothelial cells through a layer of matrigel. This effect was abolished by an inhibitor of VEGF receptor tyrosine phosphorylation or laminaran sulfate (heparanase inhibitor). It was also partially reduced by PDGF blocking mAb, whereas blocking of bFGF had no effect. Furthermore, we have demonstrated that PMP induce angiogenesis in an in vivo model, in which beads (30 μl) of 4% agarose gel containing the substances under study were transplanted subcutaneously into mice. Image analysis of the capillary area revealed the following: control beads − 0.2±0.05 mm2, VEGF + bFGF containing beads − 4.8±1.1 mm2, PMP (100 μg/ml) containing beads − 5.1±1.3 mm2, p<0.001 versus control. The latter finding was further supported by immunohistochemical staining of the skin in the vicinity of the beads for von Willebrand factor, a marker of endothelial cells (control − 4.0±3.2, VEGF+bFGF − 12±4.4, PMP − 17±6.5 capillaries per view field, p<0.05 versus control). Finally, we explored the potential effect of PMP in a rat myocardial infarction model. Ischemia was induced by LAD ligation followed by injection of either PMP or PBS into the ischemic region. Preliminary evaluation of the LAD myocardial territory in sham-operated animals revealed 157±42.0 capillaries per view field. In contrast, number of capillaries observed 3 weeks after induction of ischemia was reduced to 34±21.5. When PMP were injected into the ischemic region, there was an increase in capillary number up to 97±27.3. In conclusion, PMP induce angiogenesis in both in vitro and in vivo models. Local injection of PMP into the ischemic myocardium may improve revascularization.
The Proadhesive Properties of Multimerin in Supporting Cellular Adhesion: Evidence for RGD and Non-RGD Dependent Adhesive Mechanisms.
Authors: Adam Frederic , Zheng Shilun , Joshi Nilesh , Suehiro Youko , Kelton David S. , Sandhu Amin , Jeimy Samira , Hayward Catherine P.M.
Published: October 2019

Multimerin is a large soluble protein, with an uncertain function, found in platelets, megakaryocytes, endothelium and extracellular matrix fibers but not in plasma. The observation that multimerin contains structural features of an adhesive protein, including an Arg-Gly-Asp (RGD) sequence, led us to investigate its ability to support adhesion of platelets, megakaryocytes, endothelial cells and other cell types. Multimerin had the ability to support the adhesion of both platelets and megakaryocytes and this required cellular activation and the multimerin RGD site. Studies of normal and Glanzmann platelets indicated that multimerin interacted with the major platelet integrin receptor, αIIbβ3 and radioimmunoprecipitation analyses confirmed that multimerin bound to αIIbβ3. Multimerin also supported adhesion of endothelial cells, neutrophils and other cells including smooth muscle cells, fibroblast cells, human embryonic kidney (HEK293) and epithelial cells. Unlike platelets, these cells do not express αIIbβ3; this indicated that other integrin or non-integrin receptors could be involved in cellular adhesion to multimerin. Comparisons of cell adhesion to wild-type and RGE-multimerin indicated that unlike platelets and megakaryocytes, some other cell types (e.g. endothelial cells, smooth muscle cells and neutrophils) were capable of adhering to RGE-multimerin. This suggested that cellular adhesion to multimerin occurs by both RGD and non-RGD dependent mechanisms. Finally, unlike platelets, megakaryocytes and neutrophils, adhesion of other cell types to multimerin did not require cellular activation. In conclusion, our data indicate multimerin has fairly broad proadhesive properties, involving RGD and non-RGD dependent mechanisms, and that cellular receptors including αIIbβ3 interact with multimerin to mediate its binding to activated platelets, endothelial cells and potentially other cell types.
ADP Signaling Defect in Children with Severe Acquired Aplastic Anemia: A Potential Common Pathway for Development of Aplastic Anemia.
Authors: Ford Jason C. , Wadsworth Louis D. , Schultz Kirk R.
Published: October 2019

Severe Acquired Aplastic Anemia is a rare disease with an incidence in British Columbia of 6.9 per million/year in Eastern/Southeastern Asians, 7.3 per million/year in South Asians and 1.7 per million/year in those of White/mixed ethnic descent. Two children presented with mucosal bleeding >4 years after achieving complete remission with immune suppressive therapy (antithymocyte globulin, cyclosporin A and prednisone). Evaluation revealed normal routine coagulation profiles and normal platelet numbers but mildly prolonged bleeding times (patients 8.5 - 11.0 minutes; control 3.0 – 8.0 minutes) and abnormal platelet aggregation as measured by aggregometry (optical and luminescence) after ADP stimulation. The patients had normal aggregation and ATP secretion after stimulation with thrombin (ATP secretion only), collagen, epinephrine, arachidonic acid, and ristocetin (1.125, 1.0, and 0.5 mg/mL concentrations, optical aggregation only) but showed abnormal aggregation with ADP (2.5, 5.0, and 10 μM). An additional 4 aplastic anemia patients were tested >1 year after successful immune suppressive therapy with no bleeding history: 1 of the 4 had the identical defect in platelet aggregation and ATP release after ADP stimulation, with no other defects on aggregometry. All 3 patients with abnormal platelet aggregation were of South or East Asian background, and the 3 with normal aggregation were of Caucasian/mixed ethnicity. Of the patients with an abnormal ADP platelet response, one has subsequently developed ulcerative colitis. Based on these observations we hypothesize that some patients with severe acquired aplastic anemia have an underlying defect in a signaling pathway shared by platelets, T cells, and B cells. ADP-induced signaling in platelets is induced through the surface molecule P2Y12, which is not expressed on lymphocytes. P2Y12 signaling occurs in association with a G protein Galphai2, an important signaling molecule in T and B cells and associated with development of ulcerative colitis in Galphai2 knock out mice. The specific signaling defect in platelets is currently being elucidated in these patients, and the incidence of these defects in aplastic anemia patients must be characterized.
Heparin-Induced Thrombocytopenia in Patients with Coronary Artery Disease: A Common but Underdiagnosed Syndrome.
Authors: Kim Min Ju , Park Se Hoon , Ahn Tae Hoon , Shin Eak Kyun , Bang Soo-Mee , Cho Eun Kyung , Shin Dong Bok , Lee Jae-Hoon
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is occurring in a relatively low frequency but is sometimes problematic clinically. The aims of this study were to know the epidemiology of HIT in Korean population and to gain some insight of the physicians’ awareness of this syndrome. We obtained the records of all patients treated with unfractionated heparin (UFH) for their acute coronary syndrome at the Gachon Medical School Gil Heart Center during 2002–2003. Eligible patients were required to have checked complete blood counts at least twice in their hospital courses and to have normal baseline platelet count. Patients who had unexplained (absence of all common causes) thrombocytopenia were considered to have clinical HIT. We also reviewed the medical record of all patients with thrombocytopenia to determine whether the treating-physicians have thought and/or ordered any laboratory evaluation for immune-mediated HIT. Among 874 consecutive patients treated with UFH, 272 (31.1%) had never followed their platelet count after admission and 58 (6.6%) had baseline thrombocytopenia. Thus, 554 patients had protocol-compliant data and were included in the study. Median duration of UFH administration was 6 days (range 1 to 41) and 62 (11.1%) patients developed thrombocytopenia. Among the 442 patients who received UFH for > 96 hours, 51 (11.5%) developed thrombocytopenia. In these patients with prolonged UFH administration, 16 had a hematology consult, 30 had suspected to have clinical HIT, and only one had a diagnosis of HIT in their medical records. In 21 patients, possible other causes for thrombocytopenia were evident. Even though this is a tertiary academic hospital, serologic tests for HIT was never ordered. Keeping in mind the limitations of retrospective analyses, the present study shows that thrombocytopenia is common in patients treated with UFH and that physician’s awareness of this syndrome is limited.
The Polymorphism of Platelet Membrane Integrin α2 β1(α2807TT) Is Associated with Premature Onset of Fetal Loss.
Authors: Gerhardt Andrea , Scharf Rudiger E. , Mikat-Drozdzynski Barbara , Krüssel Jan S. , Bender Hans G. , Zotz Rainer B.
Published: October 2019

Inherited thrombophilia could increase susceptibility to adverse pregnancy outcomes such as fetal loss. We determined the G1691A mutation of the factor V gene (FVL), the G20210A mutation of the prothrombin gene, the C677T polymorphism of the methylentetrahydrofolate-reductase (MTHFR) gene, the HPA-1 polymorphism of the b subunit of the platelet integrin αIIbβ3 and the C807T polymorphism of the α subunit of integrin α2β1 in 104 women with fetal loss and 277 normal women. In a subgroup analysis of women with recurrent early fetal loss (n=34), the prevalence of the genetic markers did not differ significantly between the women with early fetal loss and the normal women. However, in this subgroup of patients, the onset of fetal loss was significantly earlier in women with the α2807TT genotype (7.1 ± 1.9 vs. 8.8 ± 1.5 weeks, p=0.001). No such significant difference was observed in carriers of the other genetic markers. In the subgroup analysis of women with late fetal loss (n=70), only the prevalence of heterozygous FVL was significantly associated with late fetal loss (odds ratio 3.2, p=0.002). There was no significant association of any genetic risk factor with premature fetal loss in the subgroup analysis of women with at least one late miscarriage. This study demonstrates a significant association of the α2807TT genotype of the platelet membrane integrin α2β1 with premature onset of early fetal loss. Based on our results it appears that this risk factor does not induce the pathomechanism but modulates the course of fetal loss. Furthermore, our study confirms the association of FVL with late fetal loss.
Localization of Protein Z (PZ) In Situ in Human Neoplastic Tissues.
Authors: Sierko Ewa , Tokajuk Piotr , Ramlau Rodryg , Zimnoch Lech , Kisiel Walter , Wojtukiewicz Marek
Published: October 2019

Tumor cells synthesize and release procoagulants that lead to multi-factorial activation of factor X and subsequent thrombin generation and fibrin formation that in turn contribute to tumor growth and metastatic spread. The activity of factor Xa is inhibited by TFPI (tissue factor pathway inhibitor). Another mechanism of direct inhibition of factor Xa involves protein Z (PZ). Protein Z is a 62-kD, vitamin K-dependent plasma nonpeptidase glycoprotein, which, in complex with PZ-dependent protease inhibitor (ZPI), inhibits factor Xa in the presence of Ca2+ and phospholipids. However, data on the presence of the PZ in tumor malignant tissue are lacking. Results obtained from randomized clinical trials indicate that inhibition of blood coagulation may improve the results of anticancer therapy in selected tumor types. The purpose of the study was to evaluate the localization of PZ in situ in colon, breast, gastric, laryngeal, pancreatic, renal, endometrial cancer, non-small cell lung cancer (NSCLC), malignant melanoma and glial neoplasms. Studies were performed on tumor fragments obtained at surgical treatment of proviously untreated patients. Immunohistochemical procedures using the ABC method employed a specific polyclonal antibody directed against human PZ antigen. Protein Z was localized in cancer cell bodies in all types of malignant tumors examined. Staining intensity was more pronounced in less differentiated cancer cells of anaplastic gliomas. In contrast, more differentiated cancer cells of gastric cancer revealed stronger staining than more malignant ones. Cancer cell bodies of NSCLC and renal cancer were characterized by weak intensity of staining for PZ. In the case of pancreatic and renal cancer, as well as malignant melanoma, the intensity of staining for PZ in cancer cell bodies was irregular: both high and low expression of PZ was observed independent of the degree of malignancy. The expression of PZ was also revealed in association with tumor infiltrating macrophages in colon cancer, NSCLC and malignant melanoma. Furthermore, PZ was localized in small blood vessels of colon, renal, endometrial, gastric cancer, NSCLC, malignant melanoma and glial neoplasms. The observed localization of PZ, particularly in association with cancer cells of all malignant tumors examined, suggests that this protein may exert regulatory effects on tumor growth and metastatic dissemination.
Experimental Study of Fibrinolytic Factors and Its Inhibitors in Rabbits’ Tissues Extract.
Authors: Xu Xiaohua , Huang Liansheng , Lu Xingguo , Xu Genbo , Zhao Xiaoying
Published: October 2019

Objective: To study the expression character of fibrinolytic factors and its inhibitors in rabbits’ tissues extract.

Methods: Tissue plasminogen activator ( tPA ), Plasminogen activator inhibitor-1 ( PAI-1 ), Plasminogen ( Plg ), Plasmin ( Pl ) and α2 Plasmin inhibitor ( α2PI ) were measured by colorimetric assay.

Results: The tissues extract in renal, small intestine, lung, brain and spleen showed strong fibrinolytic function, and expressed markedly high activity levels of tPA, Plg and Pl. But striped muscle, tongue and stomach expressed markedly high activity levels of PAI-1 and α2PI, and showed low fibrinolytic character. There were linear correlation between levels of tPA and PAI-1 ( r=−0.8076, P<0.01 ), Pl and αPI ( r=−0.3278, P<0.05 ), Plg and Pl ( r=0.6811, P<0.01 ) in tissues extract fibrinolytic targets. In relative tissues, renal cortex and renal marrow were significantly high level of tPA and low level of PAI-1, but levels of Plg and Pl in cortex renalis were higher than in renal marrow, the α 2PI level in renal marrow were higher than in renal cortex ( P<0.05~0.01 ). The levels of tPA, Plg and Pl in small intestine were higher than tin large intestine ( P<0.05~0.01 ), but the levels of PAI-1 and α 2PI in small intestine were markedly lower than in large intestine ( P<0.01 ). These indicated that fibrinolytic function in small intestine was stronger than in large intestine. Although the fibrinolytic function in muscle tissue was low, but the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than in striped muscles, and the levels of PAI-1 and α 2PI were obviously lower than in striped muscle ( P<0.05~0.01 ). These indicated that the fibrinolytic function in cardiac muscle were stronger than in the striped muscle.

Conclusion: The fibrinolytic character existed significant difference in tissues, it has profound significance in the function of heamostasis and thrombosis and physiologic function in tissues.
A Quantitative Trait Locus for Cholesterol/Low Density Lipoprotein within the Promoter of the Factor IX Gene.
Authors: Viel Kevin R. , Khachidze Manana , Buil Alfonso , Soria José Manuel , Souto Juan Carlos , Fontcuberta Jordi , Blanco-Vaca Francisco , Ordoñez-Llanos Jordi , Almasy Laura , Tom Howard
Published: October 2019

The correlation between vitamin K-dependent coagulation factors (CF) and lipids has been previously established, with Factor VII notably receiving attention in the investigation of atherosclerosis. Current evidence suggests that plasma phospholipids (PL) may enhance the procoagulant reactions and may increase the availability of fat soluble vitamin K. PL may also affect the clearance of CF. There appears to be a concomitant decrease in CF as the result of lipid lowering therapies. While investigating single nucleotide polymorphisms (SNP) of the Factor IX (fIX) gene as potential quantitative trait loci (QTL) for fIX activity level (fIX:C) in the GAIT study, we observed an apparent relationship between two SNPs, G-793A and C-698T, with plasma cholesterol concentration. As expected, a similar relationship existed with low density lipoprotein (LDL) concentrations. In the current study, we examine these fIX promoter SNPs, which exhibit near perfect linkage disequilibrium (ρ=0.998, p<0.0001), as a possible QTL for cholesterol and LDL. We performed variance components analyses using Solar. We observed significant (p<0.05) marginal associations between LDL and factor VIII activity level (fVIII:C), fIX:C, cholesterol, VLDL, triglycerides, and BMI. We observed significant marginal associations between cholesterol and fVIII:C, fIX:C, LDL, VLDL, triglycerides, lipoprotein A (Lp(a)), and BMI. There was no association for smoking status or contraceptive use. For LDL, the most parsimonious model included age, age2, sex, age2 by sex, fIX:C, and the SNP covariate (coded −1,0,1). C(−698)T was significantly associated with LDL levels (nominal p=0.0262) and these covariates accounted for 33% of the total variance of LDL, with a similar result for G(−793)A. For cholesterol, the most parsimonious model included age, age2, fIX:C, Lp(a), and the SNP covariate. C(−698)T was significantly associated with LDL levels (nominal p=9.54 X 10−3) and these covariates accounted for 40% of the total variance of cholesterol, with a similar result for G(−793)A. Interestingly, these SNPs showed no association with fIX:C. Even though an increase in fIX:C results in an increase in cholesterol in this study, it is the same unit increase regardless of which allele is present, i.e. there is no interaction. The results of a linkage analysis for markers on the X chromosome were insignificant. Though this finding is intriguing and interesting with regard to the correlation between the vitamin K-dependent coagulations factors and lipids, it is entirely possible that it is by chance alone. These findings and the correlation between lipids and the vitamin K-dependent coagulation factors merit verification in an independent study population.
Quantitative Evaluation of Factor VIII in Factor VIII Products.
Authors: Butenas Saulius , Parhami-Seren Behnaz , Gissel Matthew T. , Gomperts Edward D. , Mann Kenneth G.
Published: October 2019

Several factor VIII products, recombinant and natural, have been used for hemophilia A treatment worldwide. Typically, two activity-based assays (factor Xase and aPTT) are used for the assessment of factor VIII concentration in these products. Frequently, the results are dependent upon the assay and its modifications in different laboratories. In this study, we evaluated five pharmacologic factor VIII products (three lots of each) in three activity-based assays and in two immunoassays for the concentration and activity of factor VIII protein. Two factor VIII products were plasma-derived (Immunate and Hemofil M) and three were recombinant; two of these contained full-length factor VIII (Recombinate and Kogenate) and one was B-domainless (ReFacto). Albumin-free full-length recombinant factor VIII was used as a standard in all assays. In the factor Xase assay, all recombinant factor VIII products and Immunate at 1U/ml (indicated by manufacturer) showed activity similar to that of 0.7nM (1U/ml) standard, whereas activity of Hemofil M was 64–68% of the standard. In the aPTT assay both full-length recombinant products and Hemofil M displayed activity similar to the standard, whereas Immunate had increased (142% of standard) and ReFacto decreased (83% of standard) activity. In synthetic plasma, all three recombinant products had standard-like activity, whereas Hemofil M and Immunate were slightly more active than standard. The ELISA immunoassay revealed that the factor VIII protein content in Recombinate, Kogenate and Hemofil M corresponded to the units assigned by manufacturers (1.4–1.6x1012U/mol vs1.4x1012U/mol calculated for standard), whereas the specific activity of Immunate was 50% of that expected (0.7x1012U/mol). In contrast, the specific activity of ReFacto was almost 3-fold that of full-length factor VIII (4.0x1012U/mol). The data of this study indicate that: 1) factor VIII activity estimated in different assays gives dissimilar results; 2) the specific activity of factor VIII in various factor VIII products is different and, as a consequence, administration of an equal factor VIII activity in U/ml means the administration of different amounts of factor VIII protein.
Von Willebrand Factor Levels Do Not Increase with Age in Patients with Type 1 Von Willebrand Disease.
Authors: Tarng Hong I. , Taylor Lynne , Konkle Barbara A.
Published: October 2019

A number of inherited and acquired factors modulate von Willebrand factor antigen (VWF:Ag) levels, including blood type, race, activity and stress level, thyroid hormone status, and, in women, time in menstrual cycle. In reported studies a positive correlation between VWF:Ag and/or factor VIII levels and age has been demonstrated, with an increase of 5 – 6 IU/dL per decade (Conlan MG et al, 1993; Kamphuisen PW et al, 1998). Those studies have primarily assessed VWF and factor VIII as risk factors for ischemic heart disease, cerebrovascular disease, and venous thromboembolism. None of the subjects had von Willebrand disease (VWD). Their VWF:Ag levels were in the higher normal or elevated range. The purpose of this study was to determine whether there is a relationship between age and VWF:Ag level in patients with Type 1 VWD. We collected the data from 36 patients who were diagnosed with type 1 VWD and followed at the Penn Comprehensive Hemophilia and Thrombosis Program up to a period of 13 years (See Table 1 below). For each patient, date of birth, VWF:Ag levels with corresponding test dates were collected by reviewing the medical histories and the lab results. Test results obtained during pregnancy, DDAVP testing, or during prophylaxis or therapy for bleeding control were excluded. One year was set as the observation period, so the adjacent VWF:Ag levels that were tested less than one year were excluded from the dataset. When two test results were available on a patient within a one-year period, the lower test result was used.

To investigate whether there was a relationship between VWF:Ag levels and age, cross-sectional analyses (across each visit) and longitudinal analyses were performed using scatter plots, Spearman and Pearson correlations, and regression analysis. No significant increase in VWF:Ag levels with age was demonstrated. The fact that we did not find an increase in VWF:Ag levels over time in our patients could be due to the relatively small number of patients studied or could reflect a subtype of VWD, due to our selection criteria. Only patients with abnormal values were included. Some patients have a prior diagnosis of VWD and bleeding symptoms, but have normal values when tested. Since these patients are adults, this may be due, at least in part, to an age-related increase. Type 1 VWD may occur secondary to decreased VWF synthesis and/or clearance. It is possible that age-related effects on VWF levels will differ depending on the underlying factor(s) resulting in a lower VWF level. Further studies correlating a patient’s values longitudinally with the underlying pathophysiology of their disease would aid in our understanding of their bleeding risks over time.

Patient # Age at Last Visit, range (mean) Females (%) Race % (Cauc/AA/Other) VWF:Ag mean 36 17–70 (34) 89 78/19/3 49%
Effectiveness of DDAVP in Von Willebrand’s Disease.
Authors: Balasubramanian Lakshmi , Boggio Lisa N. , Foiles Nancy , Green David
Published: October 2019

Objective: Of the inherited bleeding disorders von Willebrand’s disease (VWD) is the most common, with a prevalence of one in 10,000. Desmopressin (1-deamino-8-D-arginine vasopressin [DDAVP]) and a variety of plasma-derived concentrates of Factor VIII (FVIII) and von Willebrand factor (VWF) have been used to treat VWD. A prospective multicenter European study recently reported the effectiveness of DDAVP to be 27% in type 1 VWD and 18% in all type 2, which suggests that DDAVP is less effective than previously thought. Here we report our experience in a retrospective analysis of patients with known VWD challenged with DDAVP.

Methods: Intranasal DDAVP was administered to 20 patients with known VWD who were studied before and 2 hours after dosing. A response to DDAVP was defined by a 2-fold increase in both FVIII concentration and ristocetin cofactor activity (VWF:RCo) as well as attainment of a level of at least 30 IU/dL. In addition, 6 subjects had bleeding time (BT) tests performed. Statistical analysis was performed using one-tailed multivariate Student’s t-test.

Results: Three of the 20 patients were excluded from the study: 2 had acquired VWD and 1 inadequate data. Of the 17 evaluable patients, 13 were type 1 and four type 2 (confirmed by multimer analyses). Overall response in the 17 patients was 59% (p<0.001); 13 subjects had FVIII levels that increased to more than 80 IU/dl, and 9 subjects had VWF:RCo levels of more than 50 IU/dl. Responses were observed in 8 of the 13 (62%, p<0.001) subjects with type 1 disorder and 2 of the 4 with type 2. The BT was >10 min prior to DDAVP and decreased significantly, to ≤7.5 min, in 5 of the 6 patients after DDAVP. Even when more stringent requirements for defining a response were used (a 3 fold increase and a level of at least 30 IU/dL of both FVIII and VWF:RCo activity, as used by the European multicenter study), 5 of 13 (39%, p=0.01) of the type 1 patients had a response. The average increase in responders in VWF:RCo was 3.33 fold and FVIII, 4.43 fold.

Conclusions: The majority of patients with VWD have a significant biological response to intranasal DDAVP; a challenge test with intranasal drug identifies responders who should be considered for treatment with this agent.
The Prevalence and Clinical Significance of Inherited Thrombophilic Risk Factors in Patients with Antiphospholipid Syndrome.
Authors: Diz-Kucukkaya Reyhan , Inanc Murat , Pekcelen Yuksel
Published: October 2019

Antiphospholipid syndrome (APS) is defined as the occurrence of thrombosis and/or recurrent fetal losses in association with the presence of antiphospholipid antibodies (APLA). Although it has been suggested that APLA have a pathogenic role in the thrombotic complications of APS, pathogenicity of APLA has not been conclusively proven. It has been speculated that other inherited or acquired thrombogenic risk factors might influence the development of thrombosis in patients with APS. In the present study, we examined the effect of well known inherited thrombophilic risk factors (inherited protein C (PC), protein S (PS) and antithrombin (AT) deficiencies; factor V Leiden (FVL) G1691A mutation, and prothrombin G20210A mutation) in the development of thrombosis in APS patients. Seventy-three definite APS patients with arterial and venous thrombosis (group 1: APS patients with thrombosis), 29 antiphospholipid antibody-positive patients with first trimester abortus and/or thrombocytopenia and no history of thrombosis (group 2: APLA-positive patients without thrombosis), and 126 healthy controls (group 3) were included into the study. PC, PS, and AT deficiencies were screened with functional assays; the presence of FVL mutation and prothrombin mutation were detected by polymerase chain reaction. PS and AT activities were found to be normal in all groups. Only a single APS patient with thrombosis had been found to have PC deficiency, PC activities were normal in both APLA-positive patients without thrombosis and healthy controls. The frequencies of FVL A allele for APS patients with thrombosis, APLA-positive patients without thrombosis, and healthy controls were 10.4%, 6.8%, and 4.9%, respectively. The frequency of FVL A allele was significantly higher in APS patients with thrombosis compared with healthy controls (10.4% vs 4.9%, p= 0.02 with chi-square test). The frequencies of prothrombin A allele for APS patients with thrombosis, APLA-positive patients without thrombosis and healthy controls were 3.4%, 0%, and 1.3%, respectively. Although the frequency of A allele was higher in APS patients with thrombosis compared with both APLA-positive patients without thrombosis and healthy controls, it was not statistically significant. Our results showed that inherited PC, PS, AT deficiencies, and prothrombin G20210A mutation are not common in patients with APS. FVL G1691A mutation may contribute to the development of thrombosis in a small group of APS patients. This study suggests that the known inherited thrombophilic risk factors are not responsible for the development of thrombosis in the majority of APS patients. Further prospective studies in larger cohorts of patients are needed to delineate the exact role of thrombophilic mutations in the development of thrombosis in APS.
Presence of a T383G Mutation in the Vitamin K Epoxide Reductase Gene (VKORC1) in a Patient Resistant to Four Different Vitamin K Antagonists.
Authors: Samama Michel-Meyer M.M. , Bodin Laurent L. , Horellou Marie Helene M.H. , Parent Florence F. , Kereveur Anne A. , Beaune Philippe P. , Loriot Marie Anne M.A. , Conard Jacqueline J.
Published: October 2019

Resistance to vitamin K antagonists is a rare disorder which until recently has not been associated to a genetic factor. The existence of inherited warfarin resistant rat strains has been related to mutations of the genes involved in the vitamin K cycle. Vitamin K dependent carboxylase was cloned in 19911, while human vitamin K epoxide reductase complex 1 (VKORC1), another enzyme implicated in vitamin K cycle, has been cloned more recently2,3. Mutations in this gene are responsible for both human and rat warfarin resistance 3. In 1997, we reported 4 a 63-year old patient with recurrent pulmonary embolism and deep vein thrombosis without known hereditary or acquired thrombophilia who was found resistant to warfarin (up to 45mg/day), fluindione (up to 80mg/day), acenocoumarol (up to 12 mg/day) and phenprocoumon (up to 30 mg/day). With phenprocoumon, long-acting vitamin K antagonist, drug concentration reached 85 mg/L (usual range 1–5 mg/L) but the INR remained around 1. Daily low molecular weight heparin (LMWH) was continued 3 months. Treatment was discontinued on 2 occasions and a recurrent thrombotic episode was observed. The patient is now receiving a single daily dose of low molecular weight heparin (80 mg Enoxaparin, body weight 120 kg, 67 IU/kg once a day) for the past 10 years without thrombotic or bleeding episodes. Osteodensitometry remains normal after 9 years of treatment. Careful biochemichal investigation had demonstrated a deficiency in vitamin K dependent carboxylation and absence of blockade of vitamin K epoxide reductase by phenprocoumon. This year, we sequenced the three exons of VKORC1 in this patient and detected a heterozygous T383G transversion resulting in a leucine to arginine substitution (L128R). This mutation has been recently identified by Rost3 et al. in an individual with warfarin resistance. It was not found in 259 control subjects that we had tested. Interestingly, another gene mutation of the VKORC1 can be responsible for a combined deficiency of vitamin K dependent clotting factors 3. In conclusion, a resistance to all vitamin K antagonists, including warfarin up to 45 mg/day is extremely rare and the mutation T383G of the VKORC gene has been reported in only one patient before the case reported here. Testing for mutation in VKORC1 will help in explaining some cases of anti vitamin K resistance.
Development of Non-Anticoagulant Derivatives of Biotechnology Derived Sulfaminoheparosan Derivatives to Investigate the Non-Anticoagulant Effects of these Agents.
Authors: Cornelli Umberto , Maddineni Jyothi , Vana Laurel , Hoppensteadt Debra , Florian-Kujawski Michelle , Fareed Jawed
Published: October 2019

It is now well known that heparin and related glycosaminoglycans produce some of their non-anticoagulant effects such as the anti-inflammatory, antiproliferative and regulatory actions independent of endogenous serpine (AT and HC-II). Sulfaminoheparosans (SAH) represent a class of biotechnology derived bacterial capsular polysaccharide (K5) derivatives which are epimerized and sulfated to mimic heparin’s biologic effects and exhibit affinity to both AT and HC-II. These SAH can be produced to exhibit molecular profile mimicking the low molecular weight heparins (LMWHs). The disaccharide unit structure of epimerized SAH is GlcA-GlcNSO36SO3(3,6)H) in comparison to heparin which is IdoA2SO3-GlcNSO36SO3. The anticoagulant and antiprotease profile of a 6 kDa SAH has been found to be similar to Tinzaparin (Maddineni et al. Clin Appl Thromb Hemost 10(1):27–37,2004). As this agent also exhibits various other biologic effects such as the release of TFPI, modulation of adhesion molecules and antiproliferative effects, it was hypothesized that the non-anticoagulant forms of this agent may exhibit some of these effects. In order to produce the non-anticoagulant derivatives in 6 kDa SAH, desulfated derivatives were prepared by removing sulfate groups on the position 2 alone (2 desulfated) and positions 2 and 6 (2,6 desulfated derivatives). Additional modifications in the 2,6 desulfated derivatives included the presence of either free amino group or N-acetyl in position 2. These modifications did not result in the molecular weight profile of the desulfated derivatives. However, in comparison to the parent 6 kDa SAH both the 2-desulfated and 2,6 desulfated derivatives were weaker in the anticoagulant (PT, PTT, Heptest) and antiprotease (anti-Xa, anti-IIa) and protease generation assays. 2,6 desulfation produced a strong decrease in the anticoagulant effects. Furthermore, the desulfation was proportional to a decrease in the affinity to AT and HC-II. None of these agents produced any activation of ADP induced platelet aggregation, however in whole blood flowcytometric studies ADP induced aggregation was augmented by 6 kDa SAH whereas the mono and disulfated derivatives produced variable effects. Heparinase I and II did not produce any digestion of any of these derivatives. Incubation of the 6 kDa SAH and the 2 desulfated and 2,6 disulfated derivatives produced varying degrees of inhibition of the Lewis-lung carcinoma cell cultures which was not dependent on degree of sulfation. These studies suggest that the non-serpine mediated effect of these agents may be independent of the sulfation pattern and the relative anticoagulant effects of these sulfaminoheparosan.
Intracellular Co-Localization of Factor VIII and Von Willebrand Factor Is Necessary for In Vivo FVIII Secretion.
Authors: Lamont Patricia A. , Ragni Margaret V.
Published: October 2019

Although the extracellular association of Factor VIII (FVIII) and Von Willebrand Factor (VWF) is well established, the intracellular interaction of FVIII and VWF is not well understood. Recently, the importance of intracellular co-localization of FVIII and VWF for in vitro FVIII secretion was demonstrated in endothelial cell lines. Whether intracellular co-localization of FVIII and VWF is required for in vivo FVIII secretion, however, is not known. We previously showed that liver transplantation leads to phenotypic cure of hemophilia A, by virtue of FVIII production in the allograft liver. Because FVIII is synthesized only in the allograft liver but not in endothelial cells of transplant recipients, and VWF is synthesized in extrahepatic tissue, this is an ideal model to study whether co-localization of FVIII and VWF is required for in vivo FVIII secretion. We, therefore, studied FVIII and VWF response after desmopression (DDAVP) infusion, administered at 0.3 mcg/kg by intravenous infusion over 30 minutes, in each of two men with severe hemophilia A (FVIII:C <0.01 U/ml) who had undergone orthotopic liver transplantation for endstage liver disease six months earlier. Both men had HIV and hepatitis C co-infection and were clinically well, with mildly elevated liver function tests, and FVIII:C levels >30% following transplantation. Coagulation studies, drawn before and after DDAVP, revealed that VWF:RCoF and VWF:Ag, but not FVIII:C, increased after DDAVP administration (see Table). The prolonged aPTT and correction in a 1:1 aPTT mix confirmed the absence of an inhibitor in these subjects. The lack of FVIII response to DDAVP supports previous in vitro work, and demonstrates for the first time that intracellular co-localization of FVIII and VWF is essential for in vivo FVIII secretion. These data also suggest that extrahepatic FVIII synthesis is necessary for in vivo response of the DDAVP releasable pool of FVIII. By contrast, co-localization does not appear to be necessary for VWF secretion. Although it is not possible to exclude that a chronic, exhaustive post-transplant increase in VWF may have limited VWF response to DDAVP, it is clear that FVIII did not increase following DDAVP. These findings have important implications for the design of gene therapies for hemophilia A and Von Willebrand Disease.

Subject Demographic Sample aPTT aPTT mix FVIII:C VWF:RCoF VWF:Ag 01-BW 32yoM Hem A Pre-DDAVP 44.4 sec 37.7 sec 0.50 U/ml 2.17 U/ml 2.42 U/ml HIV+/HCV+ Post-DDAVP 44.8 sec 37.4 sec 0.48 U/ml 2.91 U/ml 2.91 U/ml 02-PB 36yoM Hem A Pre-DDAVP 49.5 sec 38.0 sec 0.32 U/ml 1.61 U/ml 2.16 U/ml HIV+/HCV+ Post-DDAVP 50.8 sec 38.5 sec 0.30 U/ml 2.20 U/ml 2.50 U/ml