Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression

Abstract Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with ∼2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an α-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of α-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0.05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

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Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression

Blood ◽

10.1182/blood.v91.10.3800.3800_3800_3807 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3800-3807

Author(s):

Chang-jun Hu ◽

Frank A. Baglia ◽

David C.B. Mills ◽

Barbara A. Konkle ◽

Peter N. Walsh

Keyword(s):

Binding Sites ◽

Factor Xi ◽

Specific Expression ◽

Platelet Factor ◽

Platelet Surface ◽

Tissue Specific ◽

Factor Xi Deficiency ◽

Plasma Factor ◽

Granule Membrane ◽

Granule Secretion

Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with ∼2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an α-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of α-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0.05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

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Platelets and Factor XI Bypass the Contact System of Blood Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615838 ◽

1999 ◽

Vol 82 (08) ◽

pp. 234-242 ◽

Cited By ~ 10

Author(s):

Peter Walsh

Keyword(s):

Blood Coagulation ◽

Coagulation Factor ◽

Bleeding Complications ◽

Ashkenazi Jews ◽

Factor Xi ◽

Contact Phase ◽

Platelet Factor ◽

Proteolytic Activation ◽

Factor Xi Deficiency ◽

Plasma Factor

IntroductionFactor XI is a plasma glycoprotein (concentration ∼30 nM) that was first identified by Rosenthal et al1 as a plasma coagulation factor deficiency in patients with abnormal hemostasis, particularly common among Ashkenazi Jews.2,3 In spite of recent advances in our understanding of the structure of factor XI and its gene, the structure-function relationships of the protein, and the molecular genetics of factor XI deficiency, considerable confusion about the physiologic role and clinical relevance of factor XI has arisen from both clinical and biochemical observations. One problem arises from the fact that, until recently, the only known pathway for activation of factor XI involved proteolytic activation by factor XIIa and interactions with coagulation proteins of the contact phase of blood coagulation.4-7 The problem arose from the clinical observation that patients with deficiencies of factor XI are subject to bleeding complications, whereas patients with deficiencies of the contact proteins are not.2,3,8-13 A related unanswered question concerns the lack of correlation in many reported patients between plasma levels of factor XI and the severity of clinical bleeding manifestations.2,3,8,9,13-15 In addition, some patients with severe factor XI deficiency experience significant bleeding complications, whereas others appear to be hemostatically normal. These clinical and biochemical observations have motivated investigations focused on alternative mechanisms for activation of factor XI independent of contact phase protein.19-22 Additional studies have focused on the identification and characterization of platelet factor XI, which is postulated to be an alternative splicing product of the factor XI gene. Platelet factor XI is present in platelet membranes and might substitute for plasma factor XI in hemostasis and account for the absence of bleeding complications in some patients with severe plasma factor XI deficiency.14,16-18,23-26 Studies addressing these two important and related problems are presented and discussed in this chapter.

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Two-Step Regulation of Ad4BP/SF-1 Gene Transcription during Fetal Adrenal Development: Initiation by a Hox-Pbx1-Prep1 Complex and Maintenance via Autoregulation by Ad4BP/SF-1

Molecular and Cellular Biology ◽

10.1128/mcb.00222-06 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4111-4121 ◽

Cited By ~ 80

Author(s):

Mohamad Zubair ◽

Satoru Ishihara ◽

Sanae Oka ◽

Katsuzumi Okumura ◽

Ken-ichirou Morohashi

Keyword(s):

Adrenal Cortex ◽

Binding Sites ◽

Orphan Nuclear Receptor ◽

Specific Expression ◽

Steroidogenic Factor 1 ◽

Tissue Specific ◽

Adrenal Cells ◽

Tissue Specific Expression ◽

And Function ◽

Intron 4

ABSTRACT The orphan nuclear receptor Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1) is essential for the proper development and function of reproductive and steroidogenic tissues. Although the expression of Ad4BP/SF-1 is specific for those tissues, the mechanisms underlying this tissue-specific expression remain unknown. In this study, we used transgenic mouse assays to examine the regulation of the tissue-specific expression of Ad4BP/SF-1. An investigation of the entire Ad4BP/SF-1 gene locus revealed a fetal adrenal enhancer (FAdE) in intron 4 containing highly conserved binding sites for Pbx-Prep, Pbx-Hox, and Ad4BP/SF-1. Transgenic assays revealed that the Ad4 sites, together with Ad4BP/SF-1, develop an autoregulatory loop and thereby maintain transcription, while the Pbx/Prep and Pbx/Hox sites initiate transcription prior to the establishment of the autoregulatory loop. Indeed, a limited number of Hox family members were found to be expressed in the adrenal primordia. Whether a true fetal-type adrenal cortex is present in mice remained controversial, and this argument was complicated by the postnatal development of the so-called X zone. Using transgenic mice with lacZ driven by the FAdE, we clearly identified a fetal adrenal cortex in mice, and the X zone is the fetal adrenal cells accumulated at the juxtamedullary region after birth.

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A Comparison of Murine and Human Factor XI

Blood ◽

10.1182/blood.v90.3.1055.1055_1055_1064 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1055-1064 ◽

Cited By ~ 1

Author(s):

David Gailani ◽

Mao-Fu Sun ◽

Yuehui Sun

Keyword(s):

Messenger Rna ◽

Human Factor ◽

Dextran Sulfate ◽

Specific Activity ◽

Expression System ◽

Contact Activation ◽

Factor Xi ◽

Specific Expression ◽

Factor Xi Deficiency ◽

Mammalian Expression

Factor XI is a plasma glycoprotein that is required for contact activation initiated fibrin formation in vitro and for normal hemostasis in vivo. In preparation for developing a mouse model of factor XI deficiency to facilitate investigations into this protease's contributions to coagulation, we cloned the complementary DNA for murine factor XI, expressed the protein in a mammalian expression system, and compared its properties with human recombinant factor XI. The 2.8-kb murine cDNA codes for a protein of 624 amino acids with 78% homology to human factor XI. Both recombinant murine and human factor XI are 160 kD homodimers comprised of two 80 kD polypeptides connected by disulfide bonds. Murine factor XI shortens the clotting time of human factor XI deficient plasma in an activated partial thromboplastin time assay, with a specific activity 50% to 70% that of the human protein. In a purified system, murine factor XI is activated by human factor XIIa and thrombin in the presence of dextran sulfate. Murine factor XI differs from human factor XI in that it undergoes autoactivation slowly in the presence of dextran sulfate. This is due primarily to murine factor XIa preferentially cleaving a site on zymogen factor XI within the light chain, rather than the activation site between Arg371 and Val372. Northern blots of polyadenylated messenger RNA show that murine factor XI message is expressed, as expected, primarily in the liver. In contrast, messenger RNA for human factor XI was identified in liver, pancreas, and kidney. The studies show that murine and human factor XI have similar structural and enzymatic properties. However, there may be variations in tissue specific expression and subtle differences in enzyme activity across species.

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Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.

Blood ◽

10.1182/blood.v104.11.1933.1933 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1933-1933

Author(s):

Catherine P.M. Hayward ◽

Nola Fuller ◽

Shilun Zheng ◽

Frederic Adam ◽

Samira Jeimy ◽

...

Keyword(s):

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Platelet Surface ◽

Disulfide Linkage ◽

Thrombin Cleavage ◽

Factor Va ◽

Plasma Factor ◽

Large Factor ◽

V Antigen

Abstract Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

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A G>A Transition in the Non-Coding Exon 1 of the Factor XI Gene as the Cause of Factor XI Deficiency in Three Afro-Caribbean Patients.

Blood ◽

10.1182/blood.v106.11.1788.1788 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1788-1788

Author(s):

Michael J. Mitchell ◽

Letian Dai ◽

Anwar Alhaq ◽

Geoffrey F. Savidge

Keyword(s):

Base Change ◽

Clinical Severity ◽

Molecular Defect ◽

Polymorphism Analysis ◽

Bleeding Tendency ◽

Translation Efficiency ◽

Factor Xi ◽

Factor Xi Deficiency ◽

Plasma Factor ◽

Exon 1

Abstract Factor XI deficiency (MIM 264900) is an autosomal bleeding disorder of variable clinical severity. In contrast to haemophilia A or B the clinical symptoms do not correlate well with plasma levels of factor XI; it is therefore difficult to predict the bleeding tendency from either the factor level or the molecular defect. FXI deficiency is particularly common in the Ashkenazi Jews with a heterozygous frequency of 9%, associated with two common founder mutations E117X (Type II) and F283L (Type III). However, factor XI deficiency is found in all ethnic groups, with causative mutations being highly heterogeneous - mutations having been described in all exons with the exception of the non-coding exon 1. In a study of >120 ethnically diverse factor XI deficient patients, three patients of Afro-Caribbean origin were found to be heterozygous for a G>A transition at nucleotide −53 within exon 1 of the factor XI gene. All three patients showed a low FXI:C on at least 3 different occasions (SM[female] 44.3–57.1, AB[female] 42.3–51.2 and GA[male] 70.3–72.9, Range 76–136u/dl). The 2 female patients were both reported to have a lupus anticoagulant which may explain the lower levels seen, although a lupus screen was negative. No variation within the coding sequence of the factor XI gene was detected. Two of the patients were heterozygous for the −403 G>T promoter polymorphism, whilst the remaining patient was homozygous for the −403 T allele and heterozygous for the −273 C>G polymorphism. Analysis of >50 factor XI alleles in patients of Afro-Caribbean origin failed to detect this base change in individuals with normal factor XI levels. Purine-rich sequences, such as that in exon 1 affected by the −53 G>A substitution, are known to form extremely stable minihairpin loops. These sequences /structures have been shown to be important as splicing enhancers and in mRNA stability, particularly in making them more resistant to nucleases. Within the 5′ untranslated region (5′-UTR) of genes they have been demonstrated to be important in modulating translation efficiency. The -53 G>A substitution is located just 10 bases prior to the start of the factor XI mRNA and any of these mechanism could potentially explain the causative nature of this change. The -53 G>A substitution is predicted to cause ‘slippage’ within the postulated minihairpin loop, potentially making it unstable. Further work is on-going to try and prove and explain the causality of this mutation. We speculate that the -53 G>A base change affects the normal processing of factor XI mRNA and, possibly in combination with the promoter polymorphisms, results in a mildly reduced plasma factor XI level.

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FoxA Proteins Regulate H19 Endoderm Enhancer E1 and Exhibit Developmental Changes in Enhancer Binding In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9601-9609.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9601-9609 ◽

Cited By ~ 12

Author(s):

Lingyun Long ◽

Brett T. Spear

Keyword(s):

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Fetal Liver ◽

Carcinoma Cells ◽

Specific Factor ◽

Specific Expression ◽

Tissue Specific ◽

Parietal Endoderm ◽

F9 Embryonal Carcinoma Cells

ABSTRACT Multiple enhancers govern developmental and tissue-specific expression of the H19-Igf2 locus, but factors that bind these elements have not been identified. Using chromatin immunoprecipitation, we have found two FoxA binding sites in the H19 E1 enhancer. Mutating these sites diminishes E1 activity in hepatoma cells. Additional chromatin immunoprecipitations show that FoxA binds to E1 in fetal liver, where H19 is abundantly expressed, but that binding decreases in adult liver, where H19 is no longer transcribed, even though FoxA proteins are present at both times. FoxA proteins are induced when F9 embryonal carcinoma cells differentiate into visceral endoderm (VE) and parietal endoderm (PE). We show that FoxA binds E1 in VE cells, where H19 is expressed, but not in PE cells, where H19 is silent. This correlation between FoxA binding and H19 expression indicates a role for FoxA in regulating H19, including developmental activation in the yolk sac and liver and postnatal repression in the liver. This is the first demonstration of a tissue-specific factor involved in developmental control of H19 expression. These data also indicate that the presence of FoxA proteins is not sufficient for binding but that additional mechanisms must govern the accessibility of FoxA proteins to their cognate binding sites within the H19 E1 enhancer.

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Uncovering tissue-specific binding features from differential deep learning

10.1101/606269 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mike Phuycharoen ◽

Peyman Zarrineh ◽

Laure Bridoux ◽

Shilu Amin ◽

Marta Losa ◽

...

Keyword(s):

Deep Learning ◽

Binding Sites ◽

Specific Binding ◽

Specific Expression ◽

Tissue Specific ◽

Dimensional Classification ◽

Branchial Arches ◽

Gradient Based ◽

Differential Binding

ABSTRACTMotivationTranscription factors (TFs) can bind DNA in a cooperative manner, enabling a mutual increase in occupancy. Through this type of interaction, alternative binding sites can be preferentially bound in different tissues to regulate tissue-specific expression programmes. Recently, deep learning models have become state-of-the-art in various pattern analysis tasks, including applications in the field of genomics. We therefore investigate the application of convolutional neural network (CNN) models to the discovery of sequence features determining cooperative and differential TF binding across tissues.ResultsWe analyse ChIP-seq data from MEIS, TFs which are broadly expressed across mouse branchial arches, and HOXA2, which is expressed in the second and more posterior branchial arches. By developing models predictive of MEIS differential binding in all three tissues we are able to accurately predict HOXA2 co-binding sites. We evaluate transfer-like and multitask approaches to regularising the high-dimensional classification task with a larger regression dataset, allowing for creation of deeper and more accurate models. We test the performance of perturbation and gradient-based attribution methods in identifying the HOXA2 sites from differential MEIS data. Our results show that deep regularised models significantly outperform shallow CNNs as well as k-mer methods in the discovery of tissue-specific sites bound in vivo.AvailabilityFor implementation and models please visit https://doi.org/10.5281/zenodo.2635463.

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Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽

10.1182/blood.v54.5.1015.bloodjournal5451015 ◽

1979 ◽

Vol 54 (5) ◽

pp. 1015-1022 ◽

Cited By ~ 2

Author(s):

JP Miletich ◽

WH Kane ◽

SL Hofmann ◽

N Stanford ◽

PW Majerus

Keyword(s):

Binding Sites ◽

Factor Xa ◽

Coagulation Factors ◽

Severe Bleeding ◽

Factor V ◽

Platelet Factor ◽

Platelet Surface ◽

Factor Va ◽

Number Of Binding Sites ◽

Stimulation Of

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

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Activation of Factor IX by Polymorphonuclear Leukocytes from a Patient with Chronic Granulocytic Leukemia, and from a Patient with Factor XI Deficiency

10.1055/s-0039-1687401 ◽

1979 ◽

Author(s):

H.S. Kingdon ◽

J.C. Herion

Keyword(s):

Polymorphonuclear Leukocytes ◽

Cell Suspensions ◽

Factor Ix ◽

Prolonged Storage ◽

Bleeding Tendency ◽

Factor Xi ◽

Chronic Granulocytic Leukemia ◽

Factor Xi Deficiency ◽

Plasma Factor ◽

Granulocytic Leukemia

We have previously reported that preparations of polymorphonuclear leukocytes (PMNs) from normal human blood are capable of activating Factor IX in the presence of calcium ion (Thrombosis Research, 13, 501-507, 1978). In the outrent studies, we demonstrated that cell suspensions subjected to freezing and thawing wete as active or more active than the suspensions subjected to ultrasonic disruption which were used previously. Furthermore, the Factor IX activating activity survived prolonged storage of the frozen cell suspensions. PMNs from a patient with chronic granulocytic leukemia contained potent Factor IX activator, as did PMNs from a patient with severe factor XI deficiency (plasma Factor XI < 1%). Preliminary experiments suggest that the Factor IX activator may reside in the granule fraction of the cells, since Factor IX activation was not observed when strontium was substituted for calcium, we conclude that Factor XIa> is not involved in this cellular activation phenomenon. Furthermore, the presence of an alternate mechanism for activating Factor IX in the blood of the Factor XI deficient patient may explain why the bleeding tendency in this deficiency state is so much milder than in Factor IX or Factor VIII deficiencies.(Supported in part by USPHS grants HL-16633, and DE-0 2668).

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