Publications by authors named "Joel Moake"

54 Publications

Production and control of coagulation proteins for factor X activation in human endothelial cells and fibroblasts.

Sci Rep 2020 02 6;10(1):2005. Epub 2020 Feb 6.

Department of Bioengineering, Rice University, Houston, TX, USA.

Human endothelial cells (ECs) synthesize, store, and secrete von Willebrand factor multimeric strings and coagulation factor (F) VIII. It is not currently known if ECs produce other coagulation factors for active participation in coagulation. We found that 3 different types of human ECs in primary culture produce clotting factors necessary for FX activation via the intrinsic (FVIII-FIX) and extrinsic (tissue factor [TF]-FVII) coagulation pathways, as well as prothrombin. Human dermal fibroblasts were used as comparator cells. TF, FVII, FIX, FX, and prothrombin were detected in ECs, and TF, FVII, FIX, and FX were detected in fibroblasts. In addition, FVII, FIX, FX, and prothrombin were detected by fluorescent microscopy in EC cytoplasm (associated with endoplasmic reticulum and Golgi proteins). FX activation occurred on human umbilical vein EC surfaces without the addition of external coagulation proteins, proteolytic enzymes, or phospholipids. Tumour necrosis factor, which suppresses the generation of activated protein C and increases TF, augmented FX activation. Fibroblasts also produced TF, but (in contrast to ECs) were incapable of activating FX without the exogenous addition of FX and had a marked increase in FX activation following the addition of both FX and FVII. We conclude that human ECs produce their own coagulation factors that can activate cell surface FX without the addition of exogenous proteins or phospholipids.
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http://dx.doi.org/10.1038/s41598-020-59058-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005260PMC
February 2020

Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

Clin Kidney J 2018 Dec 7;11(6):791-796. Epub 2018 Mar 7.

Baylor College of Medicine, Houston, TX, USA.

A 3-month-old male infant developed an extremely severe episode of atypical hemolytic uremic syndrome (aHUS) associated with partial deficiencies of full-length complement factor H (FH; ∼15% of infant normal) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) (39% of normal) and autoantibodies reactive with both proteins. His FH and ADAMTS13 genes were normal, indicating that the partial deficiencies were acquired, probably as the result of autoantibodies against full-length FH and ADAMTS13. The child also had a homozygous deletion of the complement factor H-related (CFHR)3-CFHR1 portion in the complement factor H () gene cluster. He therefore had deficiency of CFHR proteins and autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) with an unusual early onset associated with a partial deficiency of ADAMTS13 and an anti-ADAMTS13 autoantibody. His clinical episode of aHUS responded to plasma infusion and subsequent treatment with mycophenolate and rituximab. We believe that this is the first report of DEAP-HUS in an infant with partial deficiencies in both ADAMTS13 and full-length FH acquired in association with autoantibodies to both proteins.
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http://dx.doi.org/10.1093/ckj/sfy010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275444PMC
December 2018

Correlating Conformational Dynamics with the Von Willebrand Factor Reductase Activity of Factor H Using Single Molecule Force Measurements.

J Phys Chem B 2018 11 24;122(47):10653-10658. Epub 2018 Oct 24.

Activation of proteins often involves conformational transitions, and these switches are often difficult to characterize in multidomain proteins. Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kD), is a complement control protein that regulates the activity of the alternative complement pathway. Different preparations of FH can also reduce the disulfide bonds linking large Von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In contrast, commercially available purified FH (pFH) has little or no VWF reductase activity unless the pFH is chemically modified by either ethylenediaminetetraacetic acid (EDTA) or urea. We used atomic force microscopy single molecule force measurements to investigate different forms of FH, including recombinant FH and pFH, in the presence or absence of EDTA and urea, and to correlate the conformational changes to its activities. We found that the FH conformation depends on the method used for sample preparation, which affects the VWF reductase activity of FH.
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http://dx.doi.org/10.1021/acs.jpcb.8b06153DOI Listing
November 2018

Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells.

J Biol Chem 2018 05 19;293(19):7195-7208. Epub 2018 Mar 19.

the Department of Bioengineering, Rice University, Houston, Texas 77005.

Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes , , and and decreased expression of This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (, , , , and ) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.
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http://dx.doi.org/10.1074/jbc.RA118.002639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949983PMC
May 2018

Complement Component C3 Binds to the A3 Domain of von Willebrand Factor.

TH Open 2018 ;2(3):e338-e345

Section of Cardiovascular Research, Department of Medicine, Baylor College of Medicine, Houston, Texas, United States.

von Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (~280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.
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http://dx.doi.org/10.1055/s-0038-1672189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508891PMC
January 2018

A new therapeutic strategy for atypical HUS.

Authors:
Joel L Moake

Blood 2017 07;130(3):243-244

RICE UNIVERSITY; BAYLOR COLLEGE OF MEDICINE.

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http://dx.doi.org/10.1182/blood-2017-06-783704DOI Listing
July 2017

Quantification of Von Willebrand Factor Cleavage by adamts-13 in Patients Supported by Left Ventricular Assist Devices.

ASAIO J 2017 Nov/Dec;63(6):849-853

From the *System Biology Institute, Seattle, Washington; †Bloodworks Northwest Research Institute, Seattle, Washington; ‡Division of Thrombosis Research, Department of Medicine, Baylor College of Medicine, Houston, Texas; §Texas Heart Institute, Baylor College of Medicine, Houston, Texas; ¶J.W. Cox Laboratory for Biomedical Engineering, Rice University, Houston, Texas; ‖Division of Hematology, Department of Medicine, University of Washington, School of Medicine, Seattle, Washington; and #Center for Advanced Heart Failure, University of Texas at Houston, Houston, Texas.

Patients supported by left ventricular assist devices (LVADs) often present with the loss of large von Willebrand factor (VWF) multimers. This VWF deficiency is believed to contribute to the bleeding diathesis of patients on LVAD support and is caused by excessive VWF cleavage by the metalloprotease ADAMTS-13 under high shear stress. However, only a small percentage of patients who have suffered the loss of large VWF multimers bleed. The actual rates of VWF cleavage in these patients have not been reported, primarily because of the lack of reliable detection methods. We have developed and validated a selected reaction monitoring (SRM) mass spectrometry method to quantify VWF cleavage as the ratio of the ADAMTS-13-cleaved peptide MVTGNPASDEIK to the ILAGPAGDSNVVK peptide. The rate of VWF cleavage was found to be 1.26% ± 0.36% in normal plasma. It varied significantly in patient samples, ranging from 0.23% to 2.5% of total VWF antigen, even though all patients had the loss of large VWF multimers. Von Willebrand factor cleavage was greater in post-LVAD samples from patients in whom bleeding had developed, but was mostly reduced in patients in whom thrombosis had developed. This SRM method is reliable to quantify the rate of VWF cleavage in patients on LVAD support.
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http://dx.doi.org/10.1097/MAT.0000000000000602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662488PMC
May 2018

Thrombotic thrombocytopenic purpura.

Nat Rev Dis Primers 2017 Apr 6;3:17020. Epub 2017 Apr 6.

Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, Katholieke Universiteit Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Thrombotic thrombocytopenic purpura (TTP; also known as Moschcowitz disease) is characterized by the concomitant occurrence of often severe thrombocytopenia, microangiopathic haemolytic anaemia and a variable degree of ischaemic organ damage, particularly affecting the brain, heart and kidneys. Acute TTP was almost universally fatal until the introduction of plasma therapy, which improved survival from <10% to 80-90%. However, patients who survive an acute episode are at high risk of relapse and of long-term morbidity. A timely diagnosis is vital but challenging, as TTP shares symptoms and clinical presentation with numerous conditions, including, for example, haemolytic uraemic syndrome and other thrombotic microangiopathies. The underlying pathophysiology is a severe deficiency of the activity of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), the protease that cleaves von Willebrand factor (vWF) multimeric strings. Ultra-large vWF strings remain uncleaved after endothelial cell secretion and anchorage, bind to platelets and form microthrombi, leading to the clinical manifestations of TTP. Congenital TTP (Upshaw-Schulman syndrome) is the result of homozygous or compound heterozygous mutations in ADAMTS13, whereas acquired TTP is an autoimmune disorder caused by circulating anti-ADAMTS13 autoantibodies, which inhibit the enzyme or increase its clearance. Consequently, immunosuppressive drugs, such as corticosteroids and often rituximab, supplement plasma exchange therapy in patients with acquired TTP.
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http://dx.doi.org/10.1038/nrdp.2017.20DOI Listing
April 2017

von Willebrand factor proteolysis by ADAMTS-13 in patients on left ventricular assist device support.

J Heart Lung Transplant 2017 04 13;36(4):477-479. Epub 2017 Jan 13.

BloodWorks Northwest Research Institute, Seattle, Washington; Division of Hematology, Department of Medicine, University of Washington School of Medicine, Seattle, Washington.

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http://dx.doi.org/10.1016/j.healun.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944844PMC
April 2017

Acquired von Willebrand syndrome associated with left ventricular assist device.

Blood 2016 06 3;127(25):3133-41. Epub 2016 May 3.

BloodWorks Northwest Research Institute, Seattle, WA; and Division of Hematology, Department of Medicine, School of Medicine, University of Washington, Seattle, WA.

Left ventricular assist devices (LVAD) provide cardiac support for patients with end-stage heart disease as either bridge or destination therapy, and have significantly improved the survival of these patients. Whereas earlier models were designed to mimic the human heart by producing a pulsatile flow in parallel with the patient's heart, newer devices, which are smaller and more durable, provide continuous blood flow along an axial path using an internal rotor in the blood. However, device-related hemostatic complications remain common and have negatively affected patients' recovery and quality of life. In most patients, the von Willebrand factor (VWF) rapidly loses large multimers and binds poorly to platelets and subendothelial collagen upon LVAD implantation, leading to the term acquired von Willebrand syndrome (AVWS). These changes in VWF structure and adhesive activity recover quickly upon LVAD explantation and are not observed in patients with heart transplant. The VWF defects are believed to be caused by excessive cleavage of large VWF multimers by the metalloprotease ADAMTS-13 in an LVAD-driven circulation. However, evidence that this mechanism could be the primary cause for the loss of large VWF multimers and LVAD-associated bleeding remains circumstantial. This review discusses changes in VWF reactivity found in patients on LVAD support. It specifically focuses on impacts of LVAD-related mechanical stress on VWF structural stability and adhesive reactivity in exploring multiple causes of AVWS and LVAD-associated hemostatic complications.
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http://dx.doi.org/10.1182/blood-2015-10-636480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920020PMC
June 2016

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor.

Phys Rev E 2016 Jan 21;93(1):012410. Epub 2016 Jan 21.

Department of Physics and Astronomy, Rice University, Houston, Texas 77005, USA.

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100dynes/cm^{2} for 3 min at 37^{∘}C). We conclude that under the shear conditions used (100dynes/cm^{2} for 3 min at 37^{∘}C), VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.
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http://dx.doi.org/10.1103/PhysRevE.93.012410DOI Listing
January 2016

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

J Immunol 2016 Jan 16;196(2):832-45. Epub 2015 Dec 16.

Department of Bioengineering, Rice University, Houston, TX 77005.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.
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http://dx.doi.org/10.4049/jimmunol.1500960DOI Listing
January 2016

Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

PLoS One 2015 16;10(10):e0140740. Epub 2015 Oct 16.

Department of Bioengineering, Rice University, Houston, Texas, United States of America.

The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140740PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608722PMC
June 2016

Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

PLoS One 2015 19;10(6):e0130749. Epub 2015 Jun 19.

Department of Bioengineering, Rice University, Houston, TX, 77005, United States of America.

Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130749PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474637PMC
May 2016

Ultralarge von Willebrand factor-induced platelet clumping and activation of the alternative complement pathway in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndromes.

Hematol Oncol Clin North Am 2015 Jun 12;29(3):509-24. Epub 2015 Mar 12.

Department of Bioengineering, Rice University, 6500 Main Street, Houston, TX 77030, USA. Electronic address:

The molecular linkage between ultralarge (UL) von Willebrand factor (VWF) multimers and the alternative complement pathway (AP) has recently been described. Endothelial cell (EC)-secreted and anchored ULVWF multimers (in long stringlike structures) function as both hyperadhesive sites that initiate platelet adhesion and aggregation and activating surfaces for the AP. In vitro, the active form of C3, C3b binds to the EC-anchored ULVWF multimeric strings and initiates the assembly on the strings of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b). In vivo, activation of the AP via this mechanism proceeds all the way to generation of terminal complement complexes (C5b-9).
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http://dx.doi.org/10.1016/j.hoc.2015.01.008DOI Listing
June 2015

Regulatory components of the alternative complement pathway in endothelial cell cytoplasm, factor H and factor I, are not packaged in Weibel-Palade bodies.

PLoS One 2015 24;10(3):e0121994. Epub 2015 Mar 24.

Department of Bioengineering, Rice University, Houston, Texas, United States of America; Baylor College of Medicine, Houston, Texas, United States of America.

It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF--but not factor H.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121994PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372330PMC
February 2016

Thrombotic microangiopathies and the linkage between von Willebrand factor and the alternative complement pathway.

Semin Thromb Hemost 2014 Jul 26;40(5):544-50. Epub 2014 Jun 26.

Department of Bioengineering, Rice University, Houston, Texas.

Molecular linkages between von Willebrand factor (VWF) and the alternative complement pathway (AP) have recently been discovered. Endothelial cell (EC)-anchored ultra-large (UL) VWF multimeric strings function as an activating surface for the AP. C3 (in active C3b form) binds to the EC-anchored ULVWF strings, and promotes the assembly of C3bBb (C3 convertase) and C3bBbC3b (C5 convertase). These linkages may help to explain enigmatic clinical problems related to thrombotic microangiopathies, including some cases of refractory thrombotic thrombocytopenic purpura (TTP), TTP associated with only mild-modest deficiencies of ADAMTS-13, the provocation (or exacerbation) of acute episodes in patients with the atypical hemolytic uremic syndrome, and thrombosis in paroxysmal nocturnal hemoglobinuria. Recent experiments have also demonstrated that complement factor H performs a dual role: participating in regulation of the AP by binding to EC-anchored ULVWF strings; and functioning as a reductase to decrease the size of soluble VWF multimers.
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http://dx.doi.org/10.1055/s-0034-1383547DOI Listing
July 2014

Treatment of refractory thrombotic thrombocytopenic purpura with N-acetylcysteine: a case report.

Transfusion 2014 May 9;54(5):1221-4. Epub 2013 Oct 9.

Department of Medicine, Section of Hematology Oncology, Baylor College of Medicine, Houston, Texas; The Hematology Service, Ben Taub General Hospital, Houston, Texas.

Background: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease resulting in systemic microvascular thrombosis. The disease is caused by excessive platelet (PLT) adhesion to ultra-large (UL) von Willebrand factor (VWF) multimers inadequately cleaved by the processing enzyme ADAMTS-13. While many cases respond to plasma exchange performed with or without concurrent corticosteroids, treatment of the 10% to 20% of patients with refractory disease is difficult. Experimental studies demonstrating that N-acetylcysteine (NAC) inhibits PLT binding to endothelial cell-secreted and anchored UL VWF multimers suggest that NAC may be useful in the treatment of TTP.

Case Report: A 44-year-old woman presented with malaise, confusion, chest and abdominal pain, and transient visual loss. Laboratory results and peripheral blood smear were consistent with TTP. The patient was begun on plasma exchange and corticosteroid treatment, but after 10 days the PLT count was still less than 10.0 × 10(9) /L and she developed a fever. Rituximab was initiated, but the patient's condition worsened and she became comatose. Antibiotics were initiated, but cultures remained sterile. After 3 days of coma and further clinical deterioration, treatment with NAC was begun. The patient received a loading dose of 150 mg/kg NAC intravenously (IV) over 1 hour. Within 18 hours the patient awakened abruptly and began communicating with medical personnel. Plasma exchange, corticosteroids, rituximab, and NAC infusion (150 mg/kg IV over 17 hr daily × 10 days) were continued and by Day 17 the PLT count was more than 50 × 10(9) /L. The patient fully recovered and was discharged on Day 31.

Conclusion: This is the first complete report of a TTP patient treated with NAC. NAC was a safe and effective supplementary treatment for refractory TTP in this patient.
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http://dx.doi.org/10.1111/trf.12440DOI Listing
May 2014

Interaction of Shiga toxin with the A-domains and multimers of von Willebrand Factor.

J Biol Chem 2013 Nov 4;288(46):33118-23. Epub 2013 Oct 4.

From the Department of Bioengineering, Rice University, Houston, Texas 77005.

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr(1605)-Met(1606) peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.
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http://dx.doi.org/10.1074/jbc.M113.487413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829160PMC
November 2013

Human complement factor H is a reductase for large soluble von Willebrand factor multimers--brief report.

Arterioscler Thromb Vasc Biol 2013 Nov 5;33(11):2524-8. Epub 2013 Sep 5.

From the Department of Bioengineering, Rice University, Houston, TX (L.N., J.N., J.M.); and Department of Pulmonary Medicine, M.D. Anderson Cancer Center, Houston, TX (S.F., V.A.-K.).

Objective: Ultralarge von Willebrand factor (vWF) strings are secreted by, and anchored to, stimulated human endothelial cells. A disintegrin and metalloprotease with thrombospondin domains-type 13 cleaves the ultralarge vWF strings into large soluble vWF multimers. Normal plasma contains a nonproteolytic reducing activity that subsequently rapidly diminishes the size of the large soluble vWF multimers.

Approach And Results: The vWF reductase activity was isolated from normal cryoprecipitate-poor plasma by chromatography and identified as the complement regulatory protein, factor H (FH), by mass spectroscopy, SDS-PAGE, and monospecific anti-FH antibody. Removal of FH from partially purified vWF reductase by immunoabsorption eliminated the reducing activity, and the activity was recovered in the eluates. Recombinant human FH reduced large soluble vWF multimers in a free thiol-dependent reaction that was not inhibited by a variety of protease inhibitors.

Conclusions: FH contributes to the reduction of large soluble vWF multimers.
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http://dx.doi.org/10.1161/ATVBAHA.113.302280DOI Listing
November 2013

Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.

PLoS One 2013 29;8(3):e59372. Epub 2013 Mar 29.

Department of Bioengineering, Rice University, Houston, TX, USA.

Background: Vascular endothelial cells (ECs) express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF) multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP) is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome) or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura). Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms.

Methodology/principal Findings: We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs) by real-time PCR: C3 and C5; complement factor (CF) B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative) complement pathways. We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition). We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb) on ULVWF strings.

Conclusions/significance: AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric strings. Our findings provide one possible molecular mechanism for clinical linkage between different types of thrombotic and complement-mediated disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612042PMC
September 2013

Mechanical activation of a multimeric adhesive protein through domain conformational change.

Phys Rev Lett 2013 Mar 5;110(10):108102. Epub 2013 Mar 5.

Department of Physics and Astronomy, Rice University, Houston, Texas 77005, USA.

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608271PMC
http://dx.doi.org/10.1103/PhysRevLett.110.108102DOI Listing
March 2013

Complement activation in thrombotic microangiopathies.

Br J Haematol 2013 Feb 1;160(3):404-6. Epub 2012 Nov 1.

Section of Benign Hematology, Division of Internal Medicine, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

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http://dx.doi.org/10.1111/bjh.12112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758746PMC
February 2013

Generation and breakdown of soluble ultralarge von Willebrand factor multimers.

Semin Thromb Hemost 2012 Feb 7;38(1):38-46. Epub 2012 Feb 7.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

Ultralarge von Willebrand factor (ULVWF) multimeric strings are rapidly secreted by, and anchored to, stimulated endothelial cells (EC), and are hyperadhesive to platelets until cleavage by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In ADAMTS-13-deficient familial and autoantibody-mediated thrombotic thrombocytopenic purpura (TTP), there is severely restricted cleavage of EC-anchored ULVWF-platelet strings. The small amount of active enzyme released from their EC cleaves ULVWF strings minimally just above EC surfaces, thus generating soluble ULVWF multimers that are 2.5 to 50 times longer than plasma von Willebrand factor (VWF) forms. Soluble ULVWF multimers (detected in TTP and several other disorders) are also hyperadhesive to platelets and can cause excessive platelet adhesion/aggregation. Without exogenous chemicals or extreme shear stress, soluble ULVWF multimers cannot be cleaved by ADAMTS-13 but can be de-assembled (reduced) in vitro, by a free thiol-containing molecule (>30 kD) present in the cryosupernatant fraction of plasma that is not ADAMTS-13, thrombospondin-1, albumin, cysteine, or glutathione. This reduction may prevent occlusion of the microvasculature by embolic soluble ULVWF multimers (± adherent/aggregated platelets). New inhibitors of platelet adhesion to EC-anchored ULVWF multimeric strings and soluble ULVWF include an aptamer, a nanobody, and N-acetylcysteine.
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http://dx.doi.org/10.1055/s-0031-1300950DOI Listing
February 2012

In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.

J Clin Invest 2012 Jan 12;122(1):408-18. Epub 2011 Dec 12.

Department of Bioengineering, University of California, Berkeley, California, USA.

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.
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http://dx.doi.org/10.1172/JCI58753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248292PMC
January 2012

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice.

J Clin Invest 2011 Feb 25;121(2):593-603. Epub 2011 Jan 25.

Puget Sound Blood Center, 921 Terry Ave., Seattle, Washington 98104, USA.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.
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http://dx.doi.org/10.1172/JCI41062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026714PMC
February 2011

Platelets in bloom.

Authors:
Joel Moake

Blood 2010 May;115(18):3650-1

Rice University, USA.

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http://dx.doi.org/10.1182/blood-2010-02-268714DOI Listing
May 2010

Thrombotic microangiopathies: multimers, metalloprotease, and beyond.

Authors:
Joel Moake

Clin Transl Sci 2009 Oct;2(5):366-73

Rice University, Houston, Texas, USA.

The pathophysiology of various types of thrombotic microangiopathies is coming progressively into focus. Therapeutic advances are likely to follow at a quickening pace. This discussion focuses on thrombotic thrombocytopenic purpura (TTP), the hemolytic-uremic syndrome (HUS), thrombotic microangiopathies associated with transplantation-immunosuppression or anti-angiogenesis therapy, and the preeclampsia/hemolysis-elevated liver enzymes and low platelets syndrome (HELLP).
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http://dx.doi.org/10.1111/j.1752-8062.2009.00142.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350759PMC
October 2009

Thrombotic thrombocytopenia purpura (TTP) and other thrombotic microangiopathies.

Authors:
Joel Moake

Best Pract Res Clin Haematol 2009 Dec;22(4):567-76

Rice University, Department of Bioengineering, Houston, TX 77005, USA.

Thrombotoic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterised by systemic platelet aggregation, organ ischaemia, profound thrombocytopenia (with increased marrow megakaryocytes) and fragmentation of erythrocytes. Haemolytic-uraemic syndrome (HUS) is another type of thrombotic microangiopathy accompanied by renal dysfunction. In adults, a thrombotic microangiopathy that clinically more often resembles HUS than TTP may follow: bone marrow or solid organ transplantation and immunosuppression with cyclosporine or tacrolimus; total-body irradiation; mitomycin; gemcitabine; multiple chemotherapeutic agents; or antiangiogenic/antineoplastic substances. This article discusses the thrombotic microangiopathies that have provided the most extensive molecular insights to date into pathophysiology. These are familial and acquired forms of TTP associated with deficient plasma von Willebrand factor (VWF)-cleaving metalloprotease (ADAMTS-13) activity; acquired diarrhoea-associated HUS; the thrombotic microangiopathies associated with cyclosporine/tacrolimus or bevacizumab; and the preeclampsia-HELLP (haemolysis-elevated liver enzymes-low platelets) syndrome.
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http://dx.doi.org/10.1016/j.beha.2009.07.004DOI Listing
December 2009