scholarly journals Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4139-4145 ◽  
Author(s):  
Zhenyin Tao ◽  
Yuandong Peng ◽  
Leticia Nolasco ◽  
Santiago Cal ◽  
Carlos Lopez-Otin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Peptide Bond ◽  
In Vitro Assays ◽  
Flow Conditions ◽  
Cub Domain ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Thrombospondin Motif

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Evaluation of ADAMTS-13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate

2003 ◽  
Vol 90 (12) ◽  
pp. 1204-1209 ◽  
Author(s):  
Jody Whitelock ◽  
Jing-fei Dong ◽  
Miguel Cruz
Keyword(s):  
Enzyme Activity ◽  
Von Willebrand Factor ◽  
Fluid Phase ◽  
Peptide Bond ◽  
Prolonged Incubation ◽  
Enzyme Cleavage ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryThe metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF), and is absent or severely reduced in the plasma of patients with thrombotic thrombocytopenia purpura (TTP). Under physiologic flowing conditions, the enzyme cleaves endothelial cell-derived ultra-large VWF multimers at the Y842/M843 peptide bond located in the A2 domain, where many mutations associated with Type 2A VWD cluster. These VWF mutants are more susceptible for cleavage activity, decreasing the large VWF multimers in the plasma. The susceptibility of a recombinant VWF-A2 domain to ADAMTS-13 and the potential application in detecting enzyme activity were investigated. In vitro, fluid phase cleavage of VWF by ADAMTS-13 requires denaturing conditions and prolonged incubation in order to estimate enzyme activity. We have measured ADAMTS-13 activity based on enzyme cleavage of a recombinant VWF-A2 domain under non-denaturing conditions. In our assay, enzyme activity was absent in plasma from congenital and acquired TTP patient, and blocked by each EDTA, monoclonal antibody VP-1 (peptide-specific antibody against residues 828-842 of VWF), and an ADAMTS-13 antibody purified from plasma of an acquired TTP patient. This novel recombinant VWF-A2 protein has potential utility as matrix for a rapid clinical measurement of plasma ADAMTS-13 activity.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

scholarly journals The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

2016 ◽  
Vol 116 (07) ◽  
pp. 87-95 ◽  
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Richard Farndale ◽  
Dominique Bihan ◽  
J. Sadler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Structural Features ◽  
High Shear ◽  
The Individual ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMultimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbD binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

AB0157 IGG FROM PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND SYSTEMIC SCLEROSIS HAVE AN INFLUENCE ON COAGULATION FACTORS IN HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS IN-VITRO

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1379-1379
Author(s):  
R. Hasseli ◽  
M. M. Fürst ◽  
P. Singh ◽  
U. Müller-Ladner ◽  
M. Kaps ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Tissue Factor ◽  
Mhc Class I ◽  
Lupus Erythematosus ◽  
Class I ◽  
Clotting Cascade ◽  
Von Willebrand ◽  
Willebrand Factor

Background:Endothelial cells from the microvasculature (hBMEC) of the brain show significant morphological and functional differences compared to EC from other anatomical areas. They are characterized by tight junctions, are not fenestrated and show less active transport mechanisms. On the other hand, the mitochondrial density is relatively high in hBMEC due to the high cerebral glucose metabolism.It could be already observed that interferon-α from SLE-sera induces the expression of MHC class I molecules on human dermal microendothelial cell line, but it is not known whether this also occurs on hBMEC. hBMECs can synthesize pro-inflammatory cytokines and chemokines such as IL-1β, but in lower concentrations than human umbilical vein endothelial cells.Patients suffering from systemic lupus erythematosus (SLE) or systemic sclerosis (SSc) show a wide spectrum of central nervous symptoms. Both, SLE and SSc, are characterised by different autoantibodies and endothelial vascular damage, especially in microvessels. 10-40% of patients with SLE suffer from lupus vasculopathy. Vascular dysfunction is one of the earliest pathological changes in SSc. Anti-endothelial autoantibodies (AECA) appear in SLE as well as in SSc and other connective tissue diseases. Research within the last years revealed that AECA play a critical role within the vascular pathogenesis of SLE and SSc. So far there is no evidence that AECA bind to hBMEC and it is not clear whether they have an effect on this special endothelial class.Objectives:In this project, we investigated if autoantibodies against hBMEC are detectable in SLE and SSc patients and if they have an influence on the activation of the endothelium by inducing adhesion molecules and on haemostasis by inducing factors of the clotting cascade.Methods:HiTrap Protein G HP antibody purification columns were used to purify IgG antibodies. Flow cytometry was used for analysis of autoantibodies against human cerebral microvascular endothelial cell line (hCMEC/D3). 26 sera of patients with SLE and 29 sera of patients with SSc were tested for presence of autoantibodies against hCMEC/D3. To analyse in vitro effects on hCMEC/D3, we measured changes in the expression of the following surface proteins: ICAM-1, VCAM-1, MHC class I and II, tissue factor, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA, each before and after three- and 24-hours incubation with IgG-fractions. IgG fractions of 12 SLE patients, 13 SSc patients and 13 healthy control persons (HC) were tested.Results:Autoantibodies against hCMEC/D3 were found in 21 of 26 patients with SLE (81%) and in 19 of 29 patients with SSc (66%) (p > 0.05) but not in healthy donors. After three hours incubation of hCMEC/D3 IgG-fractions, an upregulation of tissue factor by SSc-IgG (6.7% ± 5.2%) compared to HC-IgG (1.1% ± 2.8%, p < 0.01) and to SLE-IgG (1.6% ± 3.9%, p < 0.05), was detectable.There was no significant correlation between changes in surface protein expression and detection of ANA or of anti-hCMEC/D3 antibodies (p > 0.05).No change in expression of ICAM-1, VCAM-1, MHC class I and II, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA could be detected after incubation with IgG-fractions.Conclusion:Both, patients with SLE and patients with SSc showed autoantibodies against hBMEC. IgG fractions of patients with SSc, but not with SLE, induced an upregulation of tissue factor on the cell surface of hCMEC/D3. This could be an indicator for a direct pathogenic effect of AECA on hBMEC and might have an influence on haemostasis by activating the clotting cascade. Inhibition of these antibodies could reduce cerebral involvement of SSc.References:[1]Weksler BB, Subileau EA, Perriere N, et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. Faseb J 2005;19:1872-1874.Disclosure of Interests:Rebecca Hasseli: None declared, Magdalena Maria Fürst: None declared, Pratibha Singh: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Manfred Kaps: None declared, Franz Blaes: None declared, Tibo Gerriets: None declared, Marlene Tschernatsch: None declared

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 290-290 ◽  
Author(s):  
Anil K. Chauhan ◽  
Janka Kiucka ◽  
Alexander Brill ◽  
Meghan T. Walsh ◽  
Denisa D. Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Leukocyte Rolling ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adherent Leukocytes

Abstract von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

Deletion of Pro1648-Leu1650 in VWF73, the Conservative Substrate for ADAMTS13, Causes von Willebrand Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3939-3939
Author(s):  
Jian Su ◽  
Xia Bai ◽  
Ziqiang Yu ◽  
Zhaoyue Wang ◽  
Changgeng Ruan
Keyword(s):  
Von Willebrand Factor ◽  
Recombinant Expression ◽  
Point Mutations ◽  
Deletion Mutation ◽  
Von Willebrand Disease ◽  
Group I ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract The multimer distribution of Von Willebrand factor (VWF) in plasma is regulated by the specific VWF cleaving protease ADAMTS13, which cleaves at the Y1605-M1606 bond in the A2 domain of VWF under the shear stress, plays paramount roles in mediating platelet adhesion to the subendothelium during vascular damage. Quantitative deficiency or qualitative abnormity in VWF caused by the mutations in the VWF gene leads to von Willebrand disease (vWD). There exist three types of vWD. Type 1 vWD is characterized by the partial quantitative deficiency of VWF and normal multimers. Type 3 refers to complete deficiency of VWF. Type 2 vWD refers to the qualitative deficiency of VWF and is subdivided into types of 2A, 2B, 2M, 2N. Meanwhile, the subtype of 2A vWD is also subdivided into two groups regarding ADAMTS13-dependent proteolysis of VWF. Group I includes the mutations G1505R, S1506L, L1540P, V1607D, which hinder the multimer assembly and diminish the secretion of VWF while group II includes R1597W, R1597Q, G1505E, I1628T, E1628K, which make VWF more susceptible to ADAMTS13 -dependent proteolysis. All these published point mutations cluster in the A2 domain of VWF and the corresponding mutation mechanism upon VWF has been elucidated. We have identified a patient with bleeding symptoms and reduced plasma VWF antigen, factor VIII and ristocetin cofactor activity, compatible with clinical von Willebrand disease. Analysis of proband’s plasma VWF multimers in low resolution agarose gels demonstrated similar results compared to the healthy. The patient carried a heterozygous deletion mutation from position 1648 to 1650 resulting in loss of three consecutive amino acids (ProIleLeu) in the pre-pro-VWF. It has been demonstrated that the minimal substrate for ADAMTS13 is intact VWF73, a region from Asp1596 to Arg1668 of von Willebrand factor. The novel deletion mutation in this patient occurred in the intact VWF73 and its mutated effect upon cleavage by ADAMTS13 could be clarified by further experiments such as in vitro recombinant expression of mutated VWF and might strengthen our understanding of the interaction between VWF and ADAMTS13.

Effect of 3-Phenyl-2-Propene-1-ol on PGE2Release from Rat Cerebral Microvascular Endothelial Cells Stimulated by IL-1β

2006 ◽  
Vol 34 (04) ◽  
pp. 685-693 ◽  
Author(s):  
Jian-You Guo ◽  
Hai-Ru Huo ◽  
Bao-Sheng Zhao ◽  
Hong-Bin Liu ◽  
Lan-Fang Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cultured Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Antipyretic Effect ◽  
Positive Immunoreactivity ◽  
Von Willebrand ◽  
Willebrand Factor

Fever, an elevation in body temperature, is thought to be terminally mediated by prostaglandin E2(PGE2). Both Guizhi Tang (GZT) and its active fraction A (Fr.A) showed an antipyretic effect in rats. 3-Phenyl-2-propene-1-ol was one of the active compounds isolated from Fr.A. In the present study, we examined the influence of interleukin-1β (IL-1β) on prostaglandin E2(PGE2) release, and the effect of 3-phenyl-2-propene-1-ol on IL-1β-induced PGE2release from rat cerebral endothelial cells (rCMEC). Cultured rCMEC were used in the study. In vitro, cells express typical phenotypic markers of brain endothelium. Using a monoclonal antibody against von Willebrand factor, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured cells. rCMEC were incubated in M199 medium containing IL-1β in the presence or absence of 3-phenyl-2-propene-1-ol. After incubation, the conditioned media were collected and the amount of PGE2was measured by enzyme-linked immunosorbent assay (ELISA). IL-1β increased the production of PGE2in a dose- and time-dependent manner. 3-Phenyl-2-propene-1-ol significantly decreased IL-1β-induced PGE2release in a dose-dependent manner. Our results indicate that 3-phenyl-2-propene-1-ol inhibits the PGE2release from rCMEC stimulated by IL-1β, and may have an antipyretic effect.

P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2150-2156 ◽  
Author(s):  
Arnoldo Padilla ◽  
Joel L. Moake ◽  
Aubrey Bernardo ◽  
Chalmette Ball ◽  
Yongtao Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Chinese Hamster ◽  
Integrin Αvβ3 ◽  
Disastrous Consequence ◽  
Endothelial Cell Surface ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Thrombospondin Motif

Abstract von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin αvβ3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a αvβ3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF–P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.

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