scholarly journals Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma

2018 ◽  
Vol 118 (03) ◽  
pp. 471-479 ◽  
Author(s):  
Chantal Clark ◽  
Mirjam Mebius ◽  
Steven de Maat ◽  
Aloysius Tielens ◽  
Philip de Groot ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Binding Studies ◽  
Direct Binding ◽  
Self Association ◽  
A Disintegrin And Metalloproteinase ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.

Baboon Fibrinogen Adsorption and Platelet Adhesion to Polymeric Materials

1991 ◽  
Vol 65 (05) ◽  
pp. 608-617 ◽  
Author(s):  
Joseph A Chinn ◽  
Thomas A Horbett ◽  
Buddy D Ratner
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Polymeric Materials ◽  
Platelet Suspension ◽  
Perfusion Chamber ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Fibrinogen Adsorption

SummaryThe role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode’s buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and min radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA).When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma.When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well.Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i. e., serum, heat defibrinogenated plasma, and congenitally afibrinogénémie plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma.While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4862-4869 ◽  
Author(s):  
Mia Golder ◽  
Cynthia M. Pruss ◽  
Carol Hegadorn ◽  
Jeffrey Mewburn ◽  
Kimberly Laverty ◽  
...  
Keyword(s):  
Ferric Chloride ◽  
Von Willebrand Disease ◽  
Expression Vectors ◽  
Severe Thrombocytopenia ◽  
Normal Platelet ◽  
Injury Model ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

scholarly journals Activated platelets induce Weibel-Palade–body secretion and leukocyte rolling in vivo: role of P-selectin

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2334-2339 ◽  
Author(s):  
Vandana S. Dole ◽  
Wolfgang Bergmeier ◽  
Heather A. Mitchell ◽  
Sarah C. Eichenberger ◽  
Denisa D. Wagner
Keyword(s):  
Acute Inflammation ◽  
Endothelial Activation ◽  
Leukocyte Rolling ◽  
Activated Platelets ◽  
Baseline Levels ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Body Secretion

AbstractThe presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin-/- platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Endothelial P-selectin is stored with von Willebrand factor (VWF) in Weibel-Palade bodies. The release of Weibel-Palade bodies on infusion of activated platelets was indicated by both elevation of plasma VWF levels and by an increase in the in vivo staining of endothelial P-selectin. We conclude that the presence of activated platelets in circulation promotes acute inflammation by stimulating secretion of Weibel-Palade bodies and P-selectin–mediated leukocyte rolling.

scholarly journals Assessment of the Dual Role of Clumping Factor A in S. Aureus Adhesion to Endothelium in Absence and Presence of Plasma

2018 ◽  
Vol 118 (07) ◽  
pp. 1230-1241 ◽  
Author(s):  
Jorien Claes ◽  
Bartosz Ditkowski ◽  
Laurens Liesenborghs ◽  
Tiago Veloso ◽  
Jose Entenza ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Binding Protein ◽  
Protein A ◽  
Bacterial Proteins ◽  
Fibronectin Binding Protein ◽  
A Disintegrin And Metalloproteinase ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractAdhesion of Staphylococcus aureus to endothelial cells (ECs) is paramount in infective endocarditis. Bacterial proteins such as clumping factor A (ClfA) and fibronectin binding protein A (FnbpA) mediate adhesion to EC surface molecules and (sub)endothelial matrix proteins including fibrinogen (Fg), fibrin, fibronectin (Fn) and von Willebrand factor (vWF). We studied the influence of shear flow and plasma on the binding of ClfA and FnbpA (including its sub-domains A, A16+, ABC, CD) to coverslip-coated vWF, Fg/fibrin, Fn or confluent ECs, making use of Lactococcus lactis, expressing these adhesins heterologously. Global adherence profiles were similar in static and flow conditions. In the absence of plasma, L. lactis-clfA binding to Fg increased with shear forces, whereas binding to fibrin did not. The degree of adhesion of L. lactis-fnbpA to EC-bound Fn and of L. lactis-clfA to EC-bound Fg, furthermore, was similar to that of L. lactis-clfA to coated vWF domain A1, in the presence of vWF-binding protein (vWbp). Yet, in plasma, L. lactis-clfA adherence to activated EC-vWF/vWbp dropped over 10 minutes by 80% due to vWF-hydrolysis by a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 and that of L. lactis-fnbpA likewise by > 70% compared to the adhesion in absence of plasma. In contrast, plasma Fg supported high L. lactis-clfA binding to resting and activated ECs. Or, in plasma S. aureus adhesion to active endothelium occurs mainly via two complementary pathways: a rapid but short-lived vWF/vWbp pathway and a stable integrin-coupled Fg-pathway. Hence, the pharmacological inhibition of ClfA-Fg interactions may constitute a valuable additive treatment in infective endocarditis.

scholarly journals The role of ADAMTS13 testing in the diagnosis and management of thrombotic microangiopathies and thrombosis

Blood ◽  
2018 ◽  
Vol 132 (9) ◽  
pp. 903-910 ◽  
Author(s):  
Camila Masias ◽  
Spero R. Cataland
Keyword(s):  
Thrombotic Microangiopathies ◽  
Acute Setting ◽  
Artery Disease ◽  
A Disintegrin And Metalloproteinase ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Deficient Activity

Abstract ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13) is a metalloprotease responsible for cleavage of ultra-large von Willebrand factor (VWF) multimers. Severely deficient activity of the protease can trigger an acute episode of thrombotic thrombocytopenic purpura (TTP). Our understanding of the pathophysiology of TTP has allowed us to grasp the important role of ADAMTS13 in other thrombotic microangiopathies (TMAs) and thrombotic disorders, such as ischemic stroke and coronary artery disease. Through its action on VWF, ADAMTS13 can have prothrombotic and proinflammatory properties, not only when its activity is severely deficient, but also when it is only moderately low. Here, we will discuss the biology of ADAMTS13 and the different assays developed to evaluate its function in the context of TTP, in the acute setting and during follow-up. We will also discuss the latest evidence regarding the role of ADAMTS13 in other TMAs, stroke, and cardiovascular disease. This information will be useful for clinicians not only when evaluating patients who present with microangiopathic hemolytic anemia and thrombocytopenia, but also when making clinical decisions regarding the follow-up of patients with TTP.

High Density Lipoprotein and Apolipoprotein A-I Bind Von Willebrand Factor and Prevent Its Self-Association Into Thick Fibers

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 194-194
Author(s):  
Dominic W. Chung ◽  
Ying Zheng ◽  
Junmei Chen ◽  
Minhua Ling ◽  
Xiaoyun Fu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Density Lipoprotein ◽  
Density Lipoprotein ◽  
Fluid Phase ◽  
Thrombocytopenic Purpura ◽  
Apolipoprotein A ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand factor (VWF) is a large plasma protein secreted constitutively or upon activation by endothelial cells. On activated endothelium, a portion of the VWF molecules remain attached to the surface, where they self-associate to produce long hyperadhesive strands and fibers capable of capturing platelets and other blood cells if not removed by the plasma metalloprotease ADAMTS13. Failure to clear these fibers underlies the pathophysiology of many microangiopathies, most prominently thrombotic thrombocytopenic purpura, in which accumulation of VWF-platelet aggregates in the microvasculature leads to ischemia and organ failure. VWF self-association also plays a major role in the adsorption of purified VWF to surfaces. We observed that adsorption of VWF to surfaces began with contact and direct binding of VWF to the surface via hydrophobic interactions. After saturating the binding sites on the surface, VWF continued to bind to and associate with the immobilized VWF molecules via hydrophilic VWF-VWF interactions, depositing multiple layers of VWF molecules on the surface until essentially all VWF molecules were depleted from the fluid phase. Using the method of Magnani and coworkers, we eluted VWF molecules that were bound to the surface via VWF-VWF interactions with the ionic detergent SDS, and then eluted VWF molecules that were bound to the surface via VWF-surface interactions with the zwitterion detergent CHAPS. Quantification of the eluted VWF showed that self-association of VWF onto the surface accounted for >80% of the adsorptive loss. Using the disappearance of purified VWF from the fluid phase as a measurement of self-association, we fractionated human plasma by heat and identified that apolipoprotein A-I (ApoAI), the major apolipoprotein component in high density lipoprotein (HDL) particles, which is stable to heat at 100° C, stabilized and prevented VWF surface adsorption by interfering with VWF self-association. Commercial preparations of ApoAI and HDL, prepared without exposure to heat, similarly prevented adsorption of purified VWF to surfaces. Half-maximal stabilization occurred at a molar ratio of eight ApoAI molecules to each VWF subunit. We assessed the role of HDL in VWF self-association that leads to the assembly of ultra-large VWF (ULVWF) strings on the surface of phorbol myristyl acetate-stimulated endothelial cells in flow chambers. We observed that HDL reduced the number and length of platelet-decorated ULVWF strings on the endothelial surface, consistent with the ability of HDL to interfere with VWF self-association and ULVWF assembly. We also studied the role of ApoAI in the recruitment of fluid-phase VWF to hyperadhesive ULVWF fibers in a synthetic microvessel system. We observed that ApoAI directly bound to hyperadhesive transluminal ULVWF fibers under flow, and this binding completely blocked the recruitment of fluid phase VWF molecules to the immobilized ULVWF fibers. These results showed that ApoAI or HDL interacted with hyperadhesive forms of VWF and modified the adhesive properties of the ULVWF strings and fibers. Consistent with its antithrombotic properties, the level of ApoAI in patients with hyperadhesive forms of VWF, such as thrombotic thrombocytopenic purpura and sepsis, was significantly reduced. These results suggest that regulation of VWF self-association may be another mechanism by which HDL protects against cardiovascular disease and extends its protective effects from large arteries to the microvasculature. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The distal carboxyl-terminal domains of ADAMTS13 are required for regulation of in vivo thrombus formation

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5323-5329 ◽  
Author(s):  
Fumiaki Banno ◽  
Anil K. Chauhan ◽  
Koichi Kokame ◽  
Jin Yang ◽  
Shigeki Miyata ◽  
...  
Keyword(s):  
Shear Rate ◽  
Genetic Background ◽  
Thrombus Formation ◽  
Short Form ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Terminal Domains

Abstract ADAMTS13 is a multidomain protease that limits platelet thrombogenesis through the cleavage of von Willebrand factor (VWF). We previously identified 2 types of mouse Adamts13 gene: the 129/Sv-strain Adamts13 gene encodes the long-form ADAMTS13 having the same domains as human ADAMTS13, whereas the C57BL/6-strain Adamts13 gene encodes the short-form ADAMTS13 lacking the distal C-terminal domains. To assess the physiologic significance of the distal C-terminal domains of ADAMTS13, we generated and analyzed 129/Sv-genetic background congenic mice (Adamts13S/S) that carry the short-form ADAMTS13. Similar to wild-type 129/Sv mice (Adamts13L/L), Adamts13S/S did not have ultralarge VWF multimers in plasma, in contrast to 129/Sv-genetic background ADAMTS13-deficient mice (Adamts13−/−). However, in vitro thrombogenesis under flow at a shear rate of 5000 s−1 was accelerated in Adamts13S/S compared with Adamts13L/L. Both in vivo thrombus formation in ferric chloride–injured arterioles and thrombocytopenia induced by collagen plus epinephrine challenge were more dramatic in Adamts13S/S than in Adamts13L/L but less than in Adamts13−/−. These results suggested that the C-terminally truncated ADAMTS13 exhibited decreased activity in the cleavage of VWF under high shear rate. Role of the C-terminal domains may become increasingly important under prothrombotic conditions.

Adenovirus Induced Thrombocytopenia: The Role of P-Selectin and von Willebrand Factor in Virus- Mediated Platelet Clearance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 222-222 ◽  
Author(s):  
Maha Othman ◽  
Andrea Labelle ◽  
Ian Mazzetti ◽  
David Lillicrap
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Critical Role ◽  
Flow Cytometric Analysis ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Acute thrombocytopenia has been consistently reported following IV administration of adenoviral vectors (Ad) but the mechanism responsible for this phenomenon has not been elucidated. Thrombocytopenia appears 24 hours after IV administration of Ad and is vector dose dependent. In this study, we have assessed the potential roles of the adhesive proteins P-selectin and von Willebrand Factor (VWF) on the aggregation and clearance of platelets following virus administration. We have addressed the question of whether the thrombocytopenia is due to a direct effect of the virus on platelets or an indirect effect related to interaction of platelets with other proteins or cells modified by the virus. We assessed platelet count in a group of Balb/c and C57Bl/6 mice over 1 week period following Ad administration and performed a detailed examination of the events within the first 24 h after Ad injection, the period that precedes the appearance of thrombocytopenia. We examined the effect of Ad on expression of the platelet activation marker P-selectin and the formation of platelet leukocyte aggregates (PLA) by means of flowcytometry after incubation of adenovirus with mouse platelets in vitro, and following Ad administration in vivo. To assess the role of VWF in Ad-induced thrombocytopenia we measured plasma VWF levels one hour after injection of Ad. Further investigations involved comparison of platelet counts, platelet activation, and the formation of PLA in a group of VWF KO mice. All studies have been performed with a replication deficient E1/E3-deleted Ad 1x 1011 viral particles/mouse. Our in vitro studies have shown that Ad directly activates mouse platelets as shown by increased expression of P-selectin. The average index of platelet activation for platelets stimulated by Ad was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. Our in vivo studies have confirmed the development of significant thrombocytopenia in both Balb/c as well as C57Bl/6 WT mice (n=8, p=0.00001, n= 6, p=0.002) 24 hours following Ad administration. Significant P-selectin expression was documented in both strains (n=4,p=0.0003; n=3, p=0.0008 respectively) as well as significant PLA one hour following Ad (n=4, p=0.01; n=3, p=0.007). The VWF KO mice showed non-significant thrombocytopenia (n= 6, p=0.063) at 24 hours following Ad, significant P-selectin expression (n=3, p=0.0003), but no significant PLA formation at one hour (n=3 p=0.12) relative to pre-injection levels. Plasma VWF levels were significantly elevated in both Balb/c and C57Bl/6 WT mice one hour following administration of the virus (n= 3, p=0.02; n= 3, p= 0.001). The average plasma VWF levels were 48.1 U/mL at 1h compared to 5.7 U/mL pre injection in Balb /c mice and 85.9 U/mL compared to 6.1 U/mL in C57Bl/6 mice. These studies have shown that Ad can act as an inducer of mouse platelet activation and as a promoter for platelet-leukocyte association both in vitro and in vivo. We have demonstrated a role for Ad in stimulating VWF release from the endothelium, and have shown that VWF has a critical role in platelet activation and clearance following Ad administration. We conclude that P-selectin and VWF proteins are directly involved in interactions between endothelial cells, platelets and leukocytes, a complex interaction that can explain at least in part the mechanisms underlying Ad-mediated thrombocytopenia.

The Effect of von Willebrand Factor Normandy Mutations on Intracellular Co-Trafficking of Factor VIII to Storage Organelles.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 765-765
Author(s):  
Maartje van den Biggelaar ◽  
Birgit Ruhdorfer ◽  
Jan Voorberg ◽  
Koen Mertens
Keyword(s):  
Von Willebrand Factor ◽  
Solid Phase ◽  
Critical Role ◽  
Laser Scanning Microscopy ◽  
Hek293 Cells ◽  
Confocal Laser ◽  
Binding Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand Factor (VWF) serves a critical role in factor (F)VIII life-cycle. It is generally believed that FVIII and VWF assemble in the circulation after secretion from vascular endothelial cells (VWF) and hepatocytes (FVIII). However, during the last few years evidence has accumulated in favor of significant extra-hepatic FVIII sources that might be compatible with in vivo biosynthesis and co-storage of FVIII and VWF within the same cell. In agreement with this view, in vitro co-expression of FVIII and VWF results in the storage of FVIII in VWF-containing organelles. We have recently demonstrated that this intracellular co-trafficking is independent on Tyr1680 of the FVIII molecule which is required for extracellular high-affinity interaction to VWF (van den Biggelaar et al. JTH, in press). In the present study, we studied the requirements for intracellular co-trafficking of VWF and FVIII in more detail by addressing VWF variants which have been established as being deficient in extracellular FVIII binding (Normandy variants). To facilitate intracellular visualization, we made use of fluorescently tagged FVIII (FVIII-YFP) and VWF (VWF-CFP). Four different VWF-CFP Normandy variants (Thr791Met, Arg816Trp, Arg854Gln, Cys1060Arg) were created as well as a VWF-CFP variant containing an Arg763Gly replacement which prevents cleavage of the VWF propeptide from mature VWF. (Dr. C. Mazurier (Lille, France) is acknowledged for kindly providing DNA constructs containing the Thr791Met and Arg816Trp replacement). Constructs were expressed in HEK293 cells and purified from conditioned medium using immuno-affinity chromatography. Multimer analysis revealed similar high-molecular weight multimers for all Normandy variants. As expected, the Arg763Gly replacement resulted in an increased size of all multimers. Solid phase binding assays in combination with Surface Plasmon Resonance binding studies showed that binding of FVIII-YFP was strongly reduced for the variants carrying an Arg763Gly, Thr791Met and Arg816Trp replacement and only mildly reduced for the variants carrying an Arg854Gln or Cys1060Arg replacement. Confocal Laser Scanning Microscopy analysis demonstrated that expression of all variants induced the formation of pseudo-Weibel-Palade bodies in HEK293 cells. In addition, all variants were able to redirect FVIII from localization at the trans-Golgi network to VWF-containing storage organelles despite their evident reduction in affinity. These data indicate that mutations in the FVIII binding region of VWF do not affect FVIII/VWF intracellular co-trafficking. We therefore conclude that the requirements that determine extracellular complex assembly differ from those that determine intracellular co-trafficking. We propose that FVIII/VWF co-trafficking may be a result of low-affinity interactions or even indirect passive targeting. We further conclude that co-trafficking is independent of post-translational propeptide cleavage. Together with the observation that co-trafficking is independent on Tyr1680, this suggests that the interactions that determine co-trafficking take place prior to and independent of post-translational processing in the late secretory pathway. Analysis of intracellular trafficking of FVIII in VWF producing cells could prove essential in unraveling the putative role of FVIII and VWF co-storage in vivo. Knowledge of FVIII/VWF co-storage may be particularly relevant to gene therapy approaches that target the FVIII gene to VWF-producing cells.

Sign in / Sign up

Export Citation Format

Share Document