Karen Vanhoorelbeke
Research interests
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Interestsvon Willebrand Factor, Hemostasis, Thrombosis, Platelets, Platelet Aggregation, Platelet Aggregation Inhibitors, Platelet Adhesiveness, ADAMTS13, Thrombotic Thrombocytopenic Purpura, von Willebrand Diseases, glycoprotein Ib, Monoclonal Antibodies, Immunoassay, ELISA, Western Blot, Cell Culture, Electrophoresis, PCR, Cloning, gene transfer, Gene Therapy, Animal Models
Publications
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2.41Impact points
Blood platelet biochemistry.
Thrombosis research. 11/2011; 129(3):245-9.
Defects in platelet function or formation increase the risk for bleeding or thrombosis, which indicates the crucial role for platelets in maintaining haemostasis in normal life. Upon vascular injury, platelets instantly adhere to the exposed extracellular matrix which results in platelet activation ... [more] Defects in platelet function or formation increase the risk for bleeding or thrombosis, which indicates the crucial role for platelets in maintaining haemostasis in normal life. Upon vascular injury, platelets instantly adhere to the exposed extracellular matrix which results in platelet activation and aggregation and the formation a haemostatic plug that stops bleeding. To prevent excessive platelet aggregate formation that eventually would occlude the vessels, this self-amplifying process nevertheless requires a tight control. This review intends to give a comprehensive overview of the currently established main mechanisms in platelet function.
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6.07Impact points
In vivo von Willebrand factor size heterogeneity in spite of the clinical deficiency of ADAMTS-13.
Journal of thrombosis and haemostasis : JTH. 09/2011; 9(12):2506-8.
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5.33Impact points
Local elongation of endothelial cell-anchored von Willebrand factor strings precedes ADAMTS13 protein-mediated proteolysis.
The Journal of biological chemistry. 09/2011; 286(42):36361-7.
Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. B... [more] Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. By following changes in VWF string length, we demonstrated that VWF strings are cleaved multiple times, successively shortening string length in the function of time and generating fragments ranging in size from 5 to over 100 μm. These are larger than generally observed in normal plasma, indicating that further proteolysis takes place in circulation. Interestingly, in 89% of all cleavage events, VWF strings elongate precisely at the cleavage site before ADAMTS13 proteolysis. These local elongations are a general characteristic of VWF strings, independent of the presence of ADAMTS13. Furthermore, large elongations, ranging in size from 1.4 to 40 μm, occur at different sites in space and time. In conclusion, ADAMTS13-mediated proteolysis of VWF strings under flow is preceded by large elongations of the string at the cleavage site. These elongations may lead to the simultaneous exposure of many exosites, thereby facilitating ADAMTS13-mediated cleavage.
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7.19Impact points
Platelets at work in primary hemostasis.
Blood reviews. 07/2011; 25(4):155-67.
When platelet numbers are low or when their function is disabled, the risk of bleeding is high, which on the one hand indicates that in normal life vascular damage is a rather common event and that hence the role of platelets in maintaining a normal hemostasis is a continuously ongoing physiological... [more] When platelet numbers are low or when their function is disabled, the risk of bleeding is high, which on the one hand indicates that in normal life vascular damage is a rather common event and that hence the role of platelets in maintaining a normal hemostasis is a continuously ongoing physiological process. Upon vascular injury, platelets instantly adhere to the exposed extracellular matrix resulting in platelet activation and aggregation to form a hemostatic plug. This self-amplifying mechanism nevertheless requires a tight control to prevent uncontrolled platelet aggregate formation that eventually would occlude the vessel. Therefore endothelial cells produce inhibitory compounds such as prostacyclin and nitric oxide that limit the growth of the platelet thrombus to the damaged area. With this review, we intend to give an integrated survey of the platelet response to vascular injury in normal hemostasis.
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7.37Impact points
MRI assessment of blood outgrowth endothelial cell homing using cationic magnetoliposomes.
Biomaterials. 03/2011; 32(17):4140-50.
The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by mag... [more] The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated. MLs were avidly internalized by BOECs giving clear MRI contrast in phantom studies and the magnetic labeling did not affect cell proliferation, viability, morphology or homeostasis and elicited only minor reactive oxygen species levels. Intravenous injection of labeled BOECs was compared with injection of free MLs and unlabeled BOECs, resulting in homing of BOECs toward the liver and spleen, which was confirmed by histology. The MLs used offer great potential for cellular tracking studies by MRI when low levels of widely distributed cells are present. In particular, the use of these MLs will allow to evaluate the efficacy of new methods to enhance BOEC homing and integration to optimize their use as efficient vehicles for gene therapy.
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6.07Impact points
The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo.
Journal of thrombosis and haemostasis : JTH. 10/2010; 8(10):2305-12.
The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Seven C-terminally truncated murine ADAMTS13 (mADAMTS13)... [more] The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13(-/-) mouse. Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.
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7.24Impact points
Binding of von Willebrand factor to collagen and glycoprotein Ibalpha, but not to glycoprotein IIb/IIIa, contributes to ischemic stroke in mice--brief report.
Arteriosclerosis, thrombosis, and vascular biology. 10/2010; 30(10):1949-51.
To unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development. VWF(-/-) mice received gene transfer to express mutants of VWF defective either in binding to fibrillar collagen, glycoprotein (GP)Ibα or GPIIb/IIIa, and underwent 60 minutes of transient middle c... [more] To unravel crucial von Willebrand factor (VWF) interactions that are detrimental in stroke development. VWF(-/-) mice received gene transfer to express mutants of VWF defective either in binding to fibrillar collagen, glycoprotein (GP)Ibα or GPIIb/IIIa, and underwent 60 minutes of transient middle cerebral artery occlusion. In VWF(-/-) mice reconstituted with VWF mutants defective in binding to collagen or GPIbα, protection against stroke was sustained, whereas VWF lacking the GPIIb/IIIa binding site restored full susceptibility similar to normal VWF. VWF-collagen and VWF-GPIbα (but not VWF-GPIIb/IIIa) interactions are instrumental in thrombus formation after transient middle cerebral artery occlusion, and their inhibition could be a promising target for stroke treatment.
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10.56Impact points
Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus).
Blood. 09/2010; 116(12):2005-10.
Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional gene... [more] Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.
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6.07Impact points
Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura.
Journal of thrombosis and haemostasis : JTH. 09/2010; 8(9):2053-62.
ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patien... [more] ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti-ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute-phase but not remission-phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute-phase but not remission-phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose-dependently inhibited by addition of exogenous α₂ -antiplasmin. Examination of the endogenous α₂-antiplasmin antigen and activity confirmed deficiency of α₂ -antiplasmin function in acute-phase but not remission-phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase-type plasminogen activator was added to an (unrelated) congenital α₂ -antiplasmin-deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.
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4.45Impact points
Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.
Thrombosis and haemostasis. 08/2010; 104(2):392-401.
Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-thr... [more] Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.
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6.07Impact points
Active platelet-binding conformation of plasma von Willebrand factor in young women with acute myocardial infarction.
Journal of thrombosis and haemostasis : JTH. 04/2010; 8(7):1653-6.
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10.56Impact points
Human platelets produced in NOD/SCID mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis.
Blood. 10/2009;
Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. We here demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice after transplantation of unexpanded cord-blood C... [more] Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. We here demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days post-transplantation with the number of circulating human platelets peaking at 2 weeks (up to 87x10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks post-transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and TRAP (Thrombin-Receptor-Activating Peptide) activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human GPIbalpha and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.
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6.07Impact points
Multi-step binding of ADAMTS13 to VWF.
Journal of thrombosis and haemostasis : JTH. 09/2009;
Summary Background: ADAMTS13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS13 d... [more] Summary Background: ADAMTS13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS13. Methods: Immunoprecipiation of VWF-ADAMTS13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by western blotting and immunosorbent assays. Results: Co-immunoprecipitation demonstrated that ADAMTS13 binds to native multimeric VWF (K(d) of 79 +/- 11 nM) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible, and did not vary with ionic strength (I of 50 to 200). Moreover, results with ADAMTS13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS13 spacer, likely thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS13 to VWF stoichiometry of 0.0040 +/- 0.0004 (mean +/- SEM, n = 10). Conclusions: ADAMTS13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.
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10.56Impact points
Mutation of the H-bond acceptor S119 in the ADAMTS13 metalloprotease domain reduces secretion and substrate turnover in a patient with congenital thrombotic thrombocytopenic purpura.
Blood. 09/2009;
Hereditary thrombotic thrombocytopenic purpura is caused by mutations in ADAMTS13 resulting in defective processing of von Willebrand factor (VWF) which causes intravascular platelet aggregation culminating in thrombocytopenia with shistocytic anemia. In this study the functional and structural role... [more] Hereditary thrombotic thrombocytopenic purpura is caused by mutations in ADAMTS13 resulting in defective processing of von Willebrand factor (VWF) which causes intravascular platelet aggregation culminating in thrombocytopenia with shistocytic anemia. In this study the functional and structural role of a recently identified ADAMTS13 metalloprotease domain mutation S119F was investigated. Secretion from heterologous cells was hampered but not completely eliminated. Secreted S119F was active towards multimeric VWF and FRETS-VWF73 but with abnormal kinetics having a significantly reduced kcat (0.88+/-0.04s(-1) vs 2.78+/-0.11s(-1)) and slightly smaller KM (1.4+/-0.2microM vs 2.3+/-0.3microM). A computational model of the metalloprotease domain demonstrates both steric and polar interaction effects caused by S119F. Interestingly, mutant S119A has properties similar to S119F (kcat=0.82+/-0.03s(-1) and KM=1.1+/-0.1microM) allowing to assign distorted kinetics to the loss of the H-bond with conserved residue W262. We conclude that the S119-W262 H-bond is crucial for maximal turnover.
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6.07Impact points
Identification of a VWF peptide antagonist that blocks platelet adhesion under high shear conditions by selectively inhibiting the VWF-collagen interaction.
Journal of thrombosis and haemostasis : JTH. 08/2009;
Summary Background: Since the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. Objectives: We used phage display to identify potential small peptides that interfere with the VWF-collagen binding ... [more] Summary Background: Since the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. Objectives: We used phage display to identify potential small peptides that interfere with the VWF-collagen binding and might serve as lead products for the development of possible oral antithrombotic compounds. Methods: A random linear heptamer peptide library was used to select VWF-binding peptides. Results: We identified a phage clone, displaying the YDPWTPS sequence, further referred to as L7-phage, that bound to VWF in a specific and a dose-dependent manner. This L7-phage specifically inhibited the VWF-collagen interaction under both static and flow conditions. Epitope mapping using deletion mutants of VWF revealed that the L7-phage does not bind to the known collagen-binding A3-domain within VWF, but to the more carboxyterminal situated C-domain. This inhibition was not due to steric hindrance of the A3 domain-collagen interaction by the L7-phage. Indeed, a tetrabranched multi-antigen peptide (MAP) presenting four copies of the peptide, but not the scrambled MAP, also inhibited VWF-collagen interaction under conditions of high shear stress at a concentration of 148 nM. Conclusions: Based on these results, we conclude that we have identified the first peptide antagonist that binds to the VWF C-domain and by this specifically inhibits the VWF binding to collagen suppressing platelet adhesion and aggregation under high shear conditions. As a consequence, this peptide and its future derivates are potentially interesting antithrombotic agents.
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10.56Impact points
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Von Willebrand factor: drug and drug target.
Cardiovascular & hematological disorders drug targets. 04/2009; 9(1):9-20.
One of the key players in many thrombotic complications is von Willebrand factor (VWF), a large, multimeric glycoprotein that is present in plasma where it fulfils a crucial role in haemostasis. First, VWF recruits platelets to vascular lesions by acting as a linker molecule between the exposed coll... [more] One of the key players in many thrombotic complications is von Willebrand factor (VWF), a large, multimeric glycoprotein that is present in plasma where it fulfils a crucial role in haemostasis. First, VWF recruits platelets to vascular lesions by acting as a linker molecule between the exposed collagen and free-flowing platelets in the circulation. Second, by serving as a carrier protein for the coagulation factor VIII, VWF protects this anti-haemophilic factor from rapid degradation. Quantitative or qualitative defects in VWF result in the most common bleeding disorder in man, known as von Willebrand disease, illustrating the central role of VWF in haemostasis. On the other hand, a thrombotic risk emerges when over-reactive VWF molecules can bind spontaneously to platelets. It is clear that because of its pivotal role in maintaining the fine balance between bleeding and thrombosis, VWF is an attractive but delicate drug target. This review focuses on the role of VWF in both haemostasis and thrombosis with special attention to the molecule as drug and drug target respectively.
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10.56Impact points
Von Willebrand factor to the rescue.
Blood. 04/2009;
Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein with complex roles in thrombosis and hemostasis. Abnormalities in VWF give rise to a variety of bleeding complications, known as von Willebrand disease (VWD), the most common inherited bleeding disorder in man. Current treatment... [more] Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein with complex roles in thrombosis and hemostasis. Abnormalities in VWF give rise to a variety of bleeding complications, known as von Willebrand disease (VWD), the most common inherited bleeding disorder in man. Current treatment of VWD is based on the replacement of the deficient or dysfunctional protein either by endogenous release from endothelial Weibel-Palade bodies or by administration of plasma-derived VWF concentrates. During the last years, several efforts have been made to optimize existing therapies for VWD, but also to devise new approaches, such as inducing endogenous expression with interleukin-11, administering exogenous recombinant VWF, or introducing the protein via gene delivery. Clearly, the efficacy of any strategy will depend on several factors, including, for example, the quantity, activity and stability of the delivered VWF. The inherent complexity of VWF biosynthesis, which involves extensive post-translational processing, may be limiting in terms of producing active VWF outside of its native cellular sources. This review summarizes recent progress in the development of different treatment strategies for VWD, including those that are established, and those that are at the experimental stage. Potential pitfalls and benefits of each strategy are discussed.
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5.33Impact points
The novel S527F mutation in the integrin beta 3 chain induces a high affinity alpha IIbbeta 3 receptor by hindering adoption of the bent conformation.
The Journal of biological chemistry. 04/2009;
Three heterozygous mutations were identified in the genes encoding the platelet integrin receptor alphaIIbbeta3 in a patient with an ill-defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable CHO cell lines were constructed expressing rec... [more] Three heterozygous mutations were identified in the genes encoding the platelet integrin receptor alphaIIbbeta3 in a patient with an ill-defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable CHO cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing either the individual R512W, L841M or S527F mutations, both the R512W and the L841M mutations, or all three mutations. All receptors were expressed on the cell surface and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3-S527F mutation produces a constitutively active receptor. Indeed, both fibrinogen and the ligand mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-LIBS antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hinders the alphaIIbbeta3 receptor from adopting a wild-type like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe-527 in beta3 and the calf-1 domain in alphaIIb, and by decreased flexibility between I-EGF domains 2 and 3.
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1.07Impact points
ADAMTS13 in health and disease.
Acta haematologica. 02/2009; 121(2-3):183-5.
We raised a set of monoclonal antibodies (mAb) against recombinant human ADAMTS13, constructed the first antigen test fully based on mAbs and compared ADAMTS13 antigen and enzymatic activity levels in a large set of plasma samples collected from different patients and healthy controls. Assessing bot... [more] We raised a set of monoclonal antibodies (mAb) against recombinant human ADAMTS13, constructed the first antigen test fully based on mAbs and compared ADAMTS13 antigen and enzymatic activity levels in a large set of plasma samples collected from different patients and healthy controls. Assessing both ADAMTS13 antigen and activity helps to understand whether or not the protease is fully active in pathological conditions, e.g. liver cirrhosis, inflammatory bowel disease, cardiac surgery, pregnancy and oral contraceptive intake, in the neonatal state and in normal individuals. Our ADAMTS13 antigen assay showed less variability than the collagen binding-based activity assay. Antigen values correlated well with activity in normal individuals, but differed to various degrees in neonates, pregnancies at later maternal age and cardiac surgery. No discrepancies were noted in liver cirrhosis and inflammatory bowel disease, which were both associated with low plasma levels of ADAMTS13. In conclusion, parallel measurement of ADAMTS13 activity and antigen provides a new tool for understanding the behavior of the VWF-cleaving protease in health and disease.
Following (17)
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Marc De Maeyer
Katholieke Universiteit Leuven -
Jean-Paul Coutelier
Université catholique de Louvain -
Kathleen Freson
Katholieke Universiteit Leuven -
Marc F Hoylaerts
Katholieke Universiteit Leuven -
Marinee Chuah
Free University of Brussel
