Hans Deckmyn
Research interests
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InterestsGene Therapy, Stem Cell, Flow Cytometry, Cell Culture, Cloning
Other
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Languagesdutch, english, french
Publications
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3.21Impact points
New insights into von Willebrand disease and platelet function.
Seminars in thrombosis and hemostasis. 02/2012; 38(1):55-63.
Regulation of binding between von Willebrand factor (VWF) and the platelet receptor glycoprotein (GP) Ibα is one of the key steps in controlling hemostasis and thrombosis. On vascular injury at sites of high shear rates, the GPIbα interaction with subendothelial-bound VWF will initiate the tethering... [more] Regulation of binding between von Willebrand factor (VWF) and the platelet receptor glycoprotein (GP) Ibα is one of the key steps in controlling hemostasis and thrombosis. On vascular injury at sites of high shear rates, the GPIbα interaction with subendothelial-bound VWF will initiate the tethering of circulating platelets to the vessel wall. Tethered platelets subsequently roll on the damaged vessel wall, a process that is amplified by the activation of the platelet integrin αΙΙbβ3 (GPIIb/IIIa). The initial tethering to VWF is rapidly followed by platelet binding to collagen through specific receptors (GPVI and α2β1), leading to firm adhesion, activation, and additional stable bonds mediated by αΙΙbβ3. The above described interactions can result in two distinct processes: physiological hemostasis and pathological thrombosis. Furthermore, VWF carries coagulation factor VIII, which is involved in thrombin formation that in addition to activating platelets, mediates fibrin formation and has several other actions. The importance of VWF in hemostasis is well known in patients suffering from von Willebrand disease (VWD) who present with a defect in both platelet plug and fibrin formation. Type 2B VWD is of special interest as it may provide further insight into the mechanism by which VWF promotes the adhesion of platelets to a thrombogenic surface under conditions of high shear stress. The variant phenotypic manifestations in patients affected with type 2B VWD, however, have raised the question of locus heterogeneity in VWD as a consequence of, for example, additional defects in receptor or signaling proteins mediating platelet adhesion and aggregation. Indeed, quite a few polymorphisms of platelet receptors have been associated with increased bleeding in VWD. However, many aspects of the disease remain to be elucidated. For instance, thrombin and platelet procoagulant activity may be important counterplayers to determine the severity of the bleeding complications associated with VWD.
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5.33Impact points
Identification of a small molecule that modulates the platelet glycoprotein Ib - Von Willebrand factor interaction.
The Journal of biological chemistry. 01/2012;
The von Willebrand Factor (VWF) A1 - glycoprotein (GP) Ibα interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or p... [more] The von Willebrand Factor (VWF) A1 - glycoprotein (GP) Ibα interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or protein nature of the inhibitors currently in development limit oral bioavailability and clinical development. We therefore aimed to search for a small molecule protein-protein interaction inhibitor (SMPPII) interfering with the VWF-GPIbα binding. After determination of putative small molecule binding pockets on the surface of VWF-A1 and GPIbα using site finding algorithms and molecular dynamics, high-throughput molecular docking was performed on both binding partners. A selection of compounds showing good in silico docking scores into the predicted pockets was retained for testing their in vitro effect on VWF-GPIbα complex formation, by which we identified a compound that surprisingly stimulated the VWF-GPIbα binding in a ristocetin co-factor ELISA and increased platelet adhesion in whole blood to collagen under arterial shear rate, but that in contrast inhibited ristocetin-induced platelet aggregation. The selected compound is adhering to the predicted binding partner GPIbα as could be confirmed by saturation transfer difference NMR spectroscopy. We thus clearly identified a small molecule that modulates VWF-GPIbα binding, and that will now serve as a starting point for further studies and chemical modifications to fully characterize the interaction and to manipulate the compounds specific activity.
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10.56Impact points
Model systems of genetically modified platelets.
Blood. 12/2011; 119(7):1634-42.
Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are... [more] Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed anti-thrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet-specific) knockout mouse, and the transplantation of genetically modified human or murine platelet progenitor cells in myelo-conditioned mice. The various strengths and pitfalls of these animal models are illustrated by recent examples from the platelet field. Finally, we highlight the latest developments in genetic engineering techniques and their possible application in platelet research.
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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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2.60Impact points
Apparent heterogeneity in the pIII-peptide fusion protein in single-phage clones isolated from peptide libraries.
Protein engineering, design & selection : PEDS. 06/2011; 24(9):721-6.
Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinitie... [more] Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinities could be separated by stepwise elution. When these different fractions were re-applied, the respective differences in affinity were retained. However, when phage fractions with different apparent affinities were first amplified, an offspring was generated with again variable affinities. As the sequence of the peptide insert was the same, the heterogeneity must be ascribed to differences in avidity and although no direct evidence could be generated, we hypothesize that this is possibly due to phages displaying different numbers of the same peptide as a consequence of either proteolytic or packaging events during the amplification step in Escherichia coli.
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3.71Impact points
Laser-induced primary and secondary hemostasis dynamics and mechanisms in relation to selective photothermolysis of port wine stains.
Journal of dermatological science. 05/2011; 63(3):139-47.
Superficial vascular anomalies such as port wine stains are commonly treated by selective photothermolysis (SP). The endovascular laser-tissue interactions underlying SP are governed by a photothermal response (thermocoagulation of blood) and a hemodynamic response (thrombosis). Currently it is not ... [more] Superficial vascular anomalies such as port wine stains are commonly treated by selective photothermolysis (SP). The endovascular laser-tissue interactions underlying SP are governed by a photothermal response (thermocoagulation of blood) and a hemodynamic response (thrombosis). Currently it is not known whether the hemodynamic response encompasses both primary and secondary hemostasis, which platelet receptors are involved, and what the SP-induced thrombosis kinetics are in low-flow venules. To (1) define the role and kinetics of primary and secondary hemostasis in laser-induced thrombus formation and (2) determine which key platelet surface receptors are involved in the hemodynamic response. 532-nm laser-irradiated hamster dorsal skin fold venules were studied by intravital fluorescence microscopy following fluorescent labeling of platelets with 5(6)-carboxyfluorescein. Heparin and fluorescently labeled anti-glycoprotein Ib-α (GPIbα) and anti-P-selectin antibodies were administered to investigate the role of coagulation and platelet receptors, respectively. Lesional sizes were quantified by software. Laser irradiation consistently produced sub-occlusive thermal coagula. Thrombosis was triggered in all irradiated venules in a thermal coagulum-independent manner and peaked at 6.25min post-irradiation. Heparin decreased the maximum thrombus size and caused thrombosis to reach a maximum at 1.25min. Immunoblocking of GPIbα abated the extent of thrombosis, whereas immunoblocking of P-selectin had no effect. The hemodynamic response ensues the photothermal response in a thermal coagulum-independent manner and involves primary and secondary hemostasis. Primary hemostasis is mediated by constitutively expressed GPIbα but not by activation-dependent P-selectin.
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10.56Impact points
The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib.
Blood. 03/2011; 117(18):4999-5008.
The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to s... [more] The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.
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2.41Impact points
Platelet adhesion to collagen.
Thrombosis research. 01/2011; 127 Suppl 2:S26-9.
Platelets play a central role in maintaining hemostasis mainly by binding to subendothelial collagen exposed upon vascular injury, thereby initiating thrombus formation. Platelets can bind directly to the exposed collagen through two major receptors i.e. the integrin a2b1 and glycoprotein (GP) VI. H... [more] Platelets play a central role in maintaining hemostasis mainly by binding to subendothelial collagen exposed upon vascular injury, thereby initiating thrombus formation. Platelets can bind directly to the exposed collagen through two major receptors i.e. the integrin a2b1 and glycoprotein (GP) VI. However, under high shear conditions the GPIb-V-IX receptor complex and its main ligand von Willebrand Factor are additionally needed for firm platelet adhesion to the vessel wall. In this review, we summarize the current knowledge on the individual roles and structure-function relationships of these main platelet adhesion receptors.
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10.56Impact points
Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function.
Blood. 11/2010; 116(22):4646-56.
Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platele... [more] Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.
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1.89Impact points
Platelet physiology and antiplatelet agents.
Clinical chemistry and laboratory medicine : CCLM / FESCC. 11/2010; 48 Suppl 1:S3-13.
Apart from the central beneficial role platelets play in hemostasis, they are also involved in atherothrombotic diseases. Here, we review the current knowledge of platelet intracellular signal transduction pathways involved in platelet adhesion, activation, amplification of the activation signal and... [more] Apart from the central beneficial role platelets play in hemostasis, they are also involved in atherothrombotic diseases. Here, we review the current knowledge of platelet intracellular signal transduction pathways involved in platelet adhesion, activation, amplification of the activation signal and aggregation, as well as pathways limiting platelet aggregation. A thorough understanding of these pathways allows explanation of the mechanism of action of existing antiplatelet agents, but also helps to identify targets for novel drug development.
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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
Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow.
Blood. 10/2010; 117(2):651-60.
A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on p... [more] A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca(2+), morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by α(IIb)β(3) blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.
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2.41Impact points
Roles of Src-like adaptor protein 2 (SLAP-2) in GPVI-mediated platelet activation SLAP-2 and GPVI signaling.
Thrombosis research. 10/2010; 126(4):e276-85.
Glycoprotein VI (GPVI) /Fc receptor gamma (FcRγ)-chain complex is one of the collagen receptors in platelets and responsible for the majority of the intracellular signaling events through a similar pathway to immune receptors. Src-like adaptor protein 2 (SLAP-2) is a recently characterized adaptor p... [more] Glycoprotein VI (GPVI) /Fc receptor gamma (FcRγ)-chain complex is one of the collagen receptors in platelets and responsible for the majority of the intracellular signaling events through a similar pathway to immune receptors. Src-like adaptor protein 2 (SLAP-2) is a recently characterized adaptor protein predominantly expressed in hematopoietic cells. In T cells, SLAP-2 was reported to associate with several tyrosine phosphorylated proteins, and function as a negative regulator of signaling downstream of T cell antigen receptor by virtue of its interaction with the ubiquitin ligase c-Cbl. But the data regarding the presence and role of SLAP-2 proteins in platelets is limited. We describe the characterization of SLAP-2 in human platelets. Human platelets were analyzed by Western blot analysis, immunoprecipitation, and pull down assay, etc. Immunoprecipitation revealed the presence of two forms of SLAP-2 with approximately 28 kD and 25 kD, and following stimulation of GPVI, the additional form with approximately 32 kD apppeared. We have found that upon GPVI activation, SLAP-2 translocated from the Triton X-100-soluble fraction to the Triton X-100-insoluble cytoskeleton fraction, with concomitant association with Syk, c-Cbl, and LAT. SLAP-2 appears to play a role in regulating signaling pathways by bringing important signaling molecules such as c-Cbl and Syk into proximity of cytoskeletal substrates. In platelets, SLAP-2 may have function as a negative regulator of GPVI-mediated signaling by interacting with c-Cbl, being similar to that reported in T cells.
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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.
Following (30)
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Jonas Demeulemeester
Katholieke Universiteit Leuven -
Lieve Moons
Katholieke Universiteit Leuven -
Dermot Cox
Royal College of Surgeons in Ireland -
Marc F Hoylaerts
Katholieke Universiteit Leuven -
Kathleen Freson
Katholieke Universiteit Leuven
