Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the di... more Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. Methods. We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. Results. Two of these antibody fragments bound spe...
A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the e... more A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human a-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-ArgPro-Phe-Ile-Pro-Thr-Cys was synthesised. This peptide, thrombin-inhibiting peptide (TIP), is a full competitive inhibitor of thrombin with an inhibition constant (Ki) of 0.4974 mM. It lengthened the thrombin time and inhibited thrombin-induced platelet activation and the platelet release reaction, both in a dose-dependent manner. It also reduced platelet adhesion onto a human microvascular endothelial matrix in the parallel plate flow chamber under both arterial and venous shear conditions. Thus, we have selected and synthesised a cyclic heptapeptide that competes with PPACK to bin...
The multimeric analysis of von Willebrand factor (VWF) is utilised for von Willebrand factor dise... more The multimeric analysis of von Willebrand factor (VWF) is utilised for von Willebrand factor disease (VWD) subclass identification and is important for treating purposes. A highly sensitive and rapid method for the visualisation of the multimeric structure of VWF in plasma and platelets was described by Krizek et al in 2000 [1]. This method uses a western blot technique where horizontal agarose electrophoresis is followed by the transfer of the VWF onto a polyvinylidine fluoride (PVDF) membrane. The multimeric pattern of VWF is visualized by immunolocalisation and luminographic detection and no radioactivity is used. We modified this method comprehensively to increase its sensitivity and to reduce the cost and duration of the test. We used one in stead of two localisation antibodies and thereby reduced the immunolocalisation time by more than two hours. This also reduced the cost of the procedure. We further reduced the cost by using two carbon plates for blotting in stead of a blot...
Circulating microparticles in human plasma may play a significant role in thrombogenesis because ... more Circulating microparticles in human plasma may play a significant role in thrombogenesis because they carry the initiator of blood coagulation, tissue factor. Microparticles in blood are derived from diverse cell types, including erythrocytes, endothelial cells and platelets. Thrombin generation is an important part of the coagulation system and might be influenced by the presence of microparticles in the circulation. With this study, we determined the contribution of microparticles to increased thrombin generation in plasma samples received for thrombophilia workup and compare that with normal plasma. Microparticles were isolated from 50 plasma samples with increased thrombin generation and 20 plasma samples with normal thrombin generation, using filtration. Thrombin generation assay were performed by adding a low concentration of tissue factor-containing phospholipids and a fluorescence substrate for thrombin formation to plasma samples and measuring fluorescence at 1-min intervals over a period of 90 min on all samples (with and without the presence of microparticles). The peak thrombin, velocity-index and area under the curve were calculated. Microparticles contribute to the different parameters in samples with increased thrombin generation as follows: 50 ± 19% for peak thrombin, 58 ± 24% for velocity-index and 35 ± 13% for area under the curve. Microparticles did not contribute to thrombin generation in plasma samples with normal thrombin generation. Microparticles play a significant role in coagulation and contribute largely to increased thrombin generation in plasma; however, microparticles do not contribute to coagulation in the plasma of participants with normal thrombin generation.
Although type II Factor VIII (FVIII) inhibitor antibodies inhibit only partially FVIII activity, ... more Although type II Factor VIII (FVIII) inhibitor antibodies inhibit only partially FVIII activity, even when such antibodies are in very large excess over FVIII, they frequently cause severe bleedings in patients with acquired or congenital hemophilia A. During the preclinical evaluation of anti-FVIII antibodies as potential antithrombotic agents in baboons, we observed severe bleedings in animals treated with an antibody (mAb-LE2E9) inhibiting in vitro about 70% baboon FVIII. The administration of another antibody (mAb-LE2E9Q), inhibiting less than 20% baboon FVIII in vitro did not induce any significant hemoglobin drop by comparison to control animals. Surprisingly, mAb-LE2E9Q significantly reduced thrombus development in extracorporeal circulation models of venous and arterial thrombosis. The discrepancy between the levels of FVIII inhibition observed with these two antibodies and their in vivo activity prompted us to use modified types of FVIII assays to further evaluate in human ...
In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strateg... more In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strategies to prevent thrombus formation consist of the use of Aspirin®, Plavix® and integrin αIIbβ3 blockers (e.g. Reopro®) in combination with Heparin®. These drugs are associated with high bleeding risk. Several in vivo experiments have shown that neutralizing the collagen von Willebrand Factor (vWF) platelet glycoprotein (GP)Ib-IX-V axis strongly inhibits arterial thrombosis without bleeding complications, therefore, these targets are of high interest to develop new anti-thrombotic drugs. Nanobodies are antibody-derived therapeutic proteins with the structural and functional properties of naturally occurring single-chain antibodies derived from camelids. ALX-0081 is a bivalent humanized Nanobody targeting the GPIb-IX-V binding site at the A1 domain of vWF. The precursor molecule was isolated from a llama immunized with the recombinant A1 domain of vWF and then humanized and engineered into ...
SummaryThe in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistat... more SummaryThe in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dosefinding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrom...
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 1995
Cultured human umbilical cord endothelial cells are mostly used to study the response of endothel... more Cultured human umbilical cord endothelial cells are mostly used to study the response of endothelium to various stimuli. The effect of the culture process on cell function is however, not known and the function of endothelial cells from different anatomical loci differs. In view of this we developed a flow chamber to study the function of fresh endothelium. The flow chamber was designed to accommodate a five cm piece of baboon aorta in a closed circuit where six hours of perfusion of the aorta with Ringer’s lactate/dextrose buffer at 37 °C was maintained by a peristaltic pump.
Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of... more Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-...
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by microvas... more Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by microvascular platelet deposition and thrombus formation in selected organs, resulting in microangiopathic haemolytic anaemia, thrombocytopenia, neurological symptoms and renal failure. Typically a very rare disorder, TTP is being seen with increased frequency in patients infected with the human immunodeficiency virus (HIV). Deficiency of the von Willebrand factor cleavage protease, ADAMTS13, has been implicated as the cause of TTP. However, the pathophysiology of HIV-associated TTP and the thrombotic potential in these patients are not known. This article provides not only an overview of the literature regarding HIV-associated TTP, but also presents new data on this disease. We propose a mechanism for the initial onset of HIV-associated TTP that includes the release of extreme amounts of von Willebrand factor and the downregulation of ADAMTS13 and/or the production of autoantibodies to ADAMTS13.
The pivotal role that blood platelets play in haemostasis and thrombosis caused a keen interest i... more The pivotal role that blood platelets play in haemostasis and thrombosis caused a keen interest in the development of specific inhibitors of platelet function. The platelet glycoprotein (GP)IIb/IIIa receptor complex for fibrinogen is a particularly attractive target for therapeutic intervention, since it is the exclusive mediator of platelet aggregation. There are about 50,000 GPIIb/IIIa receptors on the surface of normal platelets. When they are reduced to about 10,000 there is a marked decrease in platelet aggregation, a mildly prolonged bleeding time and abolition of in vivo thrombus formation1. 16N7C2, a murine monoclonal antibody against human platelets, was developed at the Center for Thrombosis and Vascular Research at the University of Leuven. Preliminary studies showed that it blocks the GPIIb/IIIa receptors of human and baboon platelets, but that it has no effect on cat, dog, pig, rabbit, rat, hamster, mouse and guinea-pig platelets. We therefore decided to evaluate the in vivo potential of this monoclonal antibody in baboons.
Background Stroke associated with human immunodeficiency virus infection may occur through a vari... more Background Stroke associated with human immunodeficiency virus infection may occur through a variety of mechanisms. Von Willebrand factor is a marker of endothelial dysfunction, and is elevated in human immunodeficiency virus infection. High levels of von Willebrand factor, a protein involved in platelet adhesion and aggregation, and low levels of ADAMTS13, a metalloproteinase that cleaves von Willebrand factor, have been associated with an increased risk of thrombosis. Aim To investigate the role of von Willebrand factor and ADAMTS13 in the pathogenesis of human immunodeficiency virus-related stroke in young patients. Methods A case-control study ( n = 100) comprising three participant groups: human immunodeficiency virus-positive antiretroviral therapy-naïve young strokes ( n = 20), human immunodeficiency virus-negative young strokes ( n = 40), and human immunodeficiency virus-positive antiretroviral therapy-naïve nonstroke controls ( n = 40). von Willebrand factor and ADAMTS13 le...
Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the di... more Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. Methods. We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. Results. Two of these antibody fragments bound spe...
A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the e... more A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human a-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-ArgPro-Phe-Ile-Pro-Thr-Cys was synthesised. This peptide, thrombin-inhibiting peptide (TIP), is a full competitive inhibitor of thrombin with an inhibition constant (Ki) of 0.4974 mM. It lengthened the thrombin time and inhibited thrombin-induced platelet activation and the platelet release reaction, both in a dose-dependent manner. It also reduced platelet adhesion onto a human microvascular endothelial matrix in the parallel plate flow chamber under both arterial and venous shear conditions. Thus, we have selected and synthesised a cyclic heptapeptide that competes with PPACK to bin...
The multimeric analysis of von Willebrand factor (VWF) is utilised for von Willebrand factor dise... more The multimeric analysis of von Willebrand factor (VWF) is utilised for von Willebrand factor disease (VWD) subclass identification and is important for treating purposes. A highly sensitive and rapid method for the visualisation of the multimeric structure of VWF in plasma and platelets was described by Krizek et al in 2000 [1]. This method uses a western blot technique where horizontal agarose electrophoresis is followed by the transfer of the VWF onto a polyvinylidine fluoride (PVDF) membrane. The multimeric pattern of VWF is visualized by immunolocalisation and luminographic detection and no radioactivity is used. We modified this method comprehensively to increase its sensitivity and to reduce the cost and duration of the test. We used one in stead of two localisation antibodies and thereby reduced the immunolocalisation time by more than two hours. This also reduced the cost of the procedure. We further reduced the cost by using two carbon plates for blotting in stead of a blot...
Circulating microparticles in human plasma may play a significant role in thrombogenesis because ... more Circulating microparticles in human plasma may play a significant role in thrombogenesis because they carry the initiator of blood coagulation, tissue factor. Microparticles in blood are derived from diverse cell types, including erythrocytes, endothelial cells and platelets. Thrombin generation is an important part of the coagulation system and might be influenced by the presence of microparticles in the circulation. With this study, we determined the contribution of microparticles to increased thrombin generation in plasma samples received for thrombophilia workup and compare that with normal plasma. Microparticles were isolated from 50 plasma samples with increased thrombin generation and 20 plasma samples with normal thrombin generation, using filtration. Thrombin generation assay were performed by adding a low concentration of tissue factor-containing phospholipids and a fluorescence substrate for thrombin formation to plasma samples and measuring fluorescence at 1-min intervals over a period of 90 min on all samples (with and without the presence of microparticles). The peak thrombin, velocity-index and area under the curve were calculated. Microparticles contribute to the different parameters in samples with increased thrombin generation as follows: 50 ± 19% for peak thrombin, 58 ± 24% for velocity-index and 35 ± 13% for area under the curve. Microparticles did not contribute to thrombin generation in plasma samples with normal thrombin generation. Microparticles play a significant role in coagulation and contribute largely to increased thrombin generation in plasma; however, microparticles do not contribute to coagulation in the plasma of participants with normal thrombin generation.
Although type II Factor VIII (FVIII) inhibitor antibodies inhibit only partially FVIII activity, ... more Although type II Factor VIII (FVIII) inhibitor antibodies inhibit only partially FVIII activity, even when such antibodies are in very large excess over FVIII, they frequently cause severe bleedings in patients with acquired or congenital hemophilia A. During the preclinical evaluation of anti-FVIII antibodies as potential antithrombotic agents in baboons, we observed severe bleedings in animals treated with an antibody (mAb-LE2E9) inhibiting in vitro about 70% baboon FVIII. The administration of another antibody (mAb-LE2E9Q), inhibiting less than 20% baboon FVIII in vitro did not induce any significant hemoglobin drop by comparison to control animals. Surprisingly, mAb-LE2E9Q significantly reduced thrombus development in extracorporeal circulation models of venous and arterial thrombosis. The discrepancy between the levels of FVIII inhibition observed with these two antibodies and their in vivo activity prompted us to use modified types of FVIII assays to further evaluate in human ...
In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strateg... more In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strategies to prevent thrombus formation consist of the use of Aspirin®, Plavix® and integrin αIIbβ3 blockers (e.g. Reopro®) in combination with Heparin®. These drugs are associated with high bleeding risk. Several in vivo experiments have shown that neutralizing the collagen von Willebrand Factor (vWF) platelet glycoprotein (GP)Ib-IX-V axis strongly inhibits arterial thrombosis without bleeding complications, therefore, these targets are of high interest to develop new anti-thrombotic drugs. Nanobodies are antibody-derived therapeutic proteins with the structural and functional properties of naturally occurring single-chain antibodies derived from camelids. ALX-0081 is a bivalent humanized Nanobody targeting the GPIb-IX-V binding site at the A1 domain of vWF. The precursor molecule was isolated from a llama immunized with the recombinant A1 domain of vWF and then humanized and engineered into ...
SummaryThe in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistat... more SummaryThe in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dosefinding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrom...
Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie, 1995
Cultured human umbilical cord endothelial cells are mostly used to study the response of endothel... more Cultured human umbilical cord endothelial cells are mostly used to study the response of endothelium to various stimuli. The effect of the culture process on cell function is however, not known and the function of endothelial cells from different anatomical loci differs. In view of this we developed a flow chamber to study the function of fresh endothelium. The flow chamber was designed to accommodate a five cm piece of baboon aorta in a closed circuit where six hours of perfusion of the aorta with Ringer’s lactate/dextrose buffer at 37 °C was maintained by a peristaltic pump.
Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of... more Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-...
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by microvas... more Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterised by microvascular platelet deposition and thrombus formation in selected organs, resulting in microangiopathic haemolytic anaemia, thrombocytopenia, neurological symptoms and renal failure. Typically a very rare disorder, TTP is being seen with increased frequency in patients infected with the human immunodeficiency virus (HIV). Deficiency of the von Willebrand factor cleavage protease, ADAMTS13, has been implicated as the cause of TTP. However, the pathophysiology of HIV-associated TTP and the thrombotic potential in these patients are not known. This article provides not only an overview of the literature regarding HIV-associated TTP, but also presents new data on this disease. We propose a mechanism for the initial onset of HIV-associated TTP that includes the release of extreme amounts of von Willebrand factor and the downregulation of ADAMTS13 and/or the production of autoantibodies to ADAMTS13.
The pivotal role that blood platelets play in haemostasis and thrombosis caused a keen interest i... more The pivotal role that blood platelets play in haemostasis and thrombosis caused a keen interest in the development of specific inhibitors of platelet function. The platelet glycoprotein (GP)IIb/IIIa receptor complex for fibrinogen is a particularly attractive target for therapeutic intervention, since it is the exclusive mediator of platelet aggregation. There are about 50,000 GPIIb/IIIa receptors on the surface of normal platelets. When they are reduced to about 10,000 there is a marked decrease in platelet aggregation, a mildly prolonged bleeding time and abolition of in vivo thrombus formation1. 16N7C2, a murine monoclonal antibody against human platelets, was developed at the Center for Thrombosis and Vascular Research at the University of Leuven. Preliminary studies showed that it blocks the GPIIb/IIIa receptors of human and baboon platelets, but that it has no effect on cat, dog, pig, rabbit, rat, hamster, mouse and guinea-pig platelets. We therefore decided to evaluate the in vivo potential of this monoclonal antibody in baboons.
Background Stroke associated with human immunodeficiency virus infection may occur through a vari... more Background Stroke associated with human immunodeficiency virus infection may occur through a variety of mechanisms. Von Willebrand factor is a marker of endothelial dysfunction, and is elevated in human immunodeficiency virus infection. High levels of von Willebrand factor, a protein involved in platelet adhesion and aggregation, and low levels of ADAMTS13, a metalloproteinase that cleaves von Willebrand factor, have been associated with an increased risk of thrombosis. Aim To investigate the role of von Willebrand factor and ADAMTS13 in the pathogenesis of human immunodeficiency virus-related stroke in young patients. Methods A case-control study ( n = 100) comprising three participant groups: human immunodeficiency virus-positive antiretroviral therapy-naïve young strokes ( n = 20), human immunodeficiency virus-negative young strokes ( n = 40), and human immunodeficiency virus-positive antiretroviral therapy-naïve nonstroke controls ( n = 40). von Willebrand factor and ADAMTS13 le...
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