Bloodworks Research Institute

Seattle, WA, United States

Bloodworks Research Institute

Seattle, WA, United States
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Johnsen J.M.,Bloodworks Research Institute | Johnsen J.M.,University of Washington
Hematology | Year: 2015

Blood types (blood group antigens) are heritable polymorphic antigenic molecules on the surface of blood cells. These were amongst the first human Mendelian traits identified, and the genetic basis of nearly all of the hundreds of blood types is known. Clinical laboratory methods have proven useful to identify selected blood group gene variants, and use of genetic blood type information is becoming widespread. However, the breadth and complexity of clinically relevant blood group genetic variation poses challenges. With recent advances in next-generation sequencing technologies, a more comprehensive DNA sequence-based genetic blood typing approach is now feasible. This chapter introduces the practitioner to high-resolution genetic blood typing beginning with an overview of the genetics of blood group antigens, the clinical problem of allosensitization, current blood type testing methods, and then discussion of next-generation sequencing and its application to the problem of genetic blood typing.


Tersteeg C.,Katholieke University Leuven Campus Kulak Kortrijk | Roodt J.,University of the Free State | Roodt J.,Universitas Hospital | Van Rensburg W.J.,University of the Free State | And 10 more authors.
Blood | Year: 2017

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF)-cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are not always effective; therefore, new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, is a possible new treatment strategy for TTP, as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimers size and hence their prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs, using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This in vivo finding was supported by in vitro data demonstrating the VWF multimer-reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving preexisting TTP signs; thrombocytopenia, hemolytic anemia, and organ damage could not be reversed, as thrombus resolution was not achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, was effective in preventing severe TTP signs in mice, but NAC was not effective in resolving preexistent acute TTP signs in mice and baboons. © 2017 by The American Society of Hematology.


Chung D.W.,Bloodworks Research Institute | Chen J.,Bloodworks Research Institute | Ling M.,Bloodworks Research Institute | Fu X.,Bloodworks Research Institute | And 7 more authors.
Blood | Year: 2016

The ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number ofmonomers in individual VWFmultimers and on the self-Association of individual VWF multimers into larger structures. VWF self-Association is accelerated by shear stress. We observed that VWF self-Association occurs during adsorption of VWF onto surfaces, assembly of secretedVWF into hyperadhesiveVWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers.VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL andApoA-I also preventedVWF on the endotheliumfromself-Associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in selfassociated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-Association would be a new approach to treating thrombotic disorders. © 2016 by The American Society of Hematology.


PubMed | Bloodworks Research Institute and University of Washington
Type: Journal Article | Journal: Blood | Year: 2016

The ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.


Nascimbene A.,University of Texas Health Science Center at Houston | Neelamegham S.,State University of New York at Buffalo | Frazier O.H.,Baylor College of Medicine | Moake J.L.,Baylor College of Medicine | And 3 more authors.
Blood | Year: 2016

Left ventricular assist devices (LVAD) provide cardiac support for patients with endstage heart disease as either bridge or destination therapy, and have significantly improved the survival of these patients. Whereas earlier models were designed to mimic the human heart by producing a pulsatile flow in parallel with the patient's heart, newer devices, which are smaller and more durable, provide continuous blood flow along an axial path using an internal rotor in the blood. However, device-related hemostatic complications remain common and have negatively affected patients' recovery and quality of life. In most patients, the von Willebrand factor (VWF) rapidly loses large multimers and binds poorly to platelets and subendothelial collagen upon LVAD implantation, leading to the term acquired von Willebrand syndrome (AVWS). These changes in VWF structure and adhesive activity recover quickly upon LVAD explantation and are not observed in patients with heart transplant. The VWF defects are believed to be caused by excessive cleavage of large VWF multimers by the metalloprotease ADAMTS-13 in an LVAD-driven circulation. However, evidence that this mechanism could be the primary cause for the loss of large VWF multimers and LVAD-associated bleeding remains circumstantial. This review discusses changes in VWF reactivity found in patients on LVAD support. It specifically focuses on impacts of LVAD-related mechanical stress on VWF structural stability and adhesive reactivity in exploring multiple causes of AVWS and LVAD-associated hemostatic complications. © 2016 by The American Society of Hematology.


PubMed | University of Washington, University of Toronto, Indiana University, Jinja Regional Referral Hospital and 2 more.
Type: | Journal: Malaria journal | Year: 2016

Malaria is a major cause of morbidity and mortality in sub-Saharan Africa, and poor outcomes have been associated with endothelial activation. In this study, biomarkers of endothelial activation, haemostasis, and thrombosis were measured in Ugandan children with severe malaria who participated in a clinical trial, in order to investigate associations between these processes.Serum and plasma were collected from participants at baseline (day 1), and on days 2, 3, 4, and 14. Von Willebrand factor (VWF) antigen was measured in stored plasma samples from all trial participants, and its association with mortality and changes over time were analysed. VWF multimer patterns were evaluated in baseline serum samples by gel electrophoresis followed by Western blotting. Levels of angiopoietins 1 and 2, VWF antigen, total active VWF, ADAMTS13, platelet counts, apolipoprotein A1, and syndecan-1 were measured in stored serum samples from 12 survivors at baseline and day 4.VWF antigen levels were associated with mortality, and decreased over time in survivors. Baseline VWF antigen and total active VWF levels were elevated, and very large multimers were present in the baseline serum of several patients. Higher platelet counts were associated with higher angiopoietin-1 and apolipoprotein A1 levels, while lower platelet counts were associated with higher syndecan-1, a marker of endothelial damage. Higher angiopoietin-2 to angiopoietin-1 ratio and higher syndecan-1 levels were correlated with lower apolipoprotein A1 levels. There were no correlations between total active VWF, VWF antigen, or ADAMTS13 levels and the other biomarkers at baseline. Changes in biomarker levels between baseline and day 4 were not correlated.These results confirm that severe malaria is associated with endothelial activation, and suggest that endothelial activation contributes to microvascular thrombosis and endothelial damage.


Graham S.M.,University of Washington | Chen J.,Bloodworks Research Institute | Chung D.W.,Bloodworks Research Institute | Barker K.R.,University of Toronto | And 9 more authors.
Malaria Journal | Year: 2016

Background: Malaria is a major cause of morbidity and mortality in sub-Saharan Africa, and poor outcomes have been associated with endothelial activation. In this study, biomarkers of endothelial activation, haemostasis, and thrombosis were measured in Ugandan children with severe malaria who participated in a clinical trial, in order to investigate associations between these processes. Methods: Serum and plasma were collected from participants at baseline (day 1), and on days 2, 3, 4, and 14. Von Willebrand factor (VWF) antigen was measured in stored plasma samples from all trial participants, and its association with mortality and changes over time were analysed. VWF multimer patterns were evaluated in baseline serum samples by gel electrophoresis followed by Western blotting. Levels of angiopoietins 1 and 2, VWF antigen, total active VWF, ADAMTS13, platelet counts, apolipoprotein A1, and syndecan-1 were measured in stored serum samples from 12 survivors at baseline and day 4. Results: VWF antigen levels were associated with mortality, and decreased over time in survivors. Baseline VWF antigen and total active VWF levels were elevated, and very large multimers were present in the baseline serum of several patients. Higher platelet counts were associated with higher angiopoietin-1 and apolipoprotein A1 levels, while lower platelet counts were associated with higher syndecan-1, a marker of endothelial damage. Higher angiopoietin-2 to angiopoietin-1 ratio and higher syndecan-1 levels were correlated with lower apolipoprotein A1 levels. There were no correlations between total active VWF, VWF antigen, or ADAMTS13 levels and the other biomarkers at baseline. Changes in biomarker levels between baseline and day 4 were not correlated. Conclusions: These results confirm that severe malaria is associated with endothelial activation, and suggest that endothelial activation contributes to microvascular thrombosis and endothelial damage. © 2016 Graham et al.


PubMed | Shire Inc, Bloodworks Research Institute, Catholic University of Leuven and University of the Free State
Type: | Journal: Blood | Year: 2016

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF) cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are however not always effective and therefore new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, could be a possible new treatment strategy for TTP as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimer size and hence its prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This was supported by in vitro data, demonstrating the VWF-multimer reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving pre-existing TTP signs; thrombocytopenia, hemolytic anemia and organ damage could not be reversed, as thrombus resolution could not be achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, is effective in preventing severe TTP signs in mice but NAC is not effective in resolving acute TTP signs in mice and baboons.

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