Normalisation of haemoglobin and control of breakthrough haemolysis with increased frequency pegcetacoplan dosing in treated paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinura is an acquired life-threatening haemolytic condition, which is generally well controlled with terminal complement blockade with eculizumab. Whilst almost all patients treated with terminal complement inhibitors develop extravascular haemolysis, only a small proportion of these results in symptomatic anaemia limiting their activities and requiring red cell transfusion. This case highlights the potential role for the C3 inhibitor, pegcetacoplan, in controlling both intravascular and extravascular haemolysis, and is the first case to report on the use of additional doses of pegcetacoplan to control breakthrough haemolysis.

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH4) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

Misinterpretation of blood group and antibody screen leading to serious errors in RhD immunoglobulin administration: A report on first two years of data from Serious Transfusion Incident Reporting program.

The Serious Transfusion Incident Reporting program (STIR) commenced haemovigilance in relation to RhD immunoglobulin (Ig) administration in 2015. During two years of reporting, 21 reports relating to RhD Ig administration were received. Thirty-three percent (7/21) were related to omission of RhD Ig, putting women at risk of RhD alloimmunisation and adverse consequences in future pregnancies. A recent case reported to STIR highlights poor communication and misinterpretation of pathology results leading to significant morbidity from haemolysis in the fetus. STIR makes recommendations related to education of staff and communication between clinical and laboratory staff to improve the safety of patient care.

Autoregulation of von Willebrand factor function by a disulfide bond switch.

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this "mechanopresentation" remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.

Mechanisms of Platelet Dysfunction in Patients with Implantable Devices.

As treatment options in modern medicine continue to expand, physicians globally have witnessed a veritable explosion in the utility of therapeutic devices. Particularly within the spheres of cardiology and critical care medicine, a plethora of devices are now available with an ever-increasing range of clinical indications. Additionally, the advent of transcatheter-mounted devices has enabled patients unsuitable for more invasive procedures to benefit from intervention, thereby greatly expanding the cohort of device-eligible patients. However, despite advances in design and materials, as well as pharmacological prophylaxis, hemostatic complications continue to plague device recipients, contributing to morbidity and mortality. Elucidating the complex interplay between components of the hemostatic system and cardiac devices has been the subject of much recent research, with greater focus on the coagulation cascade and device-induced perturbations. However, less is known about impact of mechanical surfaces on platelets and the resultant clinical complications, both hemorrhagic and thrombotic. This review will focus on exploring the pathobiology of platelet-surface interactions, contextualized within the wider hemostatic system, with a focus on the increasingly utilized technologies of transcatheter aortic-valve implantation, ventricular assist devices, and extracorporeal membrane oxygenation.

Extracorporeal membrane oxygenation-hemostatic complications.

The use of extracorporeal membrane oxygenation (ECMO) support for cardiac and respiratory failure has increased in recent years. Improvements in ECMO oxygenator and pump technologies have aided this increase in utilization. Additionally, reports of successful outcomes in supporting patients with respiratory failure during the 2009 H1N1 pandemic and reports of ECMO during cardiopulmonary resuscitation have led to increased uptake of ECMO. Patients requiring ECMO are a heterogenous group of critically ill patients with cardiac and respiratory failure. Bleeding and thrombotic complications remain a leading cause of morbidity and mortality in patients on ECMO. In this review, we describe the mechanisms and management of hemostatic, thrombotic and hemolytic complications during ECMO support.

The evolving role of the transfusion practitioner.

Much of the recent work in transfusion practice has shifted to focus on the patient, after efforts over previous decades to ensure the quality and safety of blood products. After the commencement of hemovigilance and transfusion practice improvement programs, the introduction of transfusion practitioners (TP) into health care services and blood centers has continued to increase worldwide. Since this relatively new role was introduced, much work of the TP has focused on patient and staff education, adverse events, transfusion governance, and monitoring of transfusion practices within organizations. The complex nature of the transfusion process makes the TP an integral link in the transfusion chain. Together with hospital transfusion teams and committees, the TP works collaboratively to facilitate the transfusion change management programs and initiatives. Recently, the TP role has evolved to include an emphasis on patient blood management and, to some extent, is shaped by national standards and regulations. These established roles of the TP, together with the ever-changing field of transfusion medicine, provide new opportunities and challenges for a role that is continuing to evolve worldwide.

Evolving experience of treating antibody-mediated rejection following lung transplantation.

BACKGROUND: The importance of antibody-mediated rejection (AMR) following lung transplantation remains contentious. In particular, the diagnostic criteria suggested to define AMR, namely the presence of donor-specific antibodies (DSA), C4d immunoreactivity, histological features and allograft dysfunction are not always readily applicable or confirmatory in lung transplantation. METHODS: In a retrospective single-center study of 255 lung transplant recipients (LTR), we identified 9 patients in whom a clinical diagnosis of AMR was made within 12months of transplant, and define the immunological, histological, clinical features, as well as the therapeutic response of this cohort. RESULTS: Nine LTR with AMR underwent combination therapy with high-dose intravenous corticosteroid, intravenous immunoglobulin, plasmapheresis and rituximab. Following therapy, while the total number of the original DSA dropped by 17%, and the median value of the mean fluorescence intensity (mfi) of the originally observed DSA decreased from 5292 (IQR 1319-12,754) to 2409 (IQR 920-6825) (p<0.001), clinical outcomes were variable with a number of patients progressing to either chronic lung allograft dysfunction or death within 12month. CONCLUSION: AMR in lung transplantation remains both a diagnostic and therapeutic challenge, but when clinically suspected is associated with a variable response to therapy and poor long-term outcomes.

An acquired defect associated with abnormal signaling of the platelet collagen receptor glycoprotein VI.

INTRODUCTION: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. AIM: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. METHODS: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. RESULTS: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. CONCLUSIONS: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)ß(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

ADAMTS13 binds to CD36: a potential mechanism for platelet and endothelial localization of ADAMTS13.

BACKGROUND: ADAMTS13 cleaves ultralarge von Willebrand factor (VWF) and plays a significant role in vascular biology and thrombotic thrombocytopenic purpura. CD36, a transmembrane protein present on endothelial cells and platelets (PLTs), binds to thrombospondin via three thrombospondin type 1 repeats. ADAMTS13 contains eight thrombospondin type 1 repeats. STUDY DESIGN AND METHODS: An enzyme-linked immunoassay was used to explore the binding of recombinant human CD36 (rHuCD36) to recombinant human ADAMTS13 (rHuADAMTS13). A competition assay between rHuADAMTS13 and recombinant human (rHu)-thrombospondin-2 for binding to CD36 was then performed. Subsequently, binding of rHuADAMTS13 to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) and glycoprotein Ib/IX was assessed. To examine the functional significance of an ADAMTS13-CD36 interaction, ADAMTS13 activity measured by a fluorescence resonance energy transfer assay was investigated in the presence of either rHuCD36 or concentrated PLTs. RESULTS: rHuCD36 bound to rHuADAMTS13 in a dose-dependent fashion. rHu-thrombospondin-2 competed with ADAMTS13 for CD36 occupancy, but even high concentrations of rHu-thrombospondin-2 failed to completely block binding of rHuADAMTS13 to rHuCD36. rHuADAMTS13 bound to PLT membrane fragments expressing CD36 (PLT glycoprotein IV) in preference to PLT membrane fragments expressing glycoprotein Ib/IX. ADAMTS13 activity was not inhibited by the presence of either rHuCD36 or concentrated PLTs. CONCLUSION: rHuADAMTS13 binds to both rHuCD36 and PLT membrane CD36 in vitro. The binding of CD36 to rHuADAMTS13 with retention of its enzymatic activity is consistent with a proposed role for CD36 in localizing ADAMTS13 on the endothelial cell surface where it regulates the cleavage of VWF.