David J. Kuter, MD, Dphil
Center for Hematology, Massachusetts General Hospital Cancer Center
Professor of Medicine
Harvard Medical School
Our laboratory was one of the original laboratories to discover thrombopoietin, the platelet growth factor that regulates platelet production. Our subsequent work has focused on looking at the basic biology of thrombopoietin production and function, its general pharmacology in humans and animal models, and how it might relate to several clinical problems ranging from essential thrombocythemia to the preservation of platelets during storage.
We have found that thrombopoietin is produced constitutively in the liver and is not regulated at a translational or transcriptional level. The molecule enters the circulation and is cleared by a high affinity receptor on the platelet surface that internalizes and destroys the available thrombopoietin. This is a novel feedback loop in which the receptor number on circulating platelets directly regulates the amount of ambient cytokine in the circulation. When the platelet production rate falls, the metabolism of thrombopoietin is reduced and levels rise in an attempt to stimulate bone marrow production of megakaryocytes, the bone marrow cell that produces platelets.
We have characterized the thrombopoietin receptor on platelets. The thrombopoietin receptor on platelets has a very high binding affinity of 160 picomolar and there are between 40 and 70 receptors per platelet. The exact time course of thrombopoietin binding, internalization, and destruction by platelets has been characterized in both in vitro and in some physiologic models. These results have directly predicted the effective pharmacologic dose in the clinical trials that we have conducted with our collaborators.
Characterization of Anti-thrombopoietin Antibody
We have participated in the clinical development of thrombopoietin. In one study 1,300 volunteers were injected with a truncated version of thrombopoietin. Unfortunately some of these individuals developed neutralizing antibodies to the thrombopoietin which in turn neutralized not only the recombinant molecule injected but the endogenous molecule as well. Given the constitutive nature of thrombopoietin production, this resulted in a decline in thrombopoietin levels in humans and a consequent thrombocytopenia. A major effort of our work in the last year has been to characterize the antibody that occurs in the individuals who have developed such antibodies. A precise immunologic assay has been developed to characterize the antibody levels and the rise and fall in antibody levels determined in the 13 individuals that we have studied.
The anti-thrombopoietin antibody in some of these patients is an IgG4 subtype that does not fix complement and circulates as an antibody-thrombopoietin complex. Epitope mapping of the thrombopoietin molecule shows that the antibody binds to regions in the first 163-amino acids but does not bind to thrombopoietin peptide mimetics that have been created to circumvent this particular problem. We have continued to monitor those patients who developed thrombocytopenia in an effort to determine why they formed antibody.
Genomic and Proteomic Approaches to Patients with Essential Thrombocythemia
Our clinical research team takes care of almost 800 patients with a common myeloproliferative disease called essential thrombocythemia. This clonal disorder is characterized by a high platelet count and a predilection for both bleeding and/or thrombosis. We are currently using genomic and proteomic profiling of platelets from normal patients and those with ET. Platelet mRNA is the source of nucleic acid for the genomic screening. We have just completed analysis with the Affimetrix A2 chip comparing platelets from 30 ET and over 50 normal subjects. A large number of differentially expressed genes have been identified and are undergoing further analysis. Platelet total protein is the source for the proteomic analyses. Using 2-dimensional gel electrophoresis of whole platelet proteins or fractions thereof, several proteins have been found to be overexpressed in ET platelets and have been sequenced at the Core Harvard Proteomics Facility. We seek to identify candidate genes that are dysregulated in ET and possibly determine whether there is an etiologic genetic change in this common disorder. A secondary objective of these studies is to identify genes that will aid in the diagnosis of this condition and distinguish it from other causes of elevated platelet count.
Proteogenomic Analysis of Platelets
In addition to the studies of essential thrombocythemia above, we are currently undertaking a large program to identify the proteins in platelets and their changes in various diseases states ranging from myelodysplastic syndromes to ITP. We are using several methods of proteomic analysis to define the platelet proteome, platelet phosphoproteome, and platelet membrane proteome in normal and diseases platelets.
Platelets Undergo Apoptosis
Given our interest in thrombopoietin and its effect upon megakaryocytes, we have expanded our scope of investigation to analyze why platelets do not survive well when stored under usual blood bank conditions. This is a major problem in the blood banking industry, resulting in the loss of approximately one-fifth the total platelet product harvested in this country. Over five days in storage, platelets lose their viability and are no longer transfusable. The nature of this defect has been attributed to abnormal nutrients, temperature, and other phenomena that occur ex vivo. We have shown that like other enucleate cells, platelets contain multiple apoptotic cascades and that some of these are activated during platelet storage. Upon the addition of apoptosis inhibitors to platelets during storage, platelet apoptosis is reduced. The exact molecular mechanism of this is currently under study but similar apoptotic inhibitors may allow for the enhanced storage for platelets ex vivo in the blood bank and expand the national supply of this valued blood product.
Analyses of Novel Thrombopoietic Growth Factors
We have helped develop two novel thrombopoietins and are characterizing their mode of action in preclinical studies. The first is a peptide mimetic that prevents TPO receptor internalization and amplifies endogenous TPO signalling. The second is a small molecule that dimerizes the TPO receptor and causes activation of all TPO signalling pathways. Current analyses seek to understand the interaction of these molecules on (a) receptor internalization; (b) STAT-5 phosphorylation; and (c) TPO-TPO receptor binding affinity.