South Asian Society on Atherosclerosis and Thrombosis (SASAT)
Division of Clinical and Preventive Cardiology, Medanta Hospital
Gurugram, Haryana, IndiaForeword Jawed Fareed PhD DSc FAHA
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Manual of Blood Platelets: Morphology, Physiology and Pharmacology
First Edition: 2019
I dedicate this monograph to my mentor and associate
Regents Professor of Pediatrics and PathologyForeword
This comprehensive book represents a detailed description of platelet biology highlighting the detailed structural and functional aspects of these anuclear cells. The illustrations and material included in this book represent a descriptive account of cutting edge work carried out during a 45-year period (1970–2015) and portray some of the lead discoveries on platelet structure and function. These discoveries have impacted the understanding of platelet-related diseases and the development of newer antiplatelet drugs, adding a new dimension to the treatment of cardiovascular diseases. Platelets were discovered accidentally by Professor Bizzozero in 1865 and since then have been a subject of focused basic and clinical research by experts from different disciplines. However, the work carried out at the University of Minnesota scientists led by Professor James White represents a major cornerstone in the overall development of platelet sciences. This book is a portrayal of the outstanding basic and clinical research on the structure and function of platelets and their interactions with the endothelium and other blood cells.
Of particular note, this book is a result of a very strong professional relationship between the author, Dr Rao and then lead hematologist at the University of Minnesota Professor James White, who was truly a pioneer in platelet related sciences. Dr Rao's recognition of the importance of compiling a complete account of the outstanding work carried out by Professor White is not only timely but is an inspirational tribute to a great mentor, Professor White. The compilation of this book and recognition of the pioneering work in nearly all aspects of platelet biology with a strong focus on the ultra-structure is the most appropriate way to acknowledge a mentor for which Dr Rao is to be commended. Professor White had dedicated most of his life to the study of platelets and related disorders. He himself was a 1955 graduate of the University of Minnesota Medical School, Minnesota, USA. After his initial training, he spent over 50 years at the same institution practicing hematology and overseeing a group of clinicians and scientists working on platelets. He is and will continue to be recognized as a lead investigator who applied great scientific vision and an integrated approach to identify the role of platelets in health and disease. His work on the fine structural details of platelets as they relate to the function of these anuclear cells revolutionized our understanding of the physiology and pharmacology of platelets. This book, illustrating the different aspects of platelet structure and function and a progressive evolution of innovative approaches resulting in various discoveries truly identifies him as a great mentor and leader. His interest in electron microscopy and its application to the study of platelets and blood cells encouraged the entire University of Minnesota team to advance this technique to its heights which resulted in the production of so many interesting images included some of which are included in the book. The photo micrographs and images generated at the University of Minnesota's hematology laboratories adorn the pages of the best hematology books all over the world and some of the lead publications carry these images and their references. Dr Rao has expertly summarized the complex fields of platelet morphology, physiology, pathology and pharmacology in this monograph in a masterly fashion which is easy to read and will be of major value for the students and scholars working in the field of platelets.
This monograph is comprised of 16 chapters which are dexterously prepared with beautiful illustrations wherever applicable. The first chapter provides a very clear account of platelet morphology with specific reference to different structural components. The discussion on the structure is so well written that it can be understood by those with minimal background in hematology. The second chapter, dealing with platelet morphology and function, contains beautiful illustrations and a description of various techniques used to characterize functional morphology of platelets. Complimentary to this, the 3rd chapter interfaces platelet morphology with functional alterations in various diseases highlighting the structural components involved in some of these disorders. From an evolutionary stand point platelet dysfunction in animals is referenced to highlight some points. The 4th chapter deals with platelet physiology including the progressive changes in platelet regulatory actions. Once again the emphasis is on functional changes and the impact of structural alterations is highlighted. The 5th chapter deals with biochemical pathways involved in the platelet activation mechanism. For students working in signal transduction and other areas this will be important reading. Chapter 6 illustrates the role of calcium in platelet activation. Special emphasis is placed on modulation of ionized calcium by agonists and antagonists. The platelet activation and deactivation mechanisms are illustrated. Chapter 7 deals with the role of prostaglandins in platelet function whereas Chapter 8 discusses the calcium modulation by cyclic nucleotides.
The author has great expertise in the modulation of platelets by drugs and has masterfully described the pharmacology of antiplatelet drugs in Chapter 9. The mechanism of action of these drugs and their potential impact on treatment of diseases is also illustrated. Complimentary to this, Chapter 10 provides a comprehensive account of antiplatelet drugs in clinical trials. Of note, the work of the scientists at the University of Minnesota to develop point of care instrumentation to measure platelet response to antiplatelet drugs is highlighted. Once again stating the impact of physician/scientist collaboration at the University of Minnesota. Platelets have complex physiological processes and drug resistance is a major problem. Chapter 11 deals with aspirin resistance, a subject in which the author is an expert, and provides valuable guidelines to deal with this complication. Diabetes mellitus is associated with major platelet dysfunctions and this is beautifully illustrated in Chapter 12 in terms of the role of platelets in diabetes.
Professor Rao has a strong interest in acute coronary syndrome and is a great scholar of the pathophysiology and the pharmacologic management of this syndrome. In a very concise and masterful fashion, he has compiled Chapter 13 on platelet hyper-function as a risk factor for acute coronary events. In addition, the contribution of platelet activation of circulating blood cells and activation leading to vascular dysfunction is detailed. To address the monitoring of acute coronary events, the need for a point of care assay for monitoring antiplatelet and antithrombotic therapies is also discussed. Professor Rao rightly pointed to the fact that South Asians have a very high incidence of coronary artery disease suggesting the genetic components involved in this process. The membrane modulation concept was coined at the University of Minnesota where epinephrine mediated events were implicated. This led to the understanding of the initial mechanisms involved in the activation of platelets in health and disease. The role of alpha-adrenergic receptor antagonists and beta blockers in the modulation of this process was proposed. Chapter 15 discusses the platelet vessel wall interaction which plays an important role in the progression of hemostatic plug and atherosclerotic plaque. The interplay between endothelial cells and platelets is illustrated. To investigate these interactions experimental approaches using the annular chamber profusion system are described. This chapter also contains some beautiful illustrations on the interactions of platelets and endothelial cells. Platelet reaction time (PRT) monitoring device to monitor sheer force mediated platelet activation and clot formation was also highlighted. The final chapter is dedicated to the discussion of blood biocompatibility studies with a focused reference to the contributions of the blood biocompatibility laboratory (BBL) at the medical school of the University of Minnesota. In this chapter, integrated approaches involving various groups including the faculty at the Lillehei Heart Institute are highlighted. This provided a strong academia-industry platform and the BBL offered the opportunity for medical device companies to work together to advance stroke research and other various scientific approaches to understand blood compatibility with surfaces, in particular the role of platelets in blood biomaterial interactions. Platelet physiology and function utilizing simple instruments as a Chandler loop coupled with electron-microscopic illustration of cellular activation validated blood biomaterial interactions. Other techniques dealing with blood cells under flow conditions, exposure of blood cells to fully assembled heart walls, and platelet adhesion to biomaterials were detailed. As the field of nano-microparticles emerged, this group also worked on cardiovascular drug delivery and targeting processes.
It can be easily seen that the evolution of platelet function with reference to morphometric alterations laid the foundation for a most comprehensive, multidisciplinary research approach including nearly all basic sciences. This would not have been possible without the intimate mentor trainee relationship which was initiated by Professor Jim White over 50 years ago. Dr Rao's recognition of Professor White's unparalleled drive and leadership resulted in a very productive group which continued to advance in the field of vascular medicine, the diagnosis of bleeding and thrombotic disorders related to platelets. Platelets are now considered to be an integral component of the vascular system. There is so much more to be learned. Advances in cellular and molecular biology have transformed platelet biology and have led us to recognize these cells in health and disease. This would not have been possible without the great vision of Professor White and his ability to recruit outstanding trainees, transforming them to lead physicians and scientists who will continue to impact this area for years to come. Professor Rao worked under Professor White and, like his mentor, has trained many younger scientists in the most integrated fashion bridging basic science and clinical medicine as portrayed in this book. While this book is a true tribute to Professor White at the Lillehei Heart Institute of the University of Minnesota which will remain a pioneering institution in cardiovascular research for years to come, its publication in India has a major significance including the outstanding talents in teaching and research and the pursuit for excellence. The information has direct relevance on the public health impact, in particular, the cardiovascular disease care in South Asian countries.
Jawed Fareed PhD DSc FAHA
Professor of Pharmacology and Pathology
Loyola University School of MedicinePreface
Blood platelets play a very important role in the pathogenesis of atherosclerosis, thrombosis and stroke. In 1998, Kluwer Academic Publishers of Boston, USA contacted me and asked me to put together a Handbook on Platelet Physiology and Pharmacology. I recruited well-known national and international researchers, working in the area of atherosclerosis, thrombosis and stroke, to contribute articles for this book. I made a special request to all the contributors, to make an all-out effort to describe platelet morphology, biochemistry, physiology and pharmacology as it relates to function. When elite group of researchers develop a multidisciplinary project, by and large, they stick to their expertise and will not develop a cohesive, comprehensive story to summarize their collective experience. Our monograph was a collection of state-of-the-art topics, but failed to tell a story that summarizes what is known of platelets and how they work. Since then, I have discussed this issue many a times with my mentor, Prof James G White. We both were fascinated with the mechanisms involved in platelet activation and function. We discussed the topic, “How Platelet Works” many a times, in our long-working relationship. I wanted to put together a book on this subject with his help. However, I was not able to do it for various reasons, which are not worth discussing here. I came to know about his passing way, when I was vacationing in India this summer (www.startribune.com/obituaries/detail/145854/?). His obituary mentions, that he had over 700 articles to his credit. It made me feel bad, thinking about the missed opportunity for a collective description of our work, which lasted for forty years. Since the time, I received the news of his (Dr White's) deteriorating health from our close friend and associate Dr Gines Escolar from Barcelona, Spain (Currently resides in New Jersey, USA), I have been thinking nothing but to find a way to put together a monograph, that covers our four decades of work on Platelets. With these thoughts in mind, I approached our longtime friend (Mr Jitendar P Vij) at the Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India. They have published four of our books from 2001 to 2007, under the aegis of South Asian Society on Atherosclerosis and Thrombosis (SASAT). In spite of the fact that this is too specialized a subject and may attract only selected group of readers, they graciously accepted my request and agreed to publish this book. Electron photomicrographs that I have used in this book to illustrate platelet morphology and function, were gifted to me by late James G White. His long-term associate Ms Marlys Krumweide willingly prepared a set of slides for my use at various scientific conferences. I acknowledge my sincere thanks to them and dedicate this monograph to my mentor and associate, Regents Professor of Pediatrics and Pathology, James G White, MD. Many other illustrations were prepared for us for our presentations by the professional art and graphics department, at the University of Minnesota. Some of the illustrations, I have taken the liberty of borrowing from public domain (Internet). I also have taken liberty of using significant information from our previously published articles. The author assures the readers that the major part of the work presented in this book comes from the four decades of work done by Prof James G White, Prof Gundu HR Rao, and associates at the Thrombosis Research Laboratory, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota. In each chapter, author has provided references to published work of their own group as well as relevant references from other experts. At the end of the book, author has provided a complete bibliography of their work on platelets, for those who are interested in additional reference material to the source of information. We take this opportunity to sincerely thank all those who have contributed to our collective knowledge base.
One may wonder, why I am putting together a monograph on Blood Platelets? Recently I read a historical review on Platelets, which had over 400 references. I found only one reference to our work of 40 years and over 1000 articles collectively. This shows that either the reviewer has ignored completely our contribution to this collective knowledge or has no idea of their existence in the platelet literature. Either way, we feel that it is important to present our collective thoughts on this subject for the readers, who may be interested in looking at this topic from a different perspective. In addition to this reasoning, after reading several of these reviews, I found out, that they are like viewing a rain forest from sky. You see a canopy not individual tress. In this monograph, I will not try to catalogue all that is published on this subject. I will try to write more about how platelets work (read Platelet Story) rather than minute details, which may or may not make any sense to a beginner or for someone, who wants to get to know as to how platelet works. The monograph is compiled for the beginners, and for those who are new to this area and not for experts in this field of investigation.
Gundu HR Rao PhDAcknowledgments
I would like to acknowledge significant contributions of Dr Jonathan Gerrard of Winnipeg, Canada, as well as Dr Gines Escolar of Barcelona, Spain, for our understanding of morphology, physiology and function of blood platelets. I have included references to the work of other researchers on this topic, so that those interested in learning more about specific pathways, mechanisms, will be able to access them. I express my sincere thanks to my father-in-law, Dr TN Ramachandra Rao, a pioneer scientist in our family, for his continued encouragement of my international activities. One of his wishes was, that I should put together a “single author” book on Platelets. I am glad that his wish is being fulfilled by the publication of this monograph. I express my sincere thanks to my wife, Dr Yashoda T Rao for her patience, cooperation and encouragement. I also would like to thank our grandson, Master Akshay R Tate, for his technical support in the preparation of this manuscript. I thank Mr Jitendar P Vij (Group Chairman), Mr Ankit Vij (Group President), Ms Chetna Malhotra Vohra (Associate Director—Content Strategy) and the team at Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for taking the responsibility of publishing this book for our professional society, South Asian Society on Atherosclerosis and Thrombosis (SASAT), Division of Clinical and Preventive Cardiology, Medanta Hospital, Gurugram, Haryana, India.
In a book, that is dedicated to the memory of my mentor and associate, (Late) Prof James G White, I would be failing in my duty and responsibilities, if I do not mention the names of the other four mentors who shaped my professional career. First and the foremost, my PhD thesis adviser, (Late) Donald A Wilbur, Professor, Department of Entomology, Kansas State University, Manhattan, Kansas, Dr (Late) Neville Crawford, Professor and Head, Department of Biochemistry, Royal College of Surgeons, Lincoln Inn Fields, London, Dr (Late) Sir Vijay V Kakkar, Founder-Director, Thrombosis Research Institute, Emeritus Professor of Surgery, King's College, Denmark Hill Road, London and Dr (Late) MG Doni, Professor, Instituto di Fisiologia Umana (Institute of Human Physiology), University of Padova, Padova, Italy.Four Decades of Global Collaborative Studies
Principal collaborator for these studies was Late James G White, mentor, and associate for over four decades. I will not be able to list all the collaborators by name. Hence, I humbly request all those who worked with us, if they do not see their names mentioned under this chapter, to excuse me. I do thank each and every one of them, for their valuable contributions. Dr Jonathan M Gerrard, a Canadian clinician joined us in the early 70s as a research fellow to do his PhD. He introduced us to eicosanoids. Prof John W Eaton, a molecular anthropologist in our department, introduced us to the free radical chemistry and their role in oxidation-reduction mechanisms. Dr Douglas Peterson, who was a freelance researcher worked with us for a short period and made significant contribution to our understanding of arachidonic acid-heme interactions as well as the role of epinephrine modulation of some of the steps in (Peterson DA, et al. Science. 1982;215:71-73) agonist-mediated signal transduction events. Dr Maurie J Stuart, a Professor from Children's Hospital, Thomas Jefferson University, Philadelphia, joined us a research fellow and worked with Dr Gerrard and our research group. For the first time, she demonstrated in a drug induced animal model, that the balance between prostaglandins from platelets and vessel wall is altered in hyperglycemic animals. Furthermore, she showed that islet cell transplantation in these animals restored the prostaglandin synthesis to normal levels. Chadwick A Cox, a Biochemist from Oklahoma University Health Center, Oklahoma, joined us as an honorary faculty and worked on topics related to platelet cytoskeletal rearrangements and phosphorylation of proteins associated with activation and inactivation processes (Cox AC, et al. J Cell Biol. 1984;98:8-15). Dr Douglas Weiss, a Professor in the Veterinary College, University of Minnesota, Minnesota and Gerhard J Johnson, an Adjunct faculty, Department of Medicine, University of Minnesota, Minnesota and a staff at the Veterans Administration Hospitals, collaborated with us on studies, which used various animal models.
In the areas of clinical studies, (Late) Prof Carl J Witkop, a pioneer and expert in studies on Albinism associated disorders, shared with us an National Institutes of Health (NIH) program project research grant on this topic and helped us work on patients with Albinism (Hermansky-Pudlak syndrome) from all over the world. We also shared an NIH program project with Prof Mark Nesbit, a pediatric oncologist, on bone marrow transplantation. In the cardiovascular research area, we shared an NIH program project with Prof Robert Vernier. In the basic sciences field, we shared an NIH program project grant with Prof Ellis Benson and Prof Andreas Rosenberg on Hemoglobinopathies. We collaborated extensively with Cardiology group (Prof Stanley Einzig, Prof Carl White, Prof Robert Wilson, Prof Gladwin Das). Professor James G White and I shared NIH research grants for well over three decades (36 years) including an NIH-Merritt Award for 10 years in the 80s.
International Collaboration was a very important part of our activities. Dr Rao obtained a National Science Foundation (NSF) travel grant to visit India, in the 80s to establish bilateral research projects between the USA and India. Prof John W Eaton and Dr Rao visited India in the 80s. They visited major Medical Institutions from Kashmir in the North, to Thiruvananthapuram in the South. They gave lectures on various topics at each of this Medical Institution. Institutions visited included, Kashmir Medical College, Srinagar; Postgraduate Medical Institute, Chandigarh; All India Institute of Medical Sciences, New Delhi, National Malaria Institute, New Delhi; Postgraduate Medical Institute, Lucknow; Indian Institute of Science, Bengaluru; Postgraduate Medical Institute, Chennai; and Christian Medical College, Vellore. Dr Rao was a visiting Professor in the Department of Surgery (Prof Sir Vijay J Kakkar) and Pharmacology, King's College, Denmark Hills, London, in 1986 and in the Biochemistry Department (Prof Neville Crawford) in the Royal College of Surgeons, London, in the summer of 1987, and in the Thrombosis Research Institute (Prof VJ Kakkar), London in 1989. He taught one summer at the Institute of Human Physiology (Prof Maria Gabriella Doni), University of Padova, Padova, Italy. The Government of India, invited Dr Rao as a Senior Consultant in the year 1990, under a United Nations (UN) program called, TOKTEN (Transfer of Knowledge by Expatriate Nationals). Dr Khalilullah, Professor of Cardiology and Director of GB Pant Hospital, New Delhi, India, sponsored the application for this fellowship. Dr Rao spent 6 weeks in India visiting major Medical Institutions. Dr Rao visited India again in 1991 and 1992 under the same UN program.
With the seed money (USD 10,000) obtained from the International Society on Thrombosis and Hemostasis (ISTH), USA. Dr Rao started a professional society, South Asian Society on Atherosclerosis and Thrombosis (SASAT) in Minnesota in1993. Under the aegis of SASAT, he organized 15 international conferences on the topics of Atherosclerosis and Thrombosis in India. Dr. Rao served as senior adviser to the Presidents of North American Thrombosis Forum (Prof. Samuel Goldhaber, Harvard University: www.natfonline.org), International Union of Angiology (IUA), France.Platelet Story
I joined Late Professor James G White's group as an assistant scientist to establish an analytical laboratory, to conduct biochemical and functional studies, to follow his ultra-structural studies as well as to support his earlier findings with evidence-based biochemical investigations. Being new to this area, I was wondering as to how I can contribute anything more to his massive cumulative knowledge of the subject. I started off by setting up new analytical methods, to monitor various biochemical components of platelets (serotonin, adenine nucleotides, prostaglandins, cytosolic calcium), which were known to play a critical role in platelet physiology and function. Just at the beginning of my career in this department, Jonathan Gerrard a Canadian doctor joined our group to do his PhD (He is currently a Member of the Parliament in Manitoba). He spent over five years with us and was great moving force behind research into the emerging field of prostanoids.
Just at this time, we discovered that some specific fluorescent molecules (Quin-2 and Fura-2) could be used to monitor cytosolic free calcium. Using these techniques, we were for the first time, able to demonstrate the role of agonists in calcium mobilization from internal storage sites. We were able to demonstrate at the same time, that the antagonists were capable of lowering the elevated cytosolic calcium by activating adenylyl/guanylyl-cyclases and generating second messengers, cAMP and cGMP. These studies formed the basis for our simple platelet story. By this time, a young investigator from Barcelona, Spain, Dr Gines Escolar (Senior Consultant, Hemostasis Coagulation/Hemotherapy Hospital Clinic, Department of Hemotherapy and Hemostasis) joined us. He introduced us to, some newer techniques, to follow the platelet interactions with the vessel wall. Collective information that we gathered in these years formed the basis for the “Platelet Story”. During the four decades, that I spent at the University of Minnesota, we also were able to discover a unique phenomenon of “Membrane Modulation”, which is described in detail, in a later chapter in this book.
Investigations in our laboratory at the University of Minnesota established the existence of a novel mechanism capable of securing irreversible platelet aggregation independent of ADP secretion, prostaglandin synthesis or the generation of platelet activating factor (PAF). The new mechanism, termed membrane modulation, is associated with the platelet surface, mediated through alpha-adrenergic receptors and stimulated by exposure to epinephrine. Platelets rendered refractory to a variety of aggregating agents can be restored to a normal state of sensitivity through activation of the mechanism of membrane modulation. This included platelets treated with aspirin in vivo or in vitro. In view of the fact, that our studies demonstrated that the mechanism of platelet membrane modulation was dependent upon alpha-adrenergic receptor function, we explored the significance and nature of this dependency. Since these mechanisms seems to be critical for the restoration of the sensitivity of refractory platelets to the action of a variety of agonists, any impairment of alpha adrenergic receptor function will therefore, interfere with the mechanism of membrane modulation.
Major contributions of the Platelet Research Laboratory, Laboratory Medicine and Pathology and the Thrombosis Research Laboratory, Lillehei Heart Institute, Academic Health Center, the University of Minnesota, include; superb Electron Microscopical Studies on Platelet Ultrastructure and function of normal human platelets, platelets of patients with various hematological disorders, and platelets of various animals. In view of the fact that these were not only state-of-the-art investigations at the cellular and subcellular level, but also one-of-a-kind studies, the National Institutes of Health, USA graciously funded these studies, for well over three decades. As a part of the original investigations at the Thrombosis Research Laboratory, we did a variety of biochemical studies to explore and complement the ultrastructural findings of Prof James G White.
Our association with Prof Gines Escolar and Dr Thomas Chandy introduced us to studies on blood-vessel interaction as well as blood-biomaterial interactions. Blood compatibility studies, were supported by the Departments of Chemical Engineering (Center for Interface Engineering), and the Biomedical Research Institute, Academic Health Center, the University of Minnesota. We were able to establish a state-of-the-art device/biomaterial testing facility and serve the Institute of Engineering Medicine, University of Minnesota as well as many device material manufacturing companies in the USA. One of my close associate (Dr Daniel Ericson), who joined us as an industrial fellow from 3M company, in the late 70s, established his own research laboratory (SUBc, Inc) at Rochester, Minnesota and developed two NIH (STTR) research grants for medical device development. IBM Rochester served as a contractor for the development of this device. I was the Principal Investigator for both of these device development research proposals. Multidisciplinary experience gained at the University of Minnesota, helped me initiate collaborative programs in India in several institutions. Since our research on platelets spanned over four decades, I have included a comprehensive bibliography of our collective work for readers who want to supplement their knowledge on this subject as a separate section at the end of this book.