University of Pennsylvania ScholarlyCommons
Publicly Accessible Penn Dissertations
Fall 2010
Developing Novel Platelet-Based Targeting Strategies for Thrombolytics
Rudy E. Fuentes The University of Pennsylvania, [email protected]
Follow this and additional works at: https://repository.upenn.edu/edissertations
Part of the Therapeutics Commons
Recommended Citation Fuentes, Rudy E., "Developing Novel Platelet-Based Targeting Strategies for Thrombolytics" (2010). Publicly Accessible Penn Dissertations. 263. https://repository.upenn.edu/edissertations/263
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/263 For more information, please contact [email protected]. Developing Novel Platelet-Based Targeting Strategies for Thrombolytics
Abstract Use of plasminogen activators (PAs) as thrombolytic drugs is restricted to life threatening thrombotic settings because these therapies are associated with a high risk of bleeding. We hypothesize that platelet-delivered PAs would preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing pre-formed hemostatic clots. Two potential approaches were pursued: 1) PA- loaded platelets that release the thrombolytic from its granular stores upon activation, and 2) a thrombolytic chimeric protein that specifically binds ot human platelets and activated when the platelets are incorporated into a growing thrombus. In our first approach, we desired to develop a strategy for producing platelets ex-vivo from cultured megakaryocytes that ectopically expressed urokinase-PA (uPA). No group had successfully produced sufficient ex-vivo generated platelets before, to side step this issue we infused ex-vivo generated megakaryocytes and showed that we can achieve a significant number of donor-derived platelets from these infused megakaryocytes in a murine model. The resulting platelets were normal in size, surface markers, circulating half-life, and were functional. Infused megakaryocytes localized to the pulmonary vasculature to shed platelets. We demonstrated, beginning with megakaryocytes derived from a transgenic mouse that ectopically express and store uPA in their alpha- granules that we can interfere with thrombosis by platelets generated from these megakaryocytes. In the second approach we produced a chimeric protein by fusing a single chain variable fragment (scFv) directed to the human-αIIb (hαIIb) platelet receptor subunit, with a human thrombin activatable pro- urokinase (uPA-T). The fusion protein (anti-PLT scFv/uPA-T) bound specifically ot human and to transgenic mice platelets that expressed hαIIb, termed hαIIb+ mice, but did not bind to wildtype (WT) mouse platelets. Anti-PLT scFv/uPA-T retained its zymogenic properties until activated by thrombin. HαIIb+ mice were protected from forming occlusive thrombi for at least 10 hrs post anti-PLT/uPA-T treatment in contrast to the short functional half-life of soluble uPA-T. Thus this dissertation presents two distinct strategies that in proof of principle studies are each promising as approaches for effective and targeted platelet directed thrombolytic, which merit further study to test clinical applicability.
Degree Type Dissertation
Degree Name Doctor of Philosophy (PhD)
Graduate Group Pharmacology
First Advisor Dr. Mortimer Poncz
Keywords Targeting thrombolytics, platelet, platelet transfusions, plasminogen activators, urokinase
Subject Categories Therapeutics
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/263 DEVELOPING NOVEL PLATELET BASED TARGETING STRATEGIES
FOR THROMBOLYTICS
Rudy E. Fuentes A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 Dr. Mortimer Poncz, M.D. Supervisor of Dissertation Vladimir R. Muzykantov, M.D., Ph.D. Graduate Group Chairperson Dissertation Committee Rodney M. Camire, Ph.D. Vladimir R. Muzykantov, M.D., Ph.D. Doug Cines, M.D. Donald L. Siegel, M.D., Ph.D. Joel S. Bennett, M.D.
DEVELOPING NOVEL PLATELET BASED TARGETING
STRATEGIES FOR THROMBOLYTICS
COPYRIGHT
2010
Rudy E. Fuentes
ii
Acknowledgments
I consider myself fortunate to have been influenced by a number of brilliant people while pursuing my scientific career. The first person is Dr. George B. Stefano at the State
University of New York (SUNY) Old Westbury, who gave me an opportunity to work in his laboratory during my undergraduate studies, and as a consequence sparked my interest in scientific research. His mentorship provided me with a strong academic foundation that resulted with acceptance in the prestigious Pharmacology graduate program at the University of Pennsylvania.
During my graduate studies the most influential person responsible with the work presented in this thesis is my advisor, Dr. Mortimer Poncz. I am grateful for giving me the opportunity to work in his laboratory, and providing me with the invaluable guidance to analyze and take action on scientific matters. I am amazed at his vast knowledge in hematology, and his way of communicating and directing his students in the laboratory. I want to also recognize my committee members who have helped me greatly to advance my work with their scientific insight, Drs. Rodney M. Camire ,
Vladimir R. Muzykantov, Doug Cines, Donald L. Siegel, and Joel S. Bennett. Another person that deserves my gratitude is Dr. Sriram Krishnaswamy, who has been instrumental in providing me with thoughtful assessments and suggestions during journal club.
When I look back at my academic life, I can say that it has been a rough ride, but throughout my hardships there has been my wife Karen Fuentes, who has supported me and encourage me to be persistent in completing my degree. I would also like to thank her for providing me with the best gift in life, my son Rudy A. Fuentes, and a second one
“baby Benjamin” to be born in the first weeks of February 2011.
iii
Finally I was born in the city of La Union in El Salvador and resided in the little village of El Jicaro for the first ten years of my life, until my mother Virginia Fuentes brought all six of us (Mirna, Rosa, Nelson, Ana Ruth, and Yolanda) to the United States
as a consequence of a civil war. I am proud to say that I will be the first person with a
Ph.D. in my family, with some of my siblings achieving academic success as well. I
attribute our success to my mother’s determination in providing a better life for our
family in this country. Her perseverance has been a strong source of inspiration
throughout my life, and I thank her for being such an influential person to all of us.
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ABSTRACT
DEVELOPING NOVEL PLATELET BASED TARGETING STRATEGIES FOR
THROMBOLYTICS
Rudy E. Fuentes
Mortimer Poncz
Use of plasminogen activators (PAs) as thrombolytic drugs is restricted to life threatening thrombotic settings because these therapies are associated with a high risk of bleeding. We hypothesize that platelet delivered PAs would preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing pre formed hemostatic clots. Two potential approaches were pursued: 1) PA loaded platelets that release the thrombolytic from its granular stores upon activation, and 2) a thrombolytic chimeric protein that specifically binds to human platelets and activated when the platelets are incorporated into a growing thrombus. In our first approach, we desired to develop a strategy for producing platelets ex vivo from cultured megakaryocytes that ectopically expressed urokinase PA (uPA). No group had successfully produced sufficient ex vivo generated platelets before, to side step this issue we infused ex vivo generated megakaryocytes and showed that we can achieve a significant number of donor derived platelets from these infused megakaryocytes in a murine model. The resulting platelets were normal in size, surface markers, circulating half life, and were functional. Infused megakaryocytes localized to the pulmonary vasculature to shed platelets. We demonstrated, beginning with megakaryocytes derived from a transgenic mouse that ectopically express and store uPA in their alpha granules that we can interfere with thrombosis by platelets generated from these megakaryocytes. In the second approach
v
we produced a chimeric protein by fusing a single chain variable fragment (scFv)
directed to the human αIIb (h αIIb) platelet receptor subunit, with a human thrombin activatable pro urokinase (uPA T). The fusion protein (anti PLT scFv/uPA T) bound specifically to human and to transgenic mice platelets that expressed h αIIb, termed hαIIb + mice, but did not bind to wildtype (WT) mouse platelets. Anti PLT scFv/uPA T retained its zymogenic properties until activated by thrombin. H αIIb + mice were protected from forming occlusive thrombi for at least 10 hrs post anti PLT/uPA T treatment in contrast to the short functional half life of soluble uPA T. Thus this dissertation presents two distinct strategies that in proof of principle studies are each promising as approaches for effective and targeted platelet directed thrombolytic, which merit further study to test clinical applicability.
vi
Table of Contents
Title page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Copyright Notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
List of Illustrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1. Introduction
1.1 Background
1.1.1 Pathogenesis of thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.2 History of platelet biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.3 Megakaryocyte development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.4 Platelet development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.5 Platelets role in thrombus formation . . . . . . . . . . . . . . . . . . . . . . . 6
1.1.6 Fibrinolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.7 Key mediators of fibrinolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.8 Clinical use of PAs and their limitations . . . . . . . . . . . . . . . . . . . . 12
1.2 Dissertation objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2. Infusing mature megakaryocytes into mice yields functional platelets
2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
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2.3 Materials and Methods
2.3.1 Characterization of the mice studied . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.2 Production of mature megakaryocytes ex vivo . . . . . . . . . . . . . . . 20
2.3.3 Characterization of ex vivo derived megakaryocytes . . . . . . . . . 21
2.3.4 Isolation of platelets and megakaryocytes for infusion . . . . . . . . 21
2.3.5 Flow cytometric studies in infused hαIIb + mice . . . . . . . . . . . . . . 22
2.3.6 Infusion studies in thrombocytopenic hαIIb + mice . . . . . . . . . . . 22
2.3.7 Cremaster laser injury functional studies . . . . . . . . . . . . . . . . . . . 23
2.3.8 FeCl 3 carotid artery injury functional studies . . . . . . . . . . . . . . . . 23
2.3.9 Infused megakaryocyte fate studies . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4 Results
2.4.1 Infused megakaryocytes and platelets . . . . . . . . . . . . . . . . . . . . . . 25
2.4.2 Characterization of the in vivo derived platelets from FL cells. . 26
2.4.3 Functionality of platelets derived from infused FL cells . . . . . . . 37
2.4.4 Organ distribution studies of infused cells . . . . . . . . . . . . . . . . . . 38
2.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3. Platelet targeted pro urokinase as a novel thromboprophylaxis fibrinolytic
strategy
3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.3 Materials and methods
3.3.1 Analysis of anti hαIIb moAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3.2 Platelet aggregation of anti hαIIb moAbs . . . . . . . . . . . . . . . . . . 53
3.3.3 Construction and expression of anti PLT scFv/uPA T . . . . . . . . 53
3.3.4 Biochemical characterization of anti PLT scFv/uPA T . . . . . . . . 57
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3.3.5 Binding of anti PLT scFv/uPA T to h αIIb β3 . . . . . . . . . . . . . . . . 58
3.3.6 In vitro fibrinolysis of anti PLT scFv/uPA T . . . . . . . . . . . . . . . . 59
3.3.7 Effect of anti PLT scFv/uPA T in models of vascular
thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.3.8 Future studies with the anti PLT scFv/uPA T fusion protein . . . 60
3.4 Results
3.4.1 Binding analysis of anti platelet moAbs . . . . . . . . . . . . . . . . . . . . 63
3.4.2 Binding analysis of anti platelet scFv . . . . . . . . . . . . . . . . . . . . . . 63
3.4.3 Analysis of anti platelet scFv/uPA T . . . . . . . . . . . . . . . . . . . . . . . 67
3.4.4 Fibrinolytic activity of anti platelet scFv/uPA T in vitro . . . . . . 70
3.4.5 Thrombolytic efficacy of anti PLT scFv/uPA T in mouse
Models of vascular thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4. Summary, and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
ix
List of Tables Table 2 1. Calculation of number of alveoli capillaries blocked by infused megakaryocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Table 2 2. Supplemental movie legend . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
x
List of Illustrations Figure 1 1. Megakaryopoiesis and platelet development . . . . . . . . . . . . . . . . . . . . 5
Figure 1 2. Platelet plug formation under arterial circulation . . . . . . . . . . . . . . . 8
Figure 1 3. Fibrinolytic cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Figure 1 4. Structure of scuPA and its various cleaved forms . . . . . . . . . . . . . . . . 13
Figure 1 5. Depiction of the two strategies proposed . . . . . . . . . . . . . . . . . . . . . . . 16
Figure 2 1. Isolation and characterization of FL cells . . . . . . . . . . . . . . . . . . . . . . 27
Figure 2 2. Comparison of retro orbital Vs tail vein infusions of FL large cells 28
Figure 2 3. Flow cytometric detection analysis of infused platelets . . . . . . . . . . . 29
Figure 2 4. Flow cytometric analysis of infused WT platelets, and FL derived megakaryocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Figure 2 5. Flow cytometric analysis of infused WT platelets, FL and BM derived cells on h αIIb + mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Figure 2 6. Flow cytometric analysis of infused WT platelets and FL derived cells in thrombocytopenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Figure 2 7. Effect of ADAM17 on thrombopoiesis from in vitro grown FL cells 34
Figure 2 8. Characterization of the platelets derived from infused cells . . . . . . . 35
Figure 2 9. Characterization of platelets derived from infused FL cells . . . . . . . 36
Figure 2 10. Platelet incorporation into arterial clots after laser injury . . . . . . . 39
Figure 2 11. FeCl 3 carotid artery injury studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Figure 2 12. Organ distribution studies of infused FL cells . . . . . . . . . . . . . . . . . 41
Figure 3 1. Final construct proposed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Figure 3 2. Molecular design of uPA T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Figure 3 3. Depiction of the strategy to generate the anti PLT scFv . . . . . . . . . . 54
Figure 3 4. Depiction of the strategy to generate the anti PLT scFv/uPA T . . . . 55
xi
Figure 3 5. Binding analysis of anti hαIIb specific moAbs to human platelets . . 64
Figure 3 6. Platelet aggregation analysis of anti hαIIb specific moAbs . . . . . . . 65
Figure 3 7. Generation the anti PLT scFv/uPA T fusion construct . . . . . . . . . . . 66
Figure 3 8. ELISA analysis of moAbs and scFv induced and non induced S2 media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Figure 3 9. Characterization of anti PLT scFv . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Figure 3 10. Characterization of the anti PLT scFV/uPA Ts . . . . . . . . . . . . . . . . 71
Figure 3 11. Flow cytometric analysis of haIIbß3 binding by anti PLT scFv/uPA T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Figure 3 12. Binding of anti PLT scFv/uPA T to immobilized h αIIb β3 . . . . . . . 73
Figure 3 13. Enzymatic activity of anti PLT scFv/uPA T . . . . . . . . . . . . . . . . . . . 74
Figure 3 14. Prophylactic thrombolysis by anti PLT scFV/uPA T in a FeCl 3 carotid artery injury model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Figure 3 15. Platelet and fibrin accumulation in laser induced arteriole injuries in treated haIIb + mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
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Chapter 1
Introduction
1.1 Background
1.1.1 Pathogenesis of thrombosis
Thrombosis can occur in either the arterial or venous systems and both can be life
threatening. Acute arterial thrombosis is the proximal cause of most cases of myocardial infarction (heart attack) and of about 80% of strokes, collectively the most common cause of death in the developed world, and venous thromboembolism is the third leading cause of cardiovascular associated death (1). Thrombosis manifests itself due to an imbalance in hemostatic procoagulant and anticoagulant mechanisms that maintain normal physiological conditions. The endothelium is a major contributor to maintaining this balance by providing an antithrombotic surface that inhibits platelet adhesion and clotting. However, when the endothelium is perturbed by physical forces or by inflammation, the cells undergo programmatic biochemical changes that culminate in their transformation to a prothrombotic surface (2). During vascular injury, the coagulation system is activated with the conversion of thrombin from prothrombin by exposure of tissue factor. Thrombin is able to cleave fibrinogen to form fibrin, which contributes to the stabilization of the platelet plug. Almost concurrently, fibrinolysis goes into effect to limit and eventually remove the thrombus and recanalized the vessel. It is important to note that coagulation and fibrinolysis are two antagonistic phenomena and a balance is required amongst their respective activators and inhibitors for the 1
processes to be physiologic (3). These mechanisms involved in keeping the hemostatic balance will be discussed in more detail in the sections that follow.
1.1.2 History of platelet biology
The discovery of platelets is best attributed to a summation of work by different scientists dating back to 1842, when platelets were recognized as little globules morphologically different then red or white blood cells (4). The most powerful evidence was provided by Giulio Bizzozero in 1881 and 1882, using intravital microscopy, he described platelets anatomically as well as their adhesive properties upon damage to the vascular wall (5, 6). Interestingly, 12 years before his platelet publication, Bizzozero observed large irregular cells in the bone marrow with highly lobulated, multilobed nuclei, but their significance was unknown until William Howell with his camera lucida studies in 1890 was able to corroborate on those observations and coined the term megakaryocytes (7). In 1906, Wright suggested that blood platelets were derived from the cytoplasm of megakaryocytes noting the similarities in both cells when exposed to a new polychrome staining solution (Wright’s stain) (8), and with these studies the basic elements of thrombopoiesis were established.
1.1.3 Megakaryocyte development
During megakaryopoiesis, committed hematopoietic stem cells (HSCs) undergo several stages of differentiation resulting in mature megakaryocytes that produce and release platelets into the circulation (9). HSCs are found primarily in the bone marrow (10, 11), and during mammalian development, they can also be found in the embryonic yolk sac, fetal liver, and spleen (12). To study megakaryocytic progenitors, in vitro clonal assays were developed to analyze the cellular basis of lineage commitment and cell
2
differentiation (13, 14). Based on these studies, a few megakaryocytes have been shown
to arise in culture (Figure 1 1) from mixed lineage primitive cells that are termed colony
forming unit granulocyte erythroid monocyte megakaryocyte (CFU GEMM) (15). More
differentiated burst forming unit megakaryocytes (BFU Meg) are able to produce larger
complex colonies that include satellite collections of megakaryocytes. The colony
forming unit megakaryocyte (CFU Meg) can form 3 50 mature megakaryocytes and are
the first fully committed cells to the megakaryocyte lineage base on their unique cell
surface markers (CD41 +Fc γlow CD34 +CD38 +CD9 ) (16). Subsequent cells are morphologically closer to actual megakaryocytes then their earlier counterparts. The first of these immediate megakaryocyte precursors is the promegakaryoblast, also known as a stage I megakaryocyte. It is not able to produce colonies in vitro, but able to mature into a single megakaryocyte in culture (17). The megakaryoblast has a diploid nucleus and a diameter of 10 50 m, and the promegakaryocyte or stage II megakaryocyte is 20 80 m in diameter with developing granules in its cytoplasm (18).
Once the megakaryocyte precursors reach maturity, they have undergone a series of modifications including cytoplasm maturity, which involves the development of platelet specific proteins, organelles, and membrane systems that will also be present in platelets. They also undergo endomitosis, a process unique to megakaryocytes in mammals when they become polyploid by repeated cycles of DNA replication without undergoing cytokinesis (19). The final mature megakaryocyte is 50 100 m diameter and contains a single large polyploidy nucleus (2 256N) (20).
Regulation of megakaryopoiesis has been studied in detail, and it has been shown that several cytokines are influential in maturation and development of megakaryocyte precursors. The most influential is thrombopoietin (TPO), a potent growth factor that is heavily involved in maturation and growth of early hematopoietic cells and the
3
megakaryocyte lineage (21). Stem cell factor (SCF), also known as kit ligand, has been
shown to be influential during the early stages of megakaryopoiesis (22, 23). Interleukin
6 (IL 6) and 11 are also involved in regulation and seem to work synergistically with TPO and IL 3 (24 26).
1.1.4 Platelet development
Platelets are the final product of megakaryopoiesis and are essentially cytoplasmic portions of matured megakaryocytes. In humans, platelets have a circulating half life of
7 10 days, (27) and are produced at a steady rate of 10 11 daily with an increase of more
then 20 fold in times of heightened demand (15). It is estimated that one human
megakaryocyte can produce ~10 3 platelets and ~10 2 in mice (28).
The mechanism by which megakaryocytes release platelets in circulation is still
poorly understood in part because of the rarity (<0.1%) of megakaryocyte in normal bone
marrow. Older studies attempted to explain the mechanism by proposing models that
focused on platelet budding from the surface of megakaryocytes and cytoplasmic
fragmentation via the demarcation membrane system (29 31). Both of these models have
lost support due to inconsistent observations, and primarily because of the discovery of
TPO, which facilitated ex vivo culture systems for the study of platelet biogenesis.
Currently the proplatelet formation model is more acceptable and is closely related to
Wright’s observation in 1906, that platelets were produced from long pseudopod
extensions of megakaryocytes that then release platelets (32).
Current ex vivo studies have supported that platelets are made from nodes at the
tips of proplatelet strands (33), but these simplistic systems lack the three dimensional
organization, supportive cells and flow characteristics seen within the marrow. Direct visualization of live calvaria marrow using multiphoton intravital microscopy suggest
4
that
Figure 1 1. Megakaryopoiesis and platelet development. HSCs undergo several stages of differentiation until they commit to a myeloid progenitor cell. The CFU GEMM is of mixed lineage capable of producing mixed blood cells. The BFU Meg is also able to produce a mixture of cells as well as large megakaryocyte colonies. The CFU Meg are the first fully committed cells to the megakaryocyte lineage. Promegakaryoblast are considered a stage I megakaryocyte followed by megakaryoblast which has a developed nucleus and the promegakaryocyte are stage II megakaryocyte with developing granules in its cytoplasm. Shown are some of the cytokines involved at different stages of megakaryopoiesis.
5
that megakaryocytes release into the bloodstream large cytoplasmic fragments, not
small pseudoplatelets, that must be re organized into mature platelets within the
organism (34). Indeed, older studies based on morphologic analysis of the lungs and
quantification of megakaryocyte like polyploid nuclei in the pulmonary venous system
suggest that whole megakaryocytes travel to the lungs where they release their platelets
(35 39). These potential mechanisms involving platelet formation from megakaryocytes by cytoplasmic shedding at the bone marrow or pulmonary vasculature beds may explain why it has been challenging to produce large numbers of platelets from megakaryocytes
ex vivo.
1.1.5 Platelet role in thrombus formation
Hemostasis is the process by which the body maintains the fluidity of blood under
physiological conditions and prevents excessive blood loss after injury (40). Platelets are
essential in hemostasis by forming a monolayer that supports thrombin generation and
platelet aggregation, contributing to stable thrombus formation. The degree of
involvement by platelets in a growing clot depends on the kind of circulation, for
example arterial thrombi form under conditions of high flow are composed mainly of
platelet aggregates bound together by thin fibrin strands. In contrast, venous thrombi
form in areas of stasis and are predominantly composed of red cells, with a large amount
of interspersed fibrin and relatively few platelets (41).
Under high shear arterial circulation, endothelial derived inhibitory factors
suppress platelet activation, including prostaglandin I 2 (PGI 2), and nitric oxide (NO) as well as expression of CD39 on endothelial cells (Figure 1 2) (42 44). After vascular injury and exposure of subendothelium, a multistep complex process goes into effect, which involves a diverse array of extracellular matrix components that interact with platelets to
6
form a platelet plug. The platelet plug formation can be divided into three stages (45), starting with the “initiation” step (Figure 1 2), when platelets roll on activated
endothelial cells, a process mediated by P selectin and E selectin (46, 47). The platelets are able to bind to von Willebrand factor (VWF) via its GPIb/IX/V membrane receptor,
establishing a transient bond that slows them down and facilitate their activation (48).
The platelets also adhere to exposed collagen either directly via the Glycoprotein (GP) VI
and α2β1 integrin (49). This interaction promotes platelet activation, which leads to
morphological changes, including release of granular content and expression of activated
GPIIb/IIIa, the major platelet integrin that mediates platelet aggregation.
During the “extension” stage (Figure 1 2), additional platelets are recruited when
activated platelets release agonists, such as adenosine diphosphate (ADP) and
thromboxane A 2 (TxA 2), which leads to further activation of adherent platelets. In addition tissue factor expressed at low levels by cytokine stimulated endothelial cells promotes local generation of thrombin (50). Thrombin is able to activate platelets through cleavage of the platelet G protein linked protease activated receptors (PAR 1)
(51) as well as cleavage of GPV of the GPIb IX V complex, which allows thrombin to bind to GPIb α (52). Other surface molecules may also play an accessory role in platelet activation by thrombin.
The last stage of the platelet plug formation stabilizes the plug to prevent premature disaggregation. The stabilization is attributed to the close contact between activated platelets that allows contact dependent signaling (53). The final process of stabilizing the platelet plug involves the coagulation cascade, which is able to transform soluble fibrinogen into insoluble fibrin by thrombin, thus forming the fibrinous meshwork.
7
Figure 1 2. Platelet plug formation under arterial circulation. Platelet adhesion is started when VWF links collagen to platelets at sites of injury. The activated platelets release ADP, thromboxane and other chemicals that activate more platelets. Platelet adhesion occurs via the collagen receptors and the GPIb/IX/V complex followed by aggregation of additional platelets through GPIIb/IIIa, which forms a bridge through fibrinogen to other activated platelets.
8
1.1.6 Fibrinolysis
The fibrinolytic pathway has diverse roles including regulation of cellular activity and
tissue development, but its primary functions is the conversion of inactive proenzymes to
active forms for fibrin degradation and thus maintains blood fluidity by breaking down blood clots (54). The central protein in this process is plasminogen, which is converted by PAs into plasmin that lyse the clot. The vascular endothelium is able to release uPA
and tissue plasminogen activator (tPA) the two PAs involved in the thrombolytic process.
PAs work to activate plasminogen to plasmin (Figure 1 3), which is able to lyse fibrin by
attacking it at several sites in the molecule, leading to fibrin clot lysis. The mechanism of activation by both PAs is by binding to specific cellular receptors, uPA binds to a
glycolipid anchor membrane protein uPAR, and tPA to a type II transmembrane protein
(55).
Plasmin’s activity is controlled by an antiplasmin inhibitor, α2 antiplasmin, while tPA and uPA are regulated by forming complexes with plasminogen activator inhibitor 1
(PAI 1), which is found in both plasma and platelet a granules (56). Over 90% of PAI 1 is present in the a granules in an inactive form, which is thought to be one of the reasons why platelet rich thrombi are resistant to thrombolytic therapy (57, 58).
1.1.7 Key mediators of fibrinolysis
Plasminogen is synthesized primarily in the liver and is found in high concentration in plasma (59). Plasminogen is a 92 kDa single chain glycoprotein consisting of 791 amino acid (aa) residues with Glu as the N terminal amino acid. The structure is made up of five homologous kringle like domains that mediate the interactions of the zymogen to regulatory sites, and cell surface receptors. Cleavage by PAs between Arg561 Val562 converts plasminogen to the plasmin resulting in a heavy chain (A chain) and a
9
light
Figure 1 3. Fibrinolytic cascade. Schematic cartoon of the fibrinolytic cascade.
Shown in the center is a fibrin clot at a site of vascular injury. Also shown is activation of plasminogen by PAs to produce plasmin, which is able to degrade the fibrin clot.
10
light chain (B chain) that is enzymatically active (60).
Endothelial cells synthesize and secrete tPA as a single chain active enzyme that
circulates in plasma at relative low concentrations with a half life of 5 to 7 min (61). It is made up of 527 aa with a molecular weight (MW) of 70 kDa. tPA is composed of five
domains; 1) the N terminal region of 47 residues (4 50 aa) make the finger like domain
homologous with fibronectin; 2) aa 50 87 are homologous with the epidermal growth
factor (GFD); 3) two kringle regions (aa 87 176 and 176 262) which are homologous with
the five kringles of plasminogen; and 4) a serine proteinase domain (aa 276 527) with
the catalytic site His 322 , Asp 371 , and Ser 478 (62). The high affinity binding of tPA to fibrin
is mediated by both the finger and kringle two domains. This is important because tPA is
a poor enzyme in the absence of fibrin, but in the presence of fibrin, enhances the
activation rate of plasminogen (63).
uPA was first isolated from urine, hence the name urokinase. uPA is synthesized by vascular endothelial and smooth muscle cells, by epithelial cells, fibroblasts,
monocytes/macrophages, and also by multiple malignant tumors (64 66). Unlike tPA
that is mainly involved in fibrinolysis, uPA has additional functions in cell migration and
tissue remodeling (67). uPA is secreted as a single chain polypeptide (scuPA) with a
molecular weight of 54 kDa that consists of 411 amino acid residues (68). The structure
of uPA (Figure 1 4) is subdivided into three domains, starting with the N terminal
domain homologous to the epidermal GFD (aa 9 45) responsible for the interaction of
urokinase with the uPAR/CD87 receptor. The kringle domain (aa 46 143) contains a
sequence that interacts with the specific inhibitor PAI 1. The C terminal catalytic
domain, (aa 144 411) includes the active site made up of serine proteases amino acid
triad His204, Asp255, and Ser356 (69, 70). Each domain of the protein has a rigid
structure supported by internal disulfide bonds, while intramolecular disulfide bonds
11
maintain the orientation of amino acid residues in the active center of the proteolytic
domain. This has been confirmed by loss of enzymatic activity after destruction or
irregular formation of disulfide bonds (71). The form in which single chain uPA (scuPA) is secreted has low intrinsic activity but can be cleaved at several sites to make it active.
Matrix metalloproteinase (Figure 1 4) (MMP 3) specifically hydrolyzes the
Glu143 −Leu144 peptide bond of scuPA and in its two chain (tcuPA) derivative, yielding a
17 kDa NH 2 terminal domain comprising uPAR’s binding site and a 32 kDa COOH
terminal moiety containing the serine proteinase domain of uPA (72). Plasmin cleaves
the Lys158 Ile159 peptide bond producing fully active uPA as tcuPA, connected by
disulfide bonds. The rate of plasminogen cleavage by tcuPA is more then 200 fold higher
then the rate of cleavage by scuPA (73). This form of uPA lack the GFP domain so it is
not able to bind to its receptor uPAR, which makes it ideal for therapeutic studies since it
eliminates any side effects that result from receptor activation such as inflammation.
1.1.8 Clinical use of PAs and their limitations
One major form of treatment of occlusive thrombosis consists of pharmacological
dissolution of the blood clot by intravenous infusion of PAs that activate the fibrinolytic
system (74). Physical disruption of plaques (atherectomy) is also used as treatment
especially in peripheral arterial disease of the lower extremities (75). There are several
thrombolytic drugs in clinical use, including uPA and streptokinase that are effective
thrombolytics, but are not fibrin specific. Additionally, they also decrease circulating and
intra thrombus plasminogen, potentially leading to reduction in clot lysis (76).
Furthermore, the bacterial derived streptokinase is immunogenic, resulting in drug
resistance, fevers, and allergic reactions (77). Second generation agents such as tPA and
scuPA are more fibrin selective and decrease the depletion of circulating plasminogen
12
and
Figure 1 4. Structure of scuPA and its various cleaved forms. scuPA structure is
composed of three domains, the N terminal domain contains the GFD, responsible for
the interaction of uPA with uPAR. The kringle domain contains a sequence that interacts with the specific inhibitor PAI 1. The C terminal contains the catalytic domain with the
serine proteases active site. scuPA can be cleaved by different components to form the
active for tcuPA. Matrix metalloproteinase cleaves between Glu143 −Leu144 and plasmin
can cleave between Lys158 Ile159 resulting in the two chain derivatives.
13
and fibrinogen (78). All of these agents induce thrombolysis, and while there are
differences in fibrin binding affinity and other properties, in essence turn out to have
similar clinical efficacy and such side effects as the consumption of circulating fibrinogen
and plasminogen and the development of bleeding complications (79). These agents also
have short half lives and require intravenous (IV) administration. Local infusions of
these PAs at the site of pathologic thrombus have limited improved efficacy (80 82).
1.2 Dissertation objective
Cardiovascular disease is estimated to be responsible for 17 million deaths per year,
making it one of the largest cause of mortality worldwide (83). Current anti thrombotic
therapies reduce the risk of recurrent cardiovascular events, but increase the risk of bleeding in part because they act systemically.
An improved thrombolytic agent would have better targeting of activity to sites of
developing clots. For thromboprophylaxis use, an ideal agent needs to avoid targeting a
mature thrombus and only lyse growing thrombi. An additional useful feature would be
to have a therapeutic agent with a prolonged lifespan, preventing the development of
untoward thrombi over several days and allowing injured vessels to heal without
developing recurrent, pathologic thrombi. As proof of principle, our group has shown
that uPA ectopically expressed in platelets using a transgenic approach is highly effective
in preventing arterial and venous thrombosis and that transfusion of 5% of the platelet
mass protects WT mice from clotting without causing systemic fibrinolysis or
spontaneous bleeding (84). Based on these preliminary data, we envision achieving high
levels of megakaryocyte specific expression of uPA in human megakaryocytes derived
from in vitro differentiation of embryonic stem cells, allowing one to produce 14
modified uPA containing platelets for targeting to sites of injury (Figure 1 5). To reach
that stage, we first need to develop strategies that facilitate the production of such donor
derived platelets. One way to accomplish this is by infusing ex vivo derived mature
megakaryocytes as a source for donor derived platelets.
Another platelet centric strategy for producing effective thromboprophylaxis is by developing a recombinant modular PA that specifically binds to the platelet surface
and that is activated only at a site of active clotting (Figure 1 5). A recent study targeting
red blood cells confirmed the targeting efficiency, safety and efficacy for pulmonary
thromboprophylaxis using a mutated low molecular weight scuPA, which is specifically
activated by thrombin (85). Using the same thrombin activatable pro enzyme fused to
an anti platelet specific scFv antibody fragment would likely prolong the half life of the
mutated uPA and provide a better targeting mechanism to sites of growing thrombi. The
thrombin generated from the growing thrombi serves as a control mechanism that
activates the uPA T, which can conceivably limit adverse effects, and increase its
duration in blood, prolonging its intravascular effect, and thus permitting use as
prophylaxis.
15
(A)
(B)
Figure 1 5. Depiction of the two strategies proposed. The two approaches proposed are shown starting with (A) the expression of uPA at high levels in developing megakaryocytes and produce platelets that will stored the zymogen in their alpha granules to be release in a targeted fashion at sites of injury. The second approach (B) deals with producing a chimeric protein that will bind directly to the surface of platelets via the aIIb receptor and will deliver a thrombin activatable lmw uPA to the thrombus sites, leading to clot lysis via fibrinolysis.
16
Chapter 2
Infusing mature megakaryocytes into mice yields
functional platelets
2.1 Abstract
Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes,
remains incompletely understood. Prior studies suggest that megakaryocytes shed
platelets in the pulmonary vasculature (35, 36, 38, 39, 86). To better understand
thrombopoiesis and to develop a potential platelet transfusion strategy that is not
dependent upon donors, we examined whether megakaryocytes infused into mice shed a
significant number of platelets. We found that infused megakaryocytes led to clinically
relevant increases in platelet numbers. The released platelets were normal in size,
displayed appropriate surface markers, and had a near normal circulating half life. The
functionality of the donor derived platelets was also demonstrated in vivo. The infused
megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to
shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet
numbers.
17
2.2 Background
In 2001, platelet transfusions in the USA totaled 10,196,000 units, an increase of 12.6%
from 1999 (87). While the need for healthy platelet donors is increasing, there is a
significant donor shortage due to the growing population of patients with serious
medical illnesses associated with thrombocytopenia and hemorrhages. Platelets for
transfusions also suffer from the following concerns: variability of donor units in terms of number of platelets and functionality, risk of donor to patient infectious transmission, the short lifespan of room temperature stored platelets (necessary to maintain platelets
of normal life span), bacterial contamination during this storage, and the development
of platelet alloantibodies in multiply transfused patients. All of these problems highlight
a need for new strategies to generate platelets for infusion therapy.
Two models of thrombopoiesis have been proposed: The release of platelets
produced from megakaryocytes within the marrow space and the release of platelets by
circulating megakaryocytes within the pulmonary vasculature (39). In vitro studies
suggested that platelets form nodes at the tips of proplatelets strands (33). However,
direct visualization of live calvaria marrow using multiphoton intravital microscopy
suggested that megakaryocytes, adjacent to the vascular space, release large cytoplasmic
fragments into the bloodstream (34). These must then be re organized into mature
platelets. Older studies based on morphologic analysis of the lungs and quantification of
megakaryocyte like polyploid nuclei in the pulmonary venous system suggested that
megakaryocytes travel to the lungs where they release their platelets (35, 36, 38, 39, 86).
Final platelet formation from megakaryocyte cytoplasmic shedding at the
marrow/endothelial interface or in the pulmonary vasculature beds may explain 18
why it has been challenging to produce large numbers of platelets from megakaryocytes
grown in vitro without simulating special niches and/or flow conditions.
Derivation of platelets from megakaryocytes in culture was first reported in 1995
(88), but has been difficult to quantitatively upscale. To date the best published result from infused in vitro produced platelets used irradiated mice with low platelet counts
(~10 4/ l) (10). Even in this setting, peak donor platelet counts were 1% 2% of this low number of platelets and this miniscule increase is unlikely to be of clinical utility. Given the limited success with which platelets have been generated ex vivo, we examined whether infused megakaryocytes release platelets in vivo. We found that by infusing ex vivo generated megakaryocytes into mice, we can achieve approximately an 100 fold increase in recipient platelet count over the prior best published results, thereby reaching clinically relevant levels of donor platelet. These platelets have a slightly shorter half life than infused platelets, but are normal in size and surface markers and are functional in two clotting models. We also show that the infused megakaryocytes appear to be trapped in the pulmonary bed where they shed their cytoplasm.
2.3 Material and methods
2.3.1 Characterization of the mice studied
Donor cells and platelets were derived from C57BL/6 WT mice (Jackson Laboratory) or mUK transgenic mice that ectopically expressed murine urokinase within megakaryocytes and that had been backcrossed to the C57BL/6 background >10 times
(84). The recipient hαIIb + mice were previously described (89), and are homozygous transgenic for h αIIb (h αIIb +/+ ) that are also null for the expression of platelet m αIIb 19
(m αIIb / ). These mice platelets express h αIIb levels at ~20% of the level seen on human platelets and are functional (90). mUK and h αIIb + mice were genotyped by polymerase chain reaction (PCR) analysis of tail genomic DNA using previously described primer pairs and reaction conditions (84, 89). All animal studies were done with Institutional
Animal Utilization Committee approval at the Children’s Hospital of Philadelphia.
2.3.2 Production of mature megakaryocytes ex vivo
Most studies were done with fetal liver (FL ) derived megakaryocytes, beginning with
E14 FL cells that were homogenized to a single cell suspension by sequential passage through 18, 23, and 25 gauge needles as previously described (91). The cell suspension was passed through a 100 m nylon mesh filter and cultured in Iscove’s Modified
Dulbecco’s Medium (IMDM) with 10% calf serum, 2 mM L glutamine, 1% penicillin/streptomycin and 50 ng/ml of recombinant murine TPO (R&D Systems). After
10 days in culture, over 90% of the resultant cells were megakaryocytes as determined by acetylcholinesterase positivity (data not shown). The mature megakaryocyte population was isolated using a previously described 2 stage velocity sedimentation method for isolating megakaryocytes (92), which involves centrifugation through a 1.5% and 3% bovine serum albumin (BSA) gradient at 200 RPMs for 30 min.
Adult bone marrow (BM ) cells were obtained from femurs and tibiae of C57BL/6 mice as previously described (93). The cells from the marrow lumen were flushed with sterile Dulbecco’s Phosphate Buffered Saline 1X (PBS) (Invitrogen) using a 10 ml syringe and a 25 gauge needle. The cells were disrupted and passed through a 100 m nylon mesh filter. They were then cultured in the same media as the fetal liver cells and isolated after 8 days of culture in the same manner.
20
2.3.3 Characterization of ex vivo derived megakaryocytes
FL and BM megakaryocytes to proplatelet number was determined visually with a
hemocytometer (94). DNA ploidy of the derived megakaryocytes was assessed by flow
cytometry as previously described (95). Isolated populations were washed with PBS and
stained with 10 g/ml monoclonal fluorescein isothiocyanate (FITC) labeled anti
murine CD41 antibody (BD Bioscience) for 30 min. The cells were then rinsed twice with
PBS and resuspended in propidium iodide solution (0.1% sodium citrate with 50 g/ml
of propidium iodide) (Sigma) for 4 hrs. The cells were then incubated with 10 U/ml
RNase A (Sigma) for 90 min at room temperature (RT) and analyzed by flow cytometry
using a FACScan (Becton Dickinson).
2.3.4 Isolation of platelets and megakaryocytes for infusion
Washed platelets from whole blood were isolated from the inferior vena cava of C57BL/6
mice under direct observation and collected into acid citrate dextrose (ACD) as
previously described (96). Platelet rich plasma was collected after centrifugation at 200 g
for 5 min at RT, and prostaglandin E1 (Sigma) was added to a final concentration of 1
mM. Platelets were then pelleted by centrifugation at 800 g for 10 min at RT. The pellet was washed in calcium free Tyrode’s buffer (134 mM NaCl, 3 mM KCl, 0.3 mM NaH 2PO 4,
2 mM MgCl 2, 5 mM HEPES, 5 mM glucose, 0.1% NaHCO 3, and 1 mM EGTA, pH 6.5), and resuspended in CATCH buffer (PBS, 1.5% BSA, 1 mM adenosine, 2 mM theophylline,
0.38% sodium citrate) (all from Sigma). Platelet counts were determined using a
HemaVet counter (Triad Associates), and the appropriate amount of platelets was infused into recipient mice. Megakaryocytes were produced as described in “Isolation of platelets and megakaryocytes ex vivo”. Once the mature population of megakaryocytes was obtained from the BSA gradient, the cells were washed in CATCH buffer and
21
counted with a hemocytometer and infused either retro orbitally or by tail vein.
2.3.5 Flow cytometric studies in infused hαααIIb + mice
Before infusion of cells or platelets, whole blood was collected retro orbitally from recipient mice into a mini capillary blood collection tube (RAM Scientific) and again afterwards at time points extending from 5 min to 36 hrs. The blood was double stained with monoclonal FITC conjugated mouse, anti human specific CD41 antibody
(eBioscience) and monoclonal phycoerythrin (PE) rat, anti mouse CD41 specific antibody (BD Bioscience) for 30 min and analyzed by flow cytometry using a FACScan.
Activation of the infused platelets was assessed by 3 color whole blood flow cytometry, using a monoclonal mouse anti human CD41 labeled with peridinin chlorophyll protein conjugated with a cyanine dye (PerCP Cy5.5), PE rat anti mouse CD41, and monoclonal
FITC rat anti mouse P selectin. To examine the relative expression of membrane receptors in recipient versus donor platelets, whole blood was stained with mouse anti human CD41 (PerCP Cy5.5), PE rat anti mouse CD41, and either monoclonal antibodies
FITC labeled rat anti mouse GPIb α or rat anti mouse GPIX (Emfret Analytics).
2.3.6 Infusion studies in thrombocytopenic h αIIb + mice
Mice were subjected to a high dose of irradiation (1,000 centigrays total; two sessions, 24 hours apart). Platelet counts were initially monitored daily to determine a temporal platelet profile. Animals included in these studies had platelet counts between 1 2 x
10 8/ml on day 7 after irradiation and immediately before infusion of the cells in 200 l
CATCH buffer. Additional platelet counts were performed at 4, 24, and 48 hours after infusion.
22
2.3.7 Cremaster laser injury functional studies
Washed platelets and isolated FL derived megakaryocytes were infused into haIIb + male
mice. One hour after infusion, the mice were anesthetized using intraperitoneally
injected sodium pentobarbital (11 mg/kg, Abbott Laboratories), and maintained under
anesthesia with the same anesthetic delivered via catheterized jugular vein as described
(97). Fab fragments from the anti mouse CD41 antibody were produced using the
ImmunoPure Fab Preparation kit (Pierce Biotechnologies) and conjugated with Alexa 488
using an Alexa Fluor Protein labeling kit (Molecular Probes). These labeled fragments were injected intravenously into the cannulated jugular vein 5 min before injury. The cremaster muscle was isolated and the microvessels studied using an Olympus BX61WI microscope (Olympus) with a 40X/0.8 numeric aperture (NA) water immersion objective lens. Laser injury was induced using an SRS NL100 Nitrogen Laser system (Photonic Instruments) at 65% energy level with blood vessels ranging in size from 20 to 40 m. A visual confirmation of small extravasations was made for each studied blood vessel as an assurance that the injury was made as well as an indicator of consistent injury. Data was collected over a course of 2.5 min at 5 frames per second (f/s;
750 frames per study). No more than 5 arteriole and 5 venule injuries were done in each mouse.
2.3.8 FeCl 3 carotid artery injury functional studies
FeCl 3 induced arterial injury was performed as previously described (98) with minor changes. HαIIb + mice were anesthetized by intraperitoneal injection of pentobarbital (80 mg/kg Nembutal; Ovation Pharmaceuticals) approximately 1 hr after infusion of either
WT or mUK megakaryocytes. The carotid artery was isolated by blunt dissection, and a
Doppler flow probe (Model 0.5VB; Transonic Systems) was positioned the
23
around vessel, followed by a 1 x 2 mm 2 piece of filter paper (#1; Whatman International) soaked in 20% FeCl 3 that was placed on the artery for 3 min. The area was then flushed with PBS, and blood flow through the artery was monitored for 30 min. Total flow and
time to the first complete occlusion of >10 min were recorded.
2.3.9 Infused megakaryocyte fate studies
BrdU studies were carried out to better understand the fate of the infused
megakaryocytes. FL cells were cultured for 5 days and afterwards exposed to 10 M
BrdU (Sigma) in their growth media for 2 more days. The cells were isolated and infused
into hαIIb + mice. The mice were sacrificed, and organs, including brain, heart, liver, spleen, bone marrow and lungs were dissected at 30 min, 6 hrs, and 24 hrs. The lungs were flushed with PBS and inflated with 10% formalin solution (Sigma) to preserve alveolar architecture. All organs were fixed in formalin overnight at 4°C for immunohistological analysis. Detection of BrdU labeled nuclei was performed with a rat polyclonal anti BrdU antibody diluted 1/50 (v/v, Abcam) and a biotinylated rabbit polyclonal anti rat IgG diluted 1/200, was used as the secondary antibody. Detection of infused megakaryocyte cytoplasm in the lungs was performed by labeling with a goat polyclonal antibody against murine αIIb (Santa Cruz biotechnology) and a biotinylated
rabbit polyclonal anti goat IgG antibody, diluted 1/200 as the secondary antibody for
detection was done using a DAKO kit (EnVision).
24
2.4 Results
2.4.1 Infused megakaryocytes and platelets
To address whether infused megakaryocytes give rise to circulating platelets, we
generated FL and BM derived magakaryocytes (Figure 2 1 A). This strategy has been
described to yield a large number of mature megakaryocytes (91). Mature
megakaryocytes were separated from other cells and most of the proplatelets by a two
stage velocity sedimentation to produce a “large cell” fraction with over half of the cells
having a diameter >50 m and with only ~2.5:1 pro platelets:cell (Figure 2 1 B). The
remaining “small cell” fraction was >95% small megakaryocytes and pro platelets with
~10:1 proplatelets:cell (Figure 2 1 B). Ploidy analysis was performed on both fractions and showed that the small cell fraction had low DNA ploidy relative to the large cell fraction (Figure 2 1 C).
WT platelets were used as positive controls for studies in which one of the two cell fractions of ex vivo grown FL and BM megakaryocytes were infused into h αIIb + mice either retro orbitally or by tail vein; the two approaches gave similar outcomes (Figure 2
2). The recipient animals have only h αIIb on their platelet surface (89), which allows for flow cytometric detection analysis of the infused WT platelets using species specific anti
αIIb (CD41) antibodies (Figure 2 3). After infusion, blood derived WT platelets were detected in h αIIb + recipient mice immediately at the 5 min time point (Figure 2 4 A) with a half life of approximately 36 hours (Figure 2 5 A).
The donor platelets were present in recipient blood as expected based on the initial platelet count of the recipient mouse and an estimated blood volume of 2 ml (data not shown). After infusing the large cell fractions, donor derived platelets were seen, but with delayed kinetics: only a few donor platelets were detectable at 5 min, 25
followed by a peak at approximately 90 min (Figure 2 4 B and Figure 2 5 A). These
platelets had a slightly shorter half life of approximately 20 hrs than when donor platelets had been infused. Based on the number of large cells infused, the peak increase in platelet count and a recipient mouse blood volume of 2 ml, we calculate that each
large cell gave rise to 100 200 platelets, assuming all large cells gave rise to platelets.
Infused small cells, which were richer in proplatelets, gave rise to an immediate peak just
as infused WT platelets; however, these platelets had a significantly truncated half life of
approximately 2 hrs (Figure 2 5 A).
Infusing adult BM megakaryocytes grown in culture in the presence of TPO and
fractionated as in Figure 2 1 B, we were also able to detect circulating platelets. The
number of platelets per megakaryocyte was similar to FL derived megakaryocytes, the
half life was slightly longer at 24 hrs, but the yield of megakaryocytes from adult marrow was lower compared to FL cells. Consequently, further studies below focused on FL
derived megakaryocytes.
To simulate clinical thrombocytopenia, we irradiated mice and infused CATCH buffer, WT platelets, or FL large cells near the induced platelet nadir and observed that both platelets and megakaryocytes significantly increased the platelet count relative to
CATCH buffer over a time course of more than 24 hours (Figure 2 6).
2.4.2 Characterization of the in vivo derived platelets from FL cells
Platelets derived from the large cell fraction had a shorter half life than infused platelets.
The small cell fraction had an even more truncated half life. We would have anticipated that the platelets, especially those from the large cell fraction, (with mostly newly formed platelets) would have a longer half life than infused washed platelets. One clear possibility of why FL derived large cell platelets had a shortened half life could be that
26
these
Figure 2 1. Isolation and characterization of FL cells . (A) The strategy followed in our study is presented by which the three different products for infusion were collected: isolated WT platelets, and the large and small cell fractions obtained from FL cells grown in the presence of thrombopoietin (TPO). (B) Representative fields of the small and large cell fractions. Scale bars: 100 m. (C) Representative analysis of DNA
27
content of the small and large cell fractions.
Figure 2 2. Comparison of retro orbital Vs tail vein infusions of FL large cells . Flow cytometric percentage of infused ~10 6 FL large cells in recipient animals.
N=5 studies per arm. Mean ± 1 standard deviation (SD) are shown.
28
Figure 2 3. Flow cytometric detection analysis of infused platelets. Flow
cytometric analysis was performed on whole blood from haIIb + recipient mice infused with WT donor platelets. Gate 1 (platelets) was set up in the forward scatter H/side scatter H plot (A) representing the overall platelet population. Gate 2 included either recipient (anti haIIb + FITC) platelets (B) or donor (anti maIIb + PE) platelets (C). The percentage of the recipient versus donor platelets was calculated from the FL1xFL2 density plot that included cells satisfying both Gates 1 and 2, as shown in (D).
29
Figure 2 4. Flow cytometric analysis of infused WT platelets, and FL derived
megakaryocytes. Flow cytometric analysis on whole blood from h αIIb + recipient mice before and at different time points after retro orbital infusion of donor cells. Blood was stained with labeled species specific anti αIIb antibodies. The % of platelets in the left upper quadrant represents the recipient’s platelets, while the % in the right lower quadrant represents donor derived platelets. (A) Flow cytometry from recipient mouse before and after infusion of 10 8 platelets (positive control). (B) Flow cytometry from recipient mouse before and after infusion of 10 6 FL derived large cells.
30
Figure 2 5. Flow cytometric analysis of infused WT platelets, FL and BM
derived cells on hαααIIb + mice. (A) Flow cytometric percentage of infused 10 6 FL large
cells and (B) infused 10 6 adult BM cells. N=5 for WT platelets, 9 for FL cells, 5 for BM studies. Mean ± 1 SD are shown.
31
Figure 2 6. Flow cytometric analysis of infused WT platelets and FL derived cells in thrombocytopenic mice. Percent platelet rise in irradiated thrombocytopenic mice post infusion. N=5 per arm. Mean ± 1 SD are shown. Initial
platelet counts (10 8/ml) in the three groups were: CATCH buffer = 1.8±0.2; platelets =
1.9±0.3; and large cells = 1.0±0.2.
32
these are fetal platelets which tend to have a shorter half life then adult platelets (99). To see if the half life improves when using an adult source of megakaryocytes instead of our
fetal liver derived, we infused cultured adult BM megakaryocytes, which had a half life
perhaps as good as infused WT platelets, but clearly not longer (Figure 2 5 B). Prior
studies have suggested that a metalloproteinase, tumor necrosis factor alpha converting
enzyme (ADAM17), could potentially be present in the media of cultured megakaryocytes
and be responsible for the shorter half life, but growing the cells in the presence of either
metalloproteinase inhibitors GM6001 (Ilomastat) or TAP 1 did not improve the half
lives (Figure 2 7).
One indicator of platelet activation would be the presence of donor WT platelet
microparticles. However, the size distribution of the platelets derived from the large cell
fraction was identical to that seen for infused WT platelets (without a significant pool of
microparticles by forward scatter) (Figure 2 8). Another indicator of cell activation is the
expression of P selectin on the platelet surface (100, 101). Prior studies involving platelet
concentrate storage and transfusion have shown that this marker is increased on
activated platelets (102). Flow cytometric analysis did not support platelets derived from
infused megakaryocytes having a higher level of surface P selectin expression than
infused circulating platelets (Figure 2 9 A). Moreover, ADAM17 cleaves the glycocalicin
extracellular portion of GPIb α, inactivating the GPIb/IX receptor without altering surface receptor density (103, 104). We measured relative expression of the extracellular portion of GPIb α (Figure 2 9 B) versus GPIX (Figure 2 9 C) on platelets derived from the
infused large cell fraction as well as from donor washed platelets and found no
difference. Thus, it appears that the shortened half life of the platelets derived from
infused megakaryocytes might not be due to the effects of the previously described ex vivo metalloproteinase ADAM17.
33
Figure 2 7. Effect of ADAM17 on thrombopoiesis from in vitro grown FL
cells. WT FL cells were grown in culture in the presence of either 100 M GM6001 (A
and B) or 10 M TAP 1 (C and D) and then separated into small and large cells as in
Figure 2 2 (B), and infused into h αIIb + recipient mice to see if these ADAM17 inhibitors would improve the half life observed of platelets derived from infused large cells. N=2,
performed in duplicates.
34
Figure 2 8. Characterization of the platelets derived from infused cells . (A)
Size determination of circulating donor platelets from infused platelets or (B) small cell
or (C) large cell fractions. Shown is a representative study of the forward versus side
scatter of the whole blood samples measured with the two αIIb positive populations highlighted in blue the recipient platelets and in red for the infused platelets or derived platelets. 35
Figure 2 9. Characterization of platelets derived from infused FL cells . (A)
Representative flow cytometric analysis of P selectin on infused donor cells resulting from either infused platelets or cell fractions. (B) Representative relative levels of GPIb α extracellular domain to (C) GPIX in donor platelets from infused platelets or cell fractions.
36
2.4.3 Functionality of platelets derived from infused FL cells
Clinically, platelets are transfused not to increase platelet number, but to reverse or
prevent a bleeding diathesis due to the absence of platelets. We have used two distinct
assays to look at platelet function after infused megakaryocytes. In the cremaster laser
injury model, we looked at the incorporation of both infused platelets and platelets
derived from infused large or small cells into growing clots after laser injury to cremaster
arterioles. In all three settings, donor derived platelets readily become incorporated into
the developing thrombi and did so at least to the same extent as for WT donor platelets
(Figure 2 10 A and B).
Inclusion of platelets derived from the small cells into growing clots was less vigorous. Moreover in this setting, there was a distinct population of CD41 + cells that re
circulated, rarely becoming incorporated into a thrombus (Figure 2 10 C and D) and
Supplemental Video 1). This phenomenon was rarely seen after infusion of the large cell
fraction (Figure 2 10 B) and Supplemental Video 2), and never with infused platelets
(Figure 2 10 A), and Supplemental Videos 3).
We also examined platelet function using a FeCl 3 carotid artery injury model after infusing either CATCH buffer or FL WT large cells into the h αIIb + recipient mice. These
recipient mice have a mild bleeding diathesis in part because of the presence of only 20%
of the density of CD41 compared to WT mice (89). We found that the infused WT large
cells lead to a shortened time to development of stable occlusion (5.3 ± 0.5 versus 7.4 ±
0.9 likely due to the presence of WT donor derived platelets. We also infused large cells
grown from the FLs of mice that ectopically express urokinase and store it in their α
granules (mUK mice) similar to patients with Quebec Platelet Disorder (105). We have
already demonstrated that mUK mice are resistant to thrombosis in the FeCl 3 carotid
artery injury model and mUK platelets transfused into a WT mouse block clot
37
development (84). We reasoned that if infused large cell fraction derived mUK platelets
are functional then they would interfere with clotting in the h αIIb + recipient mice.
Indeed, h αIIb + mice infused with mUK large cells failed to form stable occlusions (Figure
2 11 A), and total blood flow over the 30 min study was markedly greater in the mUK cell
infused mice than either control groups (Figure 2 11 B).
2.4.4 Organ distribution studies of infused cells
The above studies are consistent with the concept that mature megakaryocytes get
trapped in the pulmonary vasculature and shed functional platelets. Smaller and less
mature megakaryocytes may be able to escape the pulmonary capillary bed and
recirculate or get trapped in other organs. To begin to address this model of events, we
labeled the nuclei of maturing cells in culture by the inclusion of 5 bromo 2
deoxyuridine (BrdU) in the growth media prior to infusion of the cells, and isolated various organs at intervals ranging from 30 min up to 36 hrs. The major staining was in
the lungs, and this was more intense after infusion of the large cell fraction (Figure 2 12
A). Stained nuclei were maximal at the first time point checked and were visible for up to
24 hrs, but not at 36 hrs (Figure 2 12 B). A few nuclei were visible in the red pulp of the
spleen after both small or large cell fraction infusions (Figure 2 12 C). None were present
in the liver, heart, brain or bone marrow (data not shown).
Additionally the BrdU labeled megakaryocytes appear to retain their cytoplasm
for up to 30 min post infusion in the microvessels of the lungs (Figure 2 12 B and D).
This observation would be consistent with the delay seen in peak platelet count after
infused large cell fraction as seen in Figure 2 4 (B) and Figure 2 5 (A). To confirm this
observation, we stained the tissues with an anti murine CD41 antibody and detected
positive staining cytoplasm present up to 30 min (Figure 2 12 D).
38
Figure 2 10. Platelet incorporation into arterial clots after laser injury . (A C)
Representative images of platelets incorporating into clots after infused platelets or
indicated cells. (A) Donor platelets detected using a labeled anti mαIIb antibody. (B)/(C)
Same as (A), but after large and small cells infused, respectively. (D) Sequential stills from left to right noting a re circulating m αIIb + cell (arrowhead) after small cell infusion.
(E) Summation of donor platelets incorporated into growing thrombi after either infused
platelets or cells. Twenty movies were evaluated per graph. 39
Figure 2 11. FeCl 3 carotid artery injury studies . (A) Time to stable complete vessel occlusion after infused CATCH buffer or WT large cells or mUK large cells. (B) Same as in (A), but measurement of total flow over the 30 min study. For (A) and (B), N = 5 per arm. Mean ± 1 SD shown for (B).
40
Figure 2 12. Organ distribution studies of infused FL cells . (A) Staining of lung from haIIb + mice infused with PBS small or large cells grown in BrdU (arrows point to stain nuclei). (A C) Original magnification = 50X, scale bar = 200 m. (B) Kinetics of
BrdU labeled large cells in the lungs. (C) Same as (A) but for spleen. (D) High power
(200X) of lungs: BrdU labeled nuclei (left) and m αIIb (right) 30 min post infusion of large cells. Scale bar = 50 m. Data are representative of 3 separate studies. 41
2.5 Discussion
Thrombopoiesis is a unique phenomenon developed in mammals to have circulating
anuclear subcellular fragments that can participate in clot development when the
circulatory system is perturbed (106). Inherited or acquired disorders of megakaryocyte differentiation or cytoskeletal maturation can affect thrombopoiesis and final platelet counts (107 110). Understanding thrombopoiesis is important if one is to develop ex vivo methodologies for the production of clinically relevant numbers of platelets.
While much has been learned about cytoskeletal components in thrombopoiesis,
it is still unclear where platelets are made and how megakaryocytes shed them. The
methodology used to study thrombopoiesis has to a large extent defined the potential
underlying mechanism. Studies of maturing megakaryocytes in culture suggest that a
mature megakaryocyte forms an interconnected network of “beads on a string”
proplatelets that could eventually become platelets (33). This model would support in vivo thrombopoiesis localized within the marrow space itself. Instead, multiphoton
intravital microscopy of marrow space suggests that megakaryocytes adjacent to the vascular lining shed large cytoplasmic fragments that must then secondarily release final
platelets within the vasculature (34). Our studies and prior ones that included
pulmonary histology (35, 36, 38, 39, 86) suggest a third mechanism wherein normal
sized and functional platelets are released from circulating, mature, high ploidy
megakaryocytes directly within the vascular bed. These models are not necessarily
mutually exclusive: Large cytoplasmic fragments may be the norm, while whole
megakaryocytes are less frequently released into the circulation. Both large cytoplasmic
fragments and whole megakaryocytes may shed platelets within the pulmonary bed
using mechanisms consistent with the in vitro studies, but with concurrent flow 42
and local vascular factors involved as others have proposed (111).
Intuitively, one would expect that megakaryocytes lodging in the lungs might
pose a cardiovascular challenge. However, if a mouse has 1 2 X 10 9 platelets and these have a half life of 5 days (112), and if a megakaryocyte sheds 10 2 platelets, then one would need 2 4 X 10 6 megakaryocytes each day traveling to the lungs to maintain the animal’s platelet count. We infused 10 6 megakaryocytes in most of our studies, and while
death as an endpoint was not measured (all arms of our studies had some animal losses, but no one arm was particularly harmful), infusing what is near to a daily number of
needed megakaryocytes over a 10 min period was tolerated. Since the mouse has 2.3 x
10 6 alveoli in both lungs (26), and each alveolus is traversed by many capillaries (25 100; based on human alveolar structure, corrected for the difference in volume between human and murine alveoli (113), our calculation is that each mouse lung contains approximately 10 8 capillaries, which is well with in the estimates of 4 x10 11 capillaries for
the human lung (113). Given this number of capillaries, we anticipate that we are blocking ~1% of pulmonary capillaries for up to 24 hrs from our kinetic studies in Figure
2 11 (Table 2 1).
In addition to the pulmonary vasculature having the capacity to handle an influx
of megakaryocytes, another question is whether the pulmonary vasculature offers a
special niche for platelet release. Would megakaryocytes get trapped in other vascular beds and if so would they be as efficient in thrombopoiesis? Clearly a few
megakaryocytes reached and were trapped in the splenic red pulp, but whether these
megakaryocytes can shed platelets is unclear, even though some suggest that this is also
a site for platelet production (114). Individuals with significant right to left
cardiovascular shunts are known to have lower platelets counts, which others have
proposed to be due to pulmonary bypass by circulating megakaryocytes (115). However,
43
Table 2 1. Calculation of number of alveoli capillaries blocked by infused megakaryocytes. Mice platelet content: 1 2 X 10 9 platelets (112) Mice platelet half life: 37 hrs (118) Megakaryocyte sheds 10 2 platelets (determined in this study). Calculate: 2-4 X 10 6 megakaryocytes each day traveling to the lungs to maintain the animal’s platelet count. Mice alveoli: 2.3 X 10 6 in both lungs (119) Capillaries per alveoli: 25 100; based on human alveolar structure, corrected for the difference in volume between human and murine alveoli) (113) Calculate: Infusion of = 10 6 megakaryocytes over 10 mins into the mice will block 0.4- 1.7% of the entire capillary bed for a 24 hr period.
FL derived megakaryocytes that naturally bypass the pulmonary bed can form platelets
(116, 117), suggesting that other vascular beds may be able to substitute for the
pulmonary vasculature.
We have shown that the formed platelets from infused megakaryocytes are normal in size, without a significant number of microparticles (Figure 2 8), and without notable activation (Figure 2 9 A) or extracytoplasmic cleavage of surface GPIb α (Figure
2 9 B and C). Moreover these formed platelets can be actively incorporated into growing thrombi Figure 2 10. However, why these derived platelets have a slightly shorter half life than infused, isolated platelets is unclear. We anticipated that platelets derived from megakaryocytes would be newly generated with a prolong half life.
Whether the shortened half life has to do with the in vitro culture necessary to
grow the megakaryocytes is unknown. Previously, it had been suggested that
metalloproteinase ADAM17 is present in the media and affects platelet half life (120).
Our studies showed no loss of at least one of the targeted surface marker by the
metalloproteinase and using the same inhibitors to block ADAM17 as previously
described did not alter the half life of the platelets formed from infused large 44
megakaryocytes (Figure 2 7). An alternative explanation for the shortened half life to the
in vitro culture conditions is that most of our studies were done with FL derived
megakaryocytes and fetal platelets have a shortened half life compared to adult platelets
(99). However, platelets derived from grown adult marrow megakaryocytes also had a relatively short half life than infused, donor derived platelets. There are insufficient numbers of primary megakaryocytes in isolated adult marrow to test whether these cells would have a longer half life.
Our demonstration that infused megakaryocytes can form a significant number of
functional platelets may be of clinical importance. In vitro generation of a large number
of biologically responsive platelets has remained problematic. The best published
outcome for in vitro formed platelets resulted with levels of platelets that are at least two
orders of magnitude lower compared to what we have been able to achieve with infused
megakaryocytes. Our studies open the possibility that if one can establish a cell line that
can be expanded and differentiated into mature megakaryocytes then one might be able
to infuse these megakaryocytes as a non donor dependent source of platelets. Such a line
had been described for mice, G1ME cells (121). These cells can be repeatedly derived
from a GATA 1 deficient embryonic stem cell line and that upon restoration of GATA 1
goes on to form ~50% megakaryocytes (121). Moreover, modification of such lines to
ectopically express a protein of interest during megakaryopoiesis may be useful for the
targeted delivery of such a protein to sites of vascular injury as described by us for the
delivery of coagulation factor VIII for the treatment of hemophilia A and urokinase for
targeted fibrinolysis (84, 122). This additional benefit of generating platelets in vivo
from infused modified megakaryocytes is supported by our data in Figure 2 11 that
shows that urokinase delivery to thrombi is a potential benefit of our approach using
mUK FL derived megakaryocytes that ectopically express urokinase and store this
45
protein in α granules as they mature.
In summary, we have addressed whether infused megakaryocytes can release
platelets within the pulmonary bed and demonstrated that biologically active platelets
can be formed in vivo with characteristics similar to those of normal platelets. The
process is vigorous, and enough platelets are formed so that platelet count can be boosted and hemostasis can be improved and modified by targeted delivery of ectopic
proteins. Based on our estimate of 100–200 platelets generated per infused
megakaryocyte, the challenge is to generate 10 9 mature WT or modified megakaryocytes
expressing and storing an ectopic protein of interest to achieve a 10% rise in platelet
count in an average 70 kg patient.
46
Table 2 2. Supplemental movie legend
Video 1. Incorporation of donor platelets in arteriole thrombi of h αIIb + recipient mouse after infusion of FL small cells, following laser injury. As in Figure 2 10 (C) with an injury created near the center of the video and blood flow from top to bottom on the screen. The platelets depicted in green were detected with an anti mouse CD41 antibody labeled with Alexa 488 . Incorporation of platelets from small cells into the thrombi was seen with a distinct population of CD41 + cells re circulating and rarely incorporated into the growing thrombus. Video 2. Incorporation of donor platelets in arteriole thrombi of h αIIb + recipient mouse after infusion of FL large cells, following laser injury. As in Figure 2 10 (B) with an injury created near the center of the video and blood flow from top to bottom on the screen. The platelets depicted in green were detected with an anti mouse CD41 antibody labeled with Alexa 488 . Incorporation of platelets from the large cells into the growing thrombi was detected similar to infusion of WT platelets. Video 3. Incorporation of WT donor platelets in arteriole thrombi of h αIIb + recipient mouse following laser injury. As in Figure 2 10 (A) with an injury created near the center of the video and blood flow from top to bottom on the screen. The platelets depicted in green were detected with an anti mouse CD41 antibody labeled with Alexa 488 . Incorporation of the WT infused platelets into the growing thrombi was clearly detected.
47
Chapter 3
Platelet targeted pro urokinase as a novel
thromboprophylaxis fibrinolytic strategy
3.1 Abstract
Use of plasminogen activators (PAs) is restricted to life threatening thrombotic
conditions because high concentrations are required to diffuse into clots in order to
overcome PA inhibitors and compensate for rapid clearance, that can lead to bleeding.
We hypothesized that targeting pro PAs to platelets would circumvent these obstacles
and preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots and also avoiding systemic fibrinolysis. To test
this concept, we produced a chimeric protein by fusing a scFv from an anti hαIIb monoclonal antibody (moAb) 312.8 linked to a thrombin activatable uPA T, which we reasoned would be activated preferentially at sites of active clot propagation. Anti PLT scFv/uPA T expressed in Drosophila S2 insect cells bound specifically to human and to hαIIb + mouse platelets, but not to WT. Anti PLT scFv/uPA T bound specifically to
immobilized h αIIb β3 protein with a Kd of ~80 nM, and retained its zymogenic
properties until activated specifically by thrombin. The fibrinolytic activity of anti PLT
scFv/uPA T and the non targeted counterpart uPA T were analyzed in two mouse
+ models of vascular thrombosis. In the FeCl 3 carotid artery injury model h αIIb mice were
48
protected from forming occlusive thrombi for at least 10 hrs post injection of anti PLT
scFv/uPA T whereas even five fold higher concentrations of uPA T were effective for
only 2 5 min. In the cremaster arteriole injury model h αIIb + mice show a significant
reduction in both fibrin deposition and platelet accumulation even after 5 hr of anti PLT
scFv/uPA T infusion, which was not seen after infused uPA T or PBS. These studies
support another novel approach to that in Chapter 2 using modified megakaryocytes for
prophylactic targeting drug delivery by combining a pro drug that requires activation by
thrombin with platelet delivery to sites of incipient thrombotic vascular occlusion.
3.2 Background
Thrombosis is the major cause of death and disability in the U.S. (80, 123). Occlusive
thrombi impairs blood flow to vital organs causing local oxygen deprivation, tissue
necrosis and organ dysfunction. PAs and direct thrombin inhibitors are effective acutely, but are associated with substantive risks of bleeding and are not formulated for
thromboprophylaxis. No agent prevents thrombosis in the majority of patients and all
increase the risk of bleeding, in large part because they act systemically. In theory, a
more ideal thromboprophylactic agent would have the following properties: 1) circulate
as a prodrug, 2) target incipient thrombi, while sparing hemostatic clots, and 3) remain
amenable to regulation.
PAs are not used for prophylaxis due to their rapid clearance and hemorrhagic
and neurotoxic side effects. All existing PAs are small, short lived (<30 min) molecules
capable of diffusing into hemostatic clot, which may cause bleeding. PAs can also diffuse
into the CNS where they may cause collateral damage, and therefore are not 49
suited for use as prophylaxis in post surgical or other patients at imminent risk of
thrombosis. We hypothesized that coupling PAs to a longer lived carrier that is confined
to the blood and has a natural targeting mechanism to sites of thrombus formation will block PA diffusion into hemostatic mural clots and the CNS, limiting its adverse effects, while being retained in the blood, prolonging the intravascular effect, and thus permitting use as prophylaxis. We propose using a platelet delivery strategy to target incipient or ongoing pathological clotting. To do so, we developed a recombinant fusion protein composed of a scFv of a moAb (Figure 3 1) against the haIIb subunit of the platelet integrin receptor aIIbb3 (GPIIb/IIIa, CD41a) and a thrombin activatable low molecular weight single chain urokinase plasminogen activator (lmw scuPA), which has been designated uPA T (Figure 3 2).
3.3 Material and methods
3.3.1 Analysis of anti hαIIb moAbs
Various anti platelet moAbs were analyzed to check their binding specificity to WT,
hαIIb + mice and human platelets by flow cytometry (Figure 3 5) (generously provided by
Dr. Richard Aster and Dr. Daniel Bougie from the monoclonal laboratory of the Blood
Research Institute, Milwaukee, WI). The moAb (312.8) that we end up using in our
studies has been previously characterized (124). Murine platelets were isolated from the
inferior vena cava as described in “2.3.4 Isolation of Platelets and Megakaryocytes for
Infusion”. Human platelets were isolated from 10 ml of fresh blood, and platelet rich
plasma (PRP) separated after centrifugation at 200 g for 10 min at room temperature
(RT). The platelets were then isolated from PRP in the same manner as describe for 50
xx
Figure 3 1. Final construct proposed. The anticipated final construct is shown on
the right and would consist of a single chain version of uPA that contains a thrombin
cleavage site uPA T. Upon thrombin proteolysis, uPA T would activate plasminogen to
plasmin and lead to clot lysis via fibrinolysis. The uPA T will be fused at its N terminus
to an scFv derived from a moAb (shown on left) that binds in a specific fashion to the
surface of human platelets via the αIIb receptor
51
Figure 3 2. Molecular design of uPA T. Thrombin inducible lmw scuPA was generated by deleting Phe 157 and Lys 158 , which converts the plasmin activation site into one that is cleave by thrombin after Arg 156 .
52
murine platelets. Total washed platelets were counted on a Hemavet cell counter
(Triad Associates), resuspended in aliquots with a final concentration of 3x10 8/ml.
Platelets suspensions were incubated with corresponding moAbs (0.1 mg/ml) for 1 hr at
37ºC. Unbound moAb was washed with PBS prior to incubating with a secondary
polyclonal Ab, FITC goat anti mouse IgG (BD Bioscience) for 30 min and analyzed by
flow cytometry using a FACScan.
3.3.2 Platelet aggregation of anti hαIIb moAbs
Platelet aggregation studies were performed as previously described (98), Briefly human
PRP was adjusted to 3X10 5 platelets/ l with platelet poor plasma (PPP). Samples of PRP were either untreated or incubated for 5 minutes at 37°C with 20 g/mL of moAb.
Platelet aggregation was induced by adding to PRP adenosine diphosphate (ADP) at 5
M, and light transmission was measured over time in an aggregometer (Kowa AG 10E;
Kowa) while stirring.
3.3.3 Construction and expression of anti PLT scFv/uPA T
Construction of the Anti PLT scFv/uPA T was performed following techniques used for fusing plasminogen activators with serine rich linker peptides, with some modifications
(85, 125, 126). Total RNA isolated from the moAbs hybridoma cell lines was reverse transcribed (RNeasy, Qiagen) using SMART TM technology (Clontech) employing primer
combinations described previously (127). The heavy and light chain variable fragments were clone into TOPO vectors (Invitrogen) (Figure 3 3) and positive clones were
sequenced analyzed. The fragments were constructed using standard nested PCR
methodology, in which the variable heavy (Vh) fragment was amplified with an
xxxxxxxxx
53
Figure 3 3. Depiction of the strategy to generate the anti PLT scFv. The schematic diagram depicts the strategies undertaken to produce the anti PLT scFv fragment as detailed in the Methodology. 54
Figure 3 4. Depiction of the strategy to generate the anti PLT scFv/uPA T.
The schematic diagram depicts the strategies undertaken to produce the final construct, which was used to produce the fusion protein anti PLT scFv/uPA T as detailed in the
Methodology.
55
outer primer (Vh FWD 5’ tgc atg tgc atg cca tgg cag tca ggg gga ggc 3’) and a reverse
primer that introduced a linker (GGGGS) 3 (128) at the end of the fragment (linker rev 5’ gat tgc tgg aga ttg agt gag cac gct gcc acc acc gcc gct gcc acc acc gcc gct gcc acc acc gcc ggg tgt cgt ttt ggc tga gga gac 3’). The variable light (Vl) fragment was amplified with a complementary primer to “linker rev” (linker FWD 5’ gtc tcc tca gcc aaa acg aca ccc ggc ggt ggt ggc agc ggc ggt ggt ggc agc ggc ggt ggt ggc agc gtg ctc act caa tct cca gca atc 3’) and a downstream primer (Vl rev 5’ tcc ccg cgg agt tgg tgc agc atc 3’). Joining of both fragments was completed by using the same outer primer “Vh FWD” that contains a restriction site for NcoI at the 5’ end, and the downstream primer “Vl rev” which
introduces a restriction site for SpeI at the 3’ end in order to clone into insect expression vector pMT/BiP/V5 His B (Invitrogen) (Figure 3 7 A). The anti PLT scFv PCR products were purified and digested with NcoI and SpeI restriction enzymes, (New England
BioLabs) ligated and cloned into the NcoI and SpeI sites of the insect vector. Successful cloning was confirmed by restriction analysis of plasmid and by automated DNA sequencing (NAPCORE, The Children’s Hospital of Philadelphia). We then established a stable S2 drosophila cell line (Invitrogen) expressing the anti PLT scFv (Figure 3 7 C) and analyzed the protein for binding to h αIIb from the insect media by ELISA (Figure 3
8) and flow cytometry (Figure 3 9).
To produce the complete chimeric protein construct, nested PCR was performed
(Figure 3 4) to link the two fragments scFv and uPA T. The fragment for the scFv portion was amplified with primer “Vh Fwd” and a new primer that introduce a linker
(SSSSG) 2 (128) downstream of the scFv (linker rev 5’ agt ctt ttg gcc aca ctg aaa ttt taa gcc gga aga gct act acc cga tga gga aga agt tgg tgc agc atc agc ccg ttt tat ttc ca 3’). The uPA T fragment was amplified with a complimentary long primer to the “linker rev” (5’ tgg aaa taa aac ggg ctg atg ctg cac caa ctt ctt cct cat cgg gta gta gct ctt ccg gct taa aat ttc agt gtg gcc
56
aaa aga ct 3’) and an outer primer (uPA t rev 5’ cca atg cat tgg ctc gag tca tca ctt gtc atc
gtc atc ctt gta atc gat 3’).
To join both fragments once again outer primer “Vh Fwd” which contain a
restriction site for NcoI at the 5’ end was used, and the downstream primer “uPA T Rev” which introduces a restriction site for XhoI at the 3’ end as well as a three flag tag with a
stop sequence (Figure 3 7 B). The restriction sites were introduced to clone into insect
expression vector pMT/BiP/V5 HisA. Stable drosophila cell line expressing the anti PLT
scFv/uPA T was established for production of the protein. The proteins were purified
from cell media by affinity chromatography using an M2 (anti flag) affinity column
(Sigma) followed gel filtration chromatography on Sephacryl SHR100 column
(Amersham) to greater than 95% purity, which was confirmed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS PAGE) (Figure 3 10 A and B) with a yield approximately 3 to 5 mg/L medium. Proteins were concentrated in PBS to 2
mg/mL, separated into aliquots and stored at 80°C.
3.3.4 Biochemical characterization of anti PLT scFv/uPA T Characterization of the protein was performed in the same manner as previously
described for other scFv chimeric proteins (126). The size and homogeneity of the fusion
protein was analyzed using a 4% 12% gradient SDS PAGE and Western blotting (Figure
3 10 C). For Western blot analysis, the separated proteins were transferred to a
nitrocellulose membrane (Invitrogen) blocked with tris(hydroxymethyl)aminomethane
(Tris) buffered saline containing 10% non fat milk powder and 0.1% Tween–20. The
fusion protein was detected using a 1:1000 dilution of a horseradish peroxidase (HRP)
conjugated anti flag tag moAb (Sigma). The antigen antibody complex was detected with
ECL Plus (Amersham).
The intrinsic and thrombin induced activities of anti PLT scFv/uPA T were 57
measured using casein zymography. Anti PLT scFv/uPA T, (10 ng) intact or pre
incubated with 20 nM thrombin, were mixed with non reducing Tris glycine SDS sample buffer prior to zymography. The samples were resolved on a 7.5% gel cast with 10% non fat dry milk and 20 g/ml plasminogen to detect PA activity (129). The gels were re natured in Novex zymogram renaturating buffer (Invitrogen) and developed in Novex zymogram developing buffer (Invitrogen) per the manufacturer. EDTA (5 mM) (Sigma)
3.3.5 Binding of anti PLT scFv/uPA T to hαIIbβ3
Binding specificity of the anti PLT scFv/uPA T to hαIIbb3 was analyzed with platelets using flow cytometry techniques and the methodology described in “3.3.1 Analysis of anti hαIIb monoclonal antibodies”. The specificity of the fusion protein was also analyzed with plastic immobilized h αIIbh β3 in 2 modes: by standard ELISA (Figure 3 12
A) or with 125 I labeled anti PLT scFv/uPA T (Figure 3 12 B). H αIIbh β3 protein (Enzyme
Research) was immobilized on high binding plastic 96 well plates (Corning) at 1 g/well.
Non specific binding was blocked by 5% PBS/BSA for 1 hr at 37°C then washed with
PBS/0.1% Tween20 (PBS/Tween). For standard ELISA, various concentrations of anti
PLT scFv/uPA T, uPA T, or a non specific fusion protein anti GPA scFv/uPA T were incubated for 1 hr at 37°C. After washing with PBS/Tween, an anti flag HRP conjugated moAb served as the primary antibody. The antigen antibody complex was detected with
3,3',5,5' tetramethylbenzidine (TMB) peroxidase substrate (Amersham) and absorbance was measures at 490nm. For the second method, the fusion protein was radio labeled with 125 I Na (Perkin Elmer) in Iodination tubes (Thermo Scientific). After blocking with
5% PBS/BSA and washing with PBS/Tween, various concentrations of the labeled chimeric protein (100 l/well) were incubated with h αIIbh β3 or BSA coated wells for 1 hr at 37°C. Immobilized BSA served as a negative control. Non bound material
58
was removed by washing with PBS/Tween and residual radioactivity was measured on a
γ counter (Perkin Elmer).
3.3.6 In vitro fibrinolysis of anti PLT scFv/uPA T
Bovine fibrinogen (Sigma) was dissolved in PBS to a final concentration of 3 mg/ml.
Clotting was induced by addition of CaCl 2 and thrombin (Sigma) (20 mM and 0.2 units/ml final concentrations, respectively) and the resulting solution was immediately added to 24 well cell culture plate (0.75 ml/well) where clots were formed and allowed to maturate for 30 min. Anti PLT scFv/uPA T was activated by incubation with 20 nM bovine thrombin (Amersham) for 1 hr and serial dilutions (50 l/well) were added on top of each clot at 37°C. Lytic zones were measured after 12 hr incubation.
3.3.7 Effect of anti PLT scFv/uPA T in models of vascular thrombosis
Studies were done on male h αIIb + mice as described in “2.3.7 Cremaster laser injury functional studies”. The timing, spatial and absolute incorporation of fibrin and platelets into the growing thrombi were followed using a specific anti fibrin antibody and an anti species specific αIIb Ab, respectively that do not interfere with clot growth.
For FeCl 3 studies, we used the strategy as described in “2.3.8 FeCl 3 carotid artery injury functional studies”. Briefly, 1 mg/kg of anti PLT scFv/uPA T or 1 mg/kg uPA T
+ was injected IV into h αIIb mice prior to FeCl 3 induced carotid artery injury.
Administration of PBS served as the negative control. Occlusion and total blood flow was monitored by Doppler ultrasound probe. Both proteins and PBS were injected 10 mins to
10 hrs prior to arterial injury to estimate the therapeutic window of thromboprophylaxis and blood flow after injury was monitored for over 30 min.
59
3.3.8 Future studies with the anti PLT scFv/uPA T fusion protein
Effect of anti PLT scFv/uPA T fusion protein on platelet activation. To analyze if
anti PLT scFv/uPA T promotes activation of isolated platelets, flow cytometry will be
performed as described (130) with minor modifications. Platelets from h αIIb + mice will be prepared and aliquot will be incubated with saturating concentrations of anti PLT
scFv/uPA T. Unbound protein will be removed by washing. As a positive control, a
subset of intact platelets will be stimulated with the weak activator ADP (25 mmol/L) or buffer control. Fusion protein coated platelets will be handled in the same manner.
Platelets from each experimental group will be fixed and washed. The platelets will be
stained with R phycoerythrin (PE) labeled P selectin antibody (mouse IgG, CLB), mouse
moAb PAC 1 (Becton Dickinson), which recognizes only the activated form of mouse
αIIb β3, or an equivalent concentration of unspecific IgG and binding will be measured
using a FACScan (131).
Effect of anti PLT scFv/uPA T on platelet aggregation . To further assess the
effect of the fusion proteins on platelet function, platelets from h αIIb + mice will be
incubated with saturating concentrations of anti PLT scFv/uPA T and the unbound
protein will be removed by washing. Aggregation in response to a weak agonist will be
measured using a platelet aggregometer as described (132). WT and unactivated fusion
coated h αIIb + murine platelets will serve as negative controls.
Pharmacokinetics of anti PLT scFv/uPA T vs. uPA T. Anti PLT scFv/uPA T and
uPA T will be labeled with 125 I and tracer amounts will be injected via the jugular vein of
WT and h αIIb + mice. Blood clearance and misdistribution of the injected protein 10 min,
60 min, 3, 6, 24, 48, and 72 hrs after infusion will be measured. The animals will be
sacrificed and major organs (lungs, liver, kidneys, spleen, heart, brain) will be harvested
and perfused free of blood. PRP will be isolated and centrifuged at 10 4g to separate
60
platelets from PPP. All blood fractions and organs will be analyzed for radioactivity and
distribution of injected labeled proteins will be determined (% of injected dose and % of
injected dose/g tissue).
Platelet counts will be measured to determine whether anti PLT scFv/uPA T
affects platelet lifespan. To verify that platelet half life is not affected by the chimeric
protein in vivo, we will incubate h αIIb + mouse platelets with the fluorescent dense granule marker mepacrine (quinacrine) (133) and the fusion protein subsequently infused into h αIIb + mice. Survival of the infused platelets can then be detected by flow
cytometry at different time points after infusion.
We will also determine the desorption kinetic of 125 I anti PLT scFv/uPA T from
hαIIb + platelets. Washed platelets will be incubated with 125 I anti PLT scFv/uPA T and
injected into WT or h αIIb + mice. Blood and major organs will be collected as above and
the distribution of residual radioactivity will be measured over time. The half life of the
fusion protein loaded platelets will be determined using the Vybrant CFDA SE Cell
Tracer kit (Molecular Probes) as previously described (134).
Retention of anti PLT scFv/uPA T and uPA T fibrinolytic activity during
circulation in blood. We will follow the protocol described previously for a prototype
RBC/tPA conjugate (135, 136). Briefly, equimolar amounts of anti PLT scFv/uPA T,
uPA T or PBS will be injected intravenously (IV) into anesthetized WT and h αIIb + mice.
At pre designated times determined above, aliquots of blood will be drawn in the
absence of anticoagulant, mixed rapidly with trace 125 I fibrinogen and allowed to clot.
Clots will be overlaid with PBS and incubated at 37°C for 24 hrs and the release of 125 I will be measured as above. These experiments will allow us to estimate the relationship between the dose, magnitude and duration of fibrinolytic activity of anti PLT scFv/uPA
T in the circulation.
61
Depletion of fibrinogen by circulating anti PLT scFv/uPA T or uPA T. We will
follow the protocol described previously for a prototype uPA T fusion protein directed to
endothelium (85). Equimolar amounts of anti PLT scFv/uPA T, uPA T or PBS, in
amounts based on the blood fibrinolytic activity determined above, will be injected IV
into WT and h αIIb + mice. At pre designated times based on the measured circulation
time and blood activity, blood will be drawn in citrate and PPP. Plasma fibrinogen will be
measured by ELISA using a standard curve based on purified mouse fibrinogen.
Bleeding after administration of anti PLT scFv/uPA T fusion protein . We will use
the tail re bleeding time as an initial test of hemostasis in treated WT and h αIIb + mice
(137, 138). To evaluate hemorrhagic potential, mice will be anesthetized, equal doses
(determined in ex vivo blood clot lysis experiments) of anti PLT scFv/uPA T or uPA T will be injected 10 min to 72 hrs prior to analysis. Tails will be immersed in a water bath
at 37°C for 5 min, a 5 mm terminal segment will be amputated, and the time required to
stop bleeding will be determined visually and the amount of released hemoglobin will be
measured. After the initial bleeding has stopped, re bleeding will be evaluated by
immersing the tail in 4 ml of 37°C PBS containing 14 mmol/L tri sodium citrate for 1 hr.
The number of RBC and the amount of hemoglobin released in PBS will be determined
to quantify blood loss (137, 138). To elucidate whether platelet bound anti PLT
scFv/uPA T will spare hemostatic clots better than uPA T, re bleeding will be measured
as above, with the exception of injecting the proteins after spontaneous bleeding has
ceased before evaluating re bleeding.
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3.4 Results
3.4.1 Binding analysis of anti platelet moAbs
Binding of multiple anti hαIIb moAbs (generously shared by Drs. Daniel Bougie and
Richard Aster, Blood Center of Wisconsin) was verify against WT, h αIIb + mice, and
human platelets and analyzed by flow cytometry. Many of the antibodies bound with
significant affinity to the surface of human platelets (Figure 3 5) and h αIIb + mice
platelets (not shown), but none to WT platelets (not shown). Two moAbs with high
affinity to human platelets were chosen for further analysis (Abs 184.2 and 312.8) (124).
The antibodies were analyzed to see if they interfere or enhance platelet aggregation. As
it can be seen in Figure 3 6, moAb 184.2 altered aggregation, whereas moAb 312.8 may
have slightly inhibited aggregation. Both were further studied understanding that there
level of surface aIIbb3 receptor is dense and at low moAb concentrations, most receptors
may still be available for ligand binding.
3.4.2 Binding analysis of anti platelet scFv
Construction of the fusion protein was undertaken with both moAbs (184.2 and 312.8) by isolating RNA from their hybridoma cells and performing RT PCR to form heavy and
light variable fragments by a nested PCR strategy as depicted in Figure 3 3. The resultant
heavy and light chain variable fragments were assembled to form the anti PLT scFv
(Figure 3 7 A). The purified PCR product was cloned into an insect vector and expressed
in Drosophila S2 cells and expression confirmed by immunoblotting with anti V5 HRP
moAb (Figure 3 7 B). The overall construct was formed with the same PCR strategy
(Figure 3 4) where the scFv segment was joined with a uPA T (Figure 3 7 C). Binding of
x 63
Figure 3 5. Binding analysis of anti hαIIb specific moAbs to human platelets.
Human platelets were incubated with 0.1 mg/ml of different anti platelet moAb’s and
stained with an anti mouse FITC conjugated Abs. Mean fluorescence of 10 5 platelets was measured. Only the two moAbs pursued further are noted. The binding to secondary antibody alone is shown as the gray fill graph. Details of the procedure are in the
Methodology.
64
Figure 3 6. Platelet aggregation analysis of anti hαIIb specific moAbs. The two chosen moAbs were studied further using platelet aggregation studies with human
PRP as detailed in the Methodology. Monoclonal Abs were added at 20 g/mL and ADP activation was at 5 M at point indicated (see arrows).
65
Figure 3 7. Generating the anti PLT scFv/uPA T fusion construct . The general
construct strategies are shown in Figures 3 3 and 3 4. (A) Agarose size fractionation gel
of the assembled scFvs with expected size of 735 base pairs. (B) Western blot analysis of native culture medium from S2 cells transfected with anti PLT scFv expression plasmid after induction with 0.5 mM CuSO 4. An HRP moAb against the flag tag served as the detecting antibody, showing expression of the expected scFv chimera proteins (C) Same as (A), but for the scFvs assembled with a uPA T fragment into full length anti PLT scFV/uPA T with expected size of 1640 base pairs.
66
both anti PLT scFv’s to immobilized h αIIbh β3 protein was confirmed by ELISA (Figure
3 8). When analyzing binding by flow cytometry, anti PLT scFv 312.8 bound to human
and h αIIb +, but not WT, mouse platelets (Figure 3 9 A). Anti PLT scFv 184.2 bound to human platelets with less affinity (Figure 3 9 A), and it did not bind to h αIIb + or WT mouse platelets (Figure 3 9 B and C).
3.4.3 Analysis of anti platelet scFv/uPA T
After establishing stable drosophila S2 cell lines expressing anti PLT scFv/uPA T, both proteins were purified from cell media by affinity chromatography using an M2 (anti
FLAG) affinity column (Figure 3 10 A and B). To confirm that the fusion proteins retained the antigen recognition properties of the parental moAb and scFv, flow cytometry studies were performed. Anti PLT scFv/uPA T 312.8 bound to human platelets as well as h αIIb + but not WT mice platelets (Figure 3 11). The binding was similar to what was demonstrated by its scFv. Interestingly, anti PLT scFv/uPA T 184.2 appears to have lost its affinity since it did not recognize human or h αIIb + mouse platelets, in contrast to its scFv, likely due to the chimeric construct decreasing affinity of the scFv for h αIIb (Figure 3 11). Therefore, future studies focused on the fusion protein
312.8, and is referred to below as anti PLT scFv uPA T.
To further show the specificity of anti PLT scFv/uPA T we performed the experiment on plastic immobilized h αIIb β3 in two modes: with standard ELISA (Figure 3 12 A) and 125 I labeled fusion protein (Figure 3 12 B). Both methods exhibited specific and dose dependent binding of anti PLT scFv/uPA T to the antigen with a Kd of ~80 nM.
Moreover, labeling with iodine did not influence the affinity of the fusion protein, which should simplify future pharmacokinetic experiments.
67
Figure 3 8. ELISA analysis of moAbs and scFv induced and non induced S2
media. ELISA of hαIIbh β3 protein coated platelets with the original indicated moAb or scFv S2 insect cell media. Mean ± 1 SD are shown. N = 3, each done in duplicate.
68
Figure 3 9. Characterization of anti PLT scFv. (A) Human, (B) haIIb + mouse and
(C) and WT mouse platelets were incubated with 0.1 mg/ml of 312.8 anti PLT scFv
(red), moAb 312.8 (red) or 184.2 anti PLT scFv (green), and moAb 184.2 (green) for flow cytometric studies of binding. Grey filled in curve is secondary only binding. Mean fluorescence of 10 5 platelets was measured for each curve.
69
3.4.4 Fibrinolytic activity of anti platelet scFv/uPA T in vitro
To see if anti PLT scFv/uPA T has any PA activity in its native form, we performed
casein zymography. We confirmed that anti PLT scFv/uPA T is expressed as a zymogen with negligible plasminogen conversion activity (Figure 3 13 A), which drastically increased upon thrombin activation. This result appears to be similar though less potent than that seen with the non fused thrombin activatable uPA T, which served as our positive control. Purified anti PLT scFv/uPA T migrated as a predominant single band at the predicted molecular weight of ~59 kDa and this was confirmed by Western blot
(Figure 3 13 B). Cleavage by thrombin generated two bands that under reducing conditions results in a protein fragments of ~30 kDa (Figure 3 13 B).
To estimate the fibrinolytic activity of the anti PLT scFv/uPA T upon thrombin conversion into a two chain form capable of activating plasminogen, fibrin plate analysis was performed, which revealed strong, dose dependent fibrinolytic activity of the anti
PLT scFv/uPA T, comparable with the non fused uPA T (Figure 3 13 C).
3.4.5 Thrombolytic efficacy of anti PLT scFv/uPA T in mouse models
of vascular thrombosis
We tested whether anti PLT scFv uPA T thromboprophylaxis could effectively prevent occlusive thrombi using the FeCl 3 model of carotid arterial injury in mice as previously described (139). Equimolar amounts of anti PLT scFv/uPA T, un bound uPA T, or PBS
+ were IV injected into anesthetized h αIIb mice prior to FeCl 3 carotid artery injury (98).
In preliminary data (Figure 3 14), it appears that administration of uPA T or PBS does not prevent vascular occlusion after 5 min of infusion, while injection of anti PLT scFv/uPA T was protective even 10 hrs post infusion.
We also used the cremaster arteriole injury model, which allows for in situ 70
visualization
Figure 3 10. Characterization of the anti PLT scFV/uPA Ts. (A) and (B) SDS
PAGE gels of eluted proteins stained with GelCode blue stain reagent, and (C) Western blot of the anti PLT scFv/uPA Ts using an HRP moAb against the flag tag as the
detecting antibody.
71
Figure 3 11. Flow cytometric analysis of haIIbb3 binding by anti PLT scFv/uPA T. (A) Human (A), (B) haIIb + mouse and (C) WT mouse platelets were incubated with 0.1 mg/ml of 312.8 anti PLT scFv/uPA T (red), moAb 312.8 (red) or
184.2 anti PLT scFv/uPA T (green), and moAb 184.2. Representative study of three separate flow cytometric analyses is shown. Mean fluorescence of 10 5 platelets was measured. 72
Figure 3 12. Binding of anti PLT scFv/uPA T to immobilized h αIIb β3. (A)
ELISA studies with both haIIbh β3 in wells with the various concentrations of anti PLT scFv/uPA T, uPA T, or a unrelated fusion protein, anti GPA scFv/uPA T indicated. (B)
Binding of labeled anti PLT scFv/uPA T to immobilized h αIIbh β3. Varying concentrations of labeled anti PLT scFv/uPA T were incubated with h αIIbh β3 or BSA coated wells for 1 hr. N=3, done in duplicate each. Mean ± 1 SD of binding is shown.
73
Figure 3 13. Enzymatic activity of anti PLT scFv/uPA T. The intrinsic and thrombin induced plasminogen activator activities of the anti PLT scFv/uPA T were analyzed using (A) casein zymography and (B) Western blot with a mouse anti lmw scuPA antibody (American Diagnostica) as primary antibody. (C) Fibrin plate analysis revealed a strong and dose dependent fibrinolytic activity of the anti PLT scFv/uPA T similar to uPA T.
74
Figure 3 14. Prophylactic thrombolysis by anti PLT scFV/uPA T in a FeCl 3 carotid artery injury model. Indicated products were infused at time zero and then a
FeCl 3 injury followed by a 30 min measurement of carotid artery blood flow was monitored at the indicated time points. Mean ± 1 SD are shown. N = 3.
75
visualization of thrombus development in real time to test our fusion protein. In these
studies, h αIIb + male mice were IV infused with equimolar amounts of anti PLT
scFv/uPA T, un bound uPA T or PBS 5 hrs before inducing injury. Mice infused with
either PBS (Figure 3 15 A) or uPA T (Figure 3 15 B) showed similar fibrin deposition with only a small difference in platelet accumulation. Mice infused with anti PLT scFv/uPA T fusion protein (Figure 3 15 C) showed a significant reduction in both fibrin deposition and platelet accumulation. These preliminary studies support our fusion protein as being both an effective and long lasting thromboprophylactic agent.
3.5 Discussion
Thrombotic arterial occlusion is the proximate cause of myocardial infarction and stroke.
Occlusive thrombi impair blood flow to vital organs causing local oxygen deprivation, tissue necrosis and organ dysfunction. Current PA therapy is not used for prophylaxis due to their rapid clearance, (<30 min) risk of bleeding and neurotoxic side effects.
Newly designed mutant PAs with enhanced potency and fewer side effects may improve this outcome (140 145), but their selective diffusion into occlusive clots has not been tested and none has been designed for prophylactic use. An ideal thromboprophylactic agent needs to have a prolonged lifespan in the blood, circulate as a pro drug, and target incipient thrombi while sparing hemostatic clots.
Recent studies have targeted PAs to red bloods cells (RBC) by re infusing ex vivo modified cells (139) and by IV injection of PAs fused to either Fab’ or scFv from anti RBC
(126, 146). These studies showed that the fused proteins provided prolonged thromboprophylaxis against lethal stroke and pulmonary embolism with less re 76
xxxxxxx
Figure 3 15. Platelet and fibrin accumulation in laser induced arteriole
injuries in treated haIIb + mice. Analysis of in situ laser injury in cremaster arterioles 5 hrs post treatment with (A) PBS, (B) uPA T, and (C) anti PLT scFv uPA T.
Average platelet accumulation (green) and fibrin clot accumulation (red) are shown over a 3 min window after a laser injury. N=2 mice per study with 10 injuries per mouse.
77
bleeding after tail amputation (125, 146) or intracerebral hemorrhage after traumatic brain injury (147). But RBCs are not specifically targeted to growing thrombi, especially arterial thrombi. Platelets, on the other hand, become incorporated into incipient clots, which makes them ideal for a thromboprophylaxis design. Our group has shown that
uPA ectopically expressed in platelets using a transgenic approach is safe and effective in
preventing arterial and venous thrombosis and that transfusion of 5% of the platelet
mass protects wild type mice from clotting without causing systemic fibrinolysis or
spontaneous bleeding aside from post partum hemorrhage (84).
We used a similar strategy as the RBC PA studies, utilizing platelets to deliver the
thrombin activatable mutant uPA T to newly formed clots. We hypothesize that this
approach will; 1) increase the half life of uPA T in circulation, 2) significantly increase
delivery of uPA T to sites of incipient thrombus formation, 3) limit systemic side effects
due to local activation of uPA T by thrombin, and 4) bypass mature hemostatic clots not
actively recruiting platelets with less thrombin present.
We developed a recombinant fusion protein composed of a scFv from a moAb
(Figure 3 1) against the h αIIb subunit of the platelet integrin receptor αIIb β3 fused to
thrombin activatable uPA T (Figure 3 2). The new zymogen, anti PLT scFv/uPA T,
4 bound specifically to human platelets, with a B max of ~2x10 molecules per cell, assuming
approximately 80,000 GPIIb/IIIa complexes on the surface of each platelet (90). Anti
PLT scFv/uPA T also bound to h αIIb +, but not WT, mouse platelets (Figures 3 11 and 3
12). The fusion protein exhibited pronounced activity, but only upon thrombin activation
and conversion of uPA T into its active form (Figure 3 13). To test thromboprophylaxis, we used two mouse models of acute thrombotic occlusion in response to vascular injury.
In the FeCl 3 carotid artery injury model, it appears that uPA T or PBS does not prevent vascular occlusion 5 min after administration, while anti PLT scFv/uPA T was protective
78
even after 10 hrs post infusion (Figure 3 14). When analyzing h αIIb + mice with the cremaster arteriole injury model 5 hrs after administering the anti PLT scFv/uPA T, we saw a significant reduction in both fibrin deposition and platelet accumulation compared to mice that had been treated with uPA T or PBS (Figure 3 15).
These preliminary results from the mouse models of vascular injury concur with our expectation that the anti PLT scFv/uPA T binds rapidly to the surface of platelets in hαIIb + mice, while uPA T is rapidly eliminated from circulation through hepatic
clearance. Further studies should provide us with a better picture as it relates to platelet
survival, biodistribution of the protein in the lungs, liver or spleen, and the duration of
fibrinolytic activity. It also remains to be determined whether the platelet bound fusion
protein induces bleeding or, conceivably, re bleeding, at doses that prevent clot buildup.
We propose a potentially clinically feasible means to target a pro drug using the
natural procoagulant platelet thrombin pathway to induce fibrinolysis and reduce the
need for higher dosage. If effective, the design of the fusion protein and targeting
strategy allows for adaptation to other pro drugs with diverse activities (anti
inflammatory, anti oxidant) that can be targeted to platelets or other cell types, (e.g.,
neutrophils, monocytes) that target to sites of injury in other disorders.
79
Chapter 4
Summary, and future directions
PAs assist in maintaining blood fluidity by activating plasmin to degrade fibrin clots (62,
148, 149), but they have limited clinical utility due to their rapid clearance and problematic side effects, especially untoward bleeding. Their clinical limitations make
PAs unsuitable for prophylactic therapy, which merit the need for newer drugs that are safer and better suited for effective fibrinolysis. In theory, a more ideal thromboprophylactic agent should have the following properties: 1) has a prolonged lifespan in the blood, 2) circulates as a prodrug, 3) targets incipient thrombi, while sparing hemostatic clots, and 4) remains amenable to regulation. Platelets provide the first line of defense in the process of hemostasis, by localizing and sustaining the blood loss and adhering to the site of injury. Their natural targeting function makes them an ideal candidate for therapeutic delivery of bioactive agents. We concentrated on two distinct strategies that in proof of principle studies are promising for effective and targeted platelet directed thrombolytic therapy.
In our first approach, we were interested in target delivery of uPA to the injured site by expressing the protein at high levels in developing megakaryocytes so that it is stored in platelet alpha granules and released upon activation. These findings were previously demonstrated in our laboratory with transgenic mice that ectopically express uPA within platelets (84). The uPA loaded platelets showed that they could selectively lyse emboli without inducing bleeding, systemic decrease in fibrinogen levels or increase
80
in D dimers. Expression of uPA within platelets also is the underlying mechanism in
Quebec platelet disorder, wherein patients have mild bleeding (105); supporting the
contention that platelets laden with uPA may be an effective PA with little systemic bleeding. These patients show significant post traumatic bleeding, but this is
controllable with tranexamic acid (150 153), suggesting a therapeutic strategy to treat
untoward bleeding from uPA ladened platelets as a PA strategy.
We were interested in translating these findings into clinical use beginning with ex vivo generated megakaryocytes and platelets for thrombolytic targeted therapy. No one had successfully produced sufficient ex vivo generated platelets before and it is possible that given the plastic surfaces and slow release of ex vivo generated platelets from megakaryocytes that we would have difficulty generating large numbers of non activated platelets. Because of older literature suggesting that circulating megakaryocytes might become lodged in the lungs and release platelets thereafter (35,
36, 39), we tested whether infusing ex vivo generated megakaryocytes directly would lead to released platelets within the lungs. We found that we can achieve a significant number of donor derived platelets from these infused megakaryocytes in a murine model with a delay of 3 90 minutes. While these platelets had a slightly shortened half life compared to donor derived platelets, they had many features of normal platelets and were functional in two in vivo assays. In a radiation induced thrombocytopenia model, we showed that these platelets can boost the platelet count as well as donor derived platelets and in this system, may even last better. Whether these megakaryocyte derived platelets enhanced post radiation survival has yet to be tested. In the FeCl 3 carotid artery model, we not only tested platelet efficacy, but showed that megakaryocytes derived from transgenic mice with platelet uPA expression, when infused into the recipient mouse completely prevented thrombus development. We take this as the first step in a proof of
81
principle that our approach of infusing modified megakaryocytes may be a model for the
targeted delivery of a PAs to a site of untoward thrombosis and may be useful for
targeted thromboprophylaxis.
In our second approach, we extended a strategy developed by our colleagues of
endothelial and RBCs targeting fused to a thrombin activated pro enzyme uPA T (85,
126) to platelets. They had shown targeted efficient and safe thromboprophylaxis in
several clinically relevant models including after pulmonary injury. We extended their
strategy by using a platelet delivery system with uPA T that is activated and released from the antibody by thrombin generated at the site of active clotting. Since platelets target new thrombi better than RBCs, we believe that there may be advantages for the binding of uPA T to the platelet surface over the red cell surface. Our fusion uPA T that
had an scFv derived from an anti haIIb moAb generously provided by my colleagues Drs.
Richard Aster and Daniel Bougie at the Blood Center in Wisconsin, bound specifically to
human platelets through its αIIbb3 receptor. This fusion uPA T also bound to transgenic
mice that had chimeric haIIbmb3 are their platelets, but not to WT platelets. The fusion
protein exhibited pronounced activity, but only upon thrombin activation and
conversion of uPA T into its active form. In preliminary thromboprophylactic
experiments using a mouse model of vascular injury, it appears that the fusion protein was long lasting and prevented thrombus development for at least 10 hrs post infusion
in the FeCl 3 carotid artery injury model without obvious bleeding complications, while
uPA T or PBS did not prevent vascular occlusion 5 min after administration. In
analyzing h αIIb + mice with the cremaster arteriole injury model 5 hrs after administering
of the fusion protein, we also saw a significant reduction in both fibrin deposition and
platelet accumulation compared to mice that had been treated with uPA T or PBS. These
data are preliminary, and need to be repeated.
82
For the future, the studies presented here serve as proof of principle of two novel therapeutic approaches for the delivery of uPA especially for thromboprophylaxis. Both strategies include using platelets to target uPA to incipient thrombi and hopefully to avoid more mature thrombi and enhance efficacy and selectivity of the PA. For the megakaryocyte derived platelet strategy our ultimate goal would be to establish a self replicating human cell line that upon appropriate stimulus terminally differentiates into megakaryocytes expressing uPA. Upon infusion of these cells, we anticipate efficacious release of platelets from these cells. For our second approach involving anti PLT scFv/uPA T, safety and pharmacokinetics and pharmacodynamic studies need to be completed in mice. A Phase I safety trial would be needed in a setting associated with a high risk of thrombosis that may benefit from thromboprophylaxis. This may be patients undergoing stent placement or a subgroup of such patients that are at higher risk of such thrombi, like in patients with multivessel coronary artery disease with a higher prevalence of coronary risk factors when compared to patients without (154).
If either approach proves to be clinically useful, the strategies developed here for platelet targeted therapy may be extended to other systems such as the delivery of agents that are anti inflammatory, like thrombomodulin either stored within platelets or a recombinant thrombomodulin protein designed by our colleague, Bi Sen Ding (155) at the University of Pennsylvania with a thrombin cleavage site, transforming thrombin into an anti inflammatory molecule. Our approach can also be adapted to produce anti angiogenic agents by designing platelets stored with negative angiogenic regulators such as thrombospondin 1, endostatin, and angiostatin to inhibit tumor progression. We can also target the tumor with scFvs with a cleavage site for cathepsin B, which is over expressed in many tumor cells and tumor endothelial cells. The modular design used in our scFv can be applied to other cells that can be targeted and are essential in treating
83
other malignancies such as infectious disease. For example neutrophils and monocytes can be targeted via the myeloid Fc αRI (CD89) receptor which has been identified as an effective target molecule (156), and is expressed exclusively in myeloid cells.
84
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