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Hematopoiesis Ii


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Hematopoiesis Ii

  1. 1. Hematopoiesis II JC Kostyak 10-22-09
  2. 2. Outline <ul><li>MK migration -- review </li></ul><ul><li>Mk docking </li></ul><ul><li>MK platelet production </li></ul><ul><li>Platelet lifespan </li></ul><ul><li>Platelet clearance </li></ul><ul><li>Thrombocytopenias </li></ul>
  3. 3. Megakaryocytes Must Migrate When Ready to Produce Platelets SDF-1 CXCR4 Stromal Cells
  4. 4. Megakaryocytes Require PECAM-1 for Polarity <ul><li>Last time – MKs migrate toward an SDF-1 gradient </li></ul><ul><li>PECAM-1 regulated migration via polarization </li></ul><ul><li>PECAM-1 -/- mice show a lack of cell polarity </li></ul>Dhanjal et al., Blood 2007
  5. 5. Migration Through the Basement Membrane Bone Sinusoidal Vessel Endothelial Cells MMP
  6. 6. Megakaryocyte Docking ? PECAM-1 ? MK MK Once the Mk has docked, it can begin to produce proplatelet processes ?
  7. 7. Is PECAM-1 Required for MK Docking? Dhanjal et al., Blood 2007
  8. 8. Once Docked MKs Produce Proplatelets Patel et al., JCI 2005
  9. 9. Microtubule Elongation MT form long extensions which bend and branch MT line the membrane of the newly formed platelet In order to branch MT must elongate, then fold (B) and extend due to sliding (C). This bending allows the MT to form the next platelet (as seen above). Patel et al., JCI 2005
  10. 10. MT Review Conde and Cesares, Nature 2009
  11. 11. Microtubule Sliding Mitotic Kinesin Small molecular motors use ATP
  12. 12. Kinesins and Dynein V Kinesin Retrograde MTOC Anterograde + - + - Retrograde Dynein There are many examples of each! V
  13. 13. Proplatelet Formation Originates Opposite the Nucleus Italiano et al., JCB 1999
  14. 14. The Theatrical Version
  15. 15. Kinesins Localize in the Cytosol while Dynein is Localized to the Proplatelets Patel et al., Blood 2005
  16. 16. Proplatelets Extend into BM Sinusoidal Vessels PI = Circulating platelet, BI = Bulbous tip, Er = Erythrocyte, MP = Proplatelet, SW = Sinusoidal vessel Kessel and Kardon, Tissues and Organs 1979
  17. 17. New “Reticulated” Platelets <ul><li>Platelets are anucleate </li></ul><ul><li>Contain some mRNA </li></ul><ul><li>Contain all the machinery necessary for protein synthesis </li></ul><ul><li>We can use the mRNA to track new platelets (Thiazole Orange) </li></ul><ul><li>Normal = 14-18% </li></ul>
  18. 18. The Aging Platelet <ul><li>Lifespan of approximately 10 days </li></ul><ul><li>Lose hemostatic effectiveness </li></ul><ul><ul><li>Mechanism and extent are unknown </li></ul></ul><ul><li>Recognized as Senescent and removed </li></ul><ul><ul><li>Kupffer cells in liver </li></ul></ul><ul><ul><li>Macrophages in the spleen </li></ul></ul>
  19. 19. Platelets are Cleared by Kupffer Cells in the Liver <ul><li>Specialized Macrophages </li></ul><ul><li>Phagocytes that destroy RBCs and Platelets </li></ul><ul><li>???????? </li></ul>
  20. 20. …and in the Spleen <ul><li>Macrophages in the spleen recognize senescent platelets </li></ul><ul><li>Splenectomy increases platelet lifespan (~47%) </li></ul><ul><li>Not reproducible in humans due to trauma associated with emergency splenectomy </li></ul>
  21. 21. In Vivo Biotinylation to Track Platelet Clearance NH 2 Sulfo-NHS-Biotin + +
  22. 22. IVB Procedure Tail vein injection of biotin Collect blood sample (~5ul) at various times beginning 1 hr after injection Stain with streptavidin and Thiazole Orange and analyze by flow cytometry
  23. 23. Typical IVB Data Streptavidin+ quadrant Thiazole orange+, streptavidin + <ul><li>As platelets are cleared, Strept staining decreases </li></ul><ul><li>New platelets are thiazole orange+ </li></ul><ul><li>Very few Strept+ platelets remain after 124 hours </li></ul><ul><ul><li>Strpet staining of MKs </li></ul></ul>Prislovsky et al., Exp Hematol 2008
  24. 24. Platelet Related Disorders
  25. 25. Platelets in Cardiovascular Disease <ul><li>Directly responsible for mortality </li></ul><ul><li>Normal endothelium is the most anti-thrombotic surface </li></ul><ul><li>Atherosclerotic plaque formation </li></ul><ul><li>Creates a thrombotic surface </li></ul><ul><li>Pathological thrombus formation </li></ul>R. Ross, NEJM 1986
  26. 26. Diabetes Mellitus <ul><li>Vast majority of patients die from vascular disease (75%) </li></ul><ul><li>Driven by platelet hyperreactivity </li></ul><ul><ul><li>Increased platelet volume </li></ul></ul><ul><ul><li>Increased granular content </li></ul></ul><ul><ul><li>Increased platelet count </li></ul></ul><ul><ul><li>Increased adhesion </li></ul></ul><ul><li>Increased development of atherosclerotic plaques </li></ul><ul><ul><li>Perhaps due to the hyperreactive platelet? </li></ul></ul><ul><li>Buildup of acute microvascular occlusions prior to a large infarct </li></ul><ul><ul><li>Creates small hypoxic zones prior to a larger bout of ischemia </li></ul></ul>
  27. 27. Platelets and Atherosclerotic Plaque Formation in Diabetics <ul><li>An atherosclerotic plaque </li></ul><ul><ul><li>Dead smooth muscle cells </li></ul></ul><ul><li>Monocytes are recruited by platelets </li></ul><ul><ul><li>Via cytokines </li></ul></ul><ul><li>Differentiate into macrophages </li></ul><ul><ul><li>Extravasate </li></ul></ul><ul><ul><li>Interact with smooth muscle cells </li></ul></ul><ul><li>Cause apoptosis of smooth muscle cells </li></ul><ul><li>Hyperreactive platelets increase probability of developing a plaque </li></ul>Endothelial cells PDGF monocyte
  28. 28. Clinical Approach to Disorders of Platelet Number and Function <ul><li>Immediate bleeding vs Delayed bleeding </li></ul><ul><ul><li>Primary hemostasis or coagulation </li></ul></ul><ul><li>Age of onset </li></ul><ul><ul><li>Early onset suggests inherited disoder </li></ul></ul><ul><ul><li>Late onset suggests acquired disorder </li></ul></ul><ul><li>Gender and family history </li></ul><ul><ul><li>Wiskott-Aldrich syndrome </li></ul></ul><ul><ul><li>X-linked recessive, almost exclusive to males </li></ul></ul><ul><li>Medications </li></ul>
  29. 29. Wiskott-Aldrich Syndrome <ul><li>X-linked recessive </li></ul><ul><li>Characterized by thrombocytopenia, small platelets, eczema, recurrent infection </li></ul><ul><ul><li>10,000-100,000 platelets per uL </li></ul></ul><ul><li>Mild to severe bleeding </li></ul><ul><li>Low levels of reticulated platelets </li></ul><ul><li>May present in the fetus or neonate </li></ul><ul><li>WASP is a signaling molecule which regulates the actin cytoskeleton </li></ul><ul><ul><li>WAS platelets do not aggregate </li></ul></ul>
  30. 30. Immune Thrombocytopenic Purpura <ul><li>Accelerated platelet destruction due to the production of platelet autoantibodies </li></ul><ul><li>1 per 100,000 people </li></ul><ul><li>Patient present with mild to severe hemorrhage </li></ul><ul><ul><li>Otherwise normal CBC </li></ul></ul><ul><li>One readily measurable marker – platelet count </li></ul><ul><li>Viral infection is thought to serve as an initiator </li></ul><ul><li>Best treatment is to raise the platelet count </li></ul><ul><ul><li>IVIG (intravenous immunoglobulins) </li></ul></ul><ul><ul><li>Binds the host antibody rendering it unable to bind its antigen </li></ul></ul>
  31. 31. Bernard-Soulier Syndrome <ul><li>Autosomal recessive </li></ul><ul><li>Thrombocytopenia perhaps due to decreased platelet survival </li></ul><ul><li>Megakaryocyte demarcation membranes are abnormal and fragmented </li></ul><ul><li>Platelets are giant (~20uM) </li></ul><ul><li>Moderate to severe bleeding is associated </li></ul><ul><li>Platelet transfusions are possible </li></ul><ul><li>GP 1b-IX-V complex is affected </li></ul><ul><ul><li>Binds vWF and is necessary for attachment to the endothelium </li></ul></ul>
  32. 32. Glanzmann Thrombasthenia <ul><li>Autosomal recessive </li></ul><ul><li>Moderate to severe bleeding disorder </li></ul><ul><li>Platelet count is normal </li></ul><ul><li>Defect in the integrin α IIb β 3 </li></ul><ul><li>Platelets will not aggregate (will not form a thrombus) </li></ul><ul><li>Platelets will not spread to cover a wound </li></ul><ul><li>Good target for gene therapy </li></ul><ul><li>Main form of treatment is a platelet transfusion </li></ul><ul><ul><li>Problematic due to # of platelets required and immune rejection </li></ul></ul>
  33. 33. Platelet Transfusions <ul><li>Biggest obstacle is getting enough platelets to perform the transfusion </li></ul><ul><li>Platelets can be isolated and stored in anti-coagulants </li></ul><ul><li>If stored for more than 3 days the recipient has a decreased chance for recovery </li></ul><ul><li>Platelet ½ life drop to 3 days </li></ul><ul><li>Very difficult to store platelets </li></ul><ul><ul><li>Unagitated platelets die quickly </li></ul></ul><ul><ul><li>Some platelets activate </li></ul></ul><ul><ul><li>Storage temperature 20 º -24 º C </li></ul></ul>
  34. 34. HIV <ul><li>HIV patients develop chronic immune thrombocytopenic purpura </li></ul><ul><li>Platelet counts can drop to as little as 16,000 per uL </li></ul><ul><ul><li>Due to increased destruction </li></ul></ul><ul><li>Splenectomy is good option for these patients </li></ul><ul><li>AZT increases platelet counts as well </li></ul><ul><li>AIDs patients have more polyploid megakaryocytes </li></ul><ul><ul><li>So no defect in megakaryopoiesis </li></ul></ul><ul><li>However, platelet production is severely inhibited </li></ul><ul><ul><li>Proplatelet production is perturbed </li></ul></ul><ul><ul><li>Not due to HIV infection of the Mks </li></ul></ul>
  35. 35. Cardiopulmonary Bypass <ul><li>2000 per day performed </li></ul><ul><li>“ Bypass” partially occluded arteries with a vein </li></ul><ul><li>Need a thrombocytopenic environment to reduce thrombus formation </li></ul><ul><ul><li>Mild thrombocytopenia occurs upon surgery, mechanism unknow </li></ul></ul><ul><ul><li>Dilute the blood with isotonic solution </li></ul></ul><ul><li>Excessive bleeding post-op </li></ul><ul><ul><li>Up to 1L in 24 hours </li></ul></ul><ul><ul><li>Prolonged bleeding up to 72 hours </li></ul></ul><ul><li>Both create severe thrombocytopenia following surgery </li></ul><ul><li>Heparin-induced thrombocytopenia </li></ul>
  36. 36. Thrombocytopenia in Pregnancy <ul><li>Found in 7% of pregnant women </li></ul><ul><li>Fetus can develop thrombocytopenia which is directly related to the mother </li></ul><ul><li>No way to test the fetal platelet count </li></ul><ul><li>Petechiae develop near mucus membranes </li></ul><ul><li>Increased morbidity to the mother during C-section </li></ul><ul><li>Increased risk to fetus during natural birth </li></ul><ul><li>Generally not treated </li></ul>