Hematopoiesis II

JC Kostyak

10-22-09

Outline

• MK migration -- review

• Mk docking

• MK platelet production

• Platelet lifespan

• Platelet clearance

• Thrombocytopenias

Megakaryocytes Must Migrate When Ready to Produce Platelets

SDF-1

CXCR4

Stromal Cells

Megakaryocytes Require PECAM-1 for Polarity

• Last time – MKs migrate toward an SDF-1 gradient

• PECAM-1 regulated migration via polarization• PECAM-1-/- mice show a lack of cell polarity

Dhanjal et al., Blood 2007

Migration Through the Basement Membrane

Bone Sinusoidal VesselEndothelial

Cells

MMP

Megakaryocyte Docking

?

?

PECAM-1 ?MK

MK

Once the Mk has docked, it can begin to produce proplatelet processes

Is PECAM-1 Required for MK Docking?

Dhanjal et al., Blood 2007

Once Docked MKs Produce Proplatelets

Patel et al., JCI 2005

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

MT Review

Conde and Cesares, Nature 2009

Microtubule Sliding

www.Stanford.edu

Mitotic Kinesin

Small molecular motors use ATP

Kinesins and Dynein

V

Kinesin

Retrograde

MTOC

Anterograde

+

-

+

-

Retro

grade

V

Dynein

There are many examples of each!

Proplatelet Formation Originates

Opposite the Nucleus

Italiano et al., JCB 1999

The Theatrical Version

Kinesins Localize in the Cytosol while Dynein is Localized to the Proplatelets

Patel et al., Blood 2005

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

New “Reticulated” Platelets

• Platelets are anucleate• Contain some mRNA• Contain all the machinery

necessary for protein synthesis

• We can use the mRNA to track new platelets (Thiazole Orange)

• Normal = 14-18%

The Aging Platelet

• Lifespan of approximately 10 days

• Lose hemostatic effectiveness– Mechanism and extent are unknown

• Recognized as Senescent and removed– Kupffer cells in liver– Macrophages in the spleen

Platelets are Cleared by Kupffer Cells in the Liver

• Specialized Macrophages• Phagocytes that destroy RBCs and Platelets• ????????

…and in the Spleen

• Macrophages in the spleen recognize senescent platelets

• Splenectomy increases platelet lifespan (~47%)

• Not reproducible in humans due to trauma associated with emergency splenectomy

In Vivo Biotinylation to Track Platelet Clearance

NH2

Sulfo-NHS-Biotin

+ +

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

Typical IVB Data

Streptavidin+ quadrant

Thiazole orange+,

streptavidin +

•As platelets are cleared, Strept staining decreases

•New platelets are thiazole orange+

•Very few Strept+ platelets remain after 124 hours

•Strpet staining of MKs

Prislovsky et al., Exp Hematol 2008

Platelet Related Disorders

Platelets in Cardiovascular Disease

• Directly responsible for mortality

• Normal endothelium is the most anti-thrombotic surface

• Atherosclerotic plaque formation

• Creates a thrombotic surface

• Pathological thrombus formation R. Ross, NEJM 1986

Diabetes Mellitus

• Vast majority of patients die from vascular disease (75%)

• Driven by platelet hyperreactivity– Increased platelet volume– Increased granular content– Increased platelet count– Increased adhesion

• Increased development of atherosclerotic plaques– Perhaps due to the hyperreactive platelet?

• Buildup of acute microvascular occlusions prior to a large infarct– Creates small hypoxic zones prior to a larger bout of

ischemia

Platelets and Atherosclerotic Plaque Formation in Diabetics

• An atherosclerotic plaque– Dead smooth muscle cells

• Monocytes are recruited by platelets– Via cytokines

• Differentiate into macrophages– Extravasate– Interact with smooth muscle

cells

• Cause apoptosis of smooth muscle cells

• Hyperreactive platelets increase probability of developing a plaque

Endothelial cells

PDGF

monocyte

Clinical Approach to Disorders of Platelet Number and Function

• Immediate bleeding vs Delayed bleeding– Primary hemostasis or coagulation

• Age of onset– Early onset suggests inherited disoder– Late onset suggests acquired disorder

• Gender and family history– Wiskott-Aldrich syndrome– X-linked recessive, almost exclusive to males

• Medications

Wiskott-Aldrich Syndrome

• X-linked recessive• Characterized by thrombocytopenia, small

platelets, eczema, recurrent infection– 10,000-100,000 platelets per uL

• Mild to severe bleeding• Low levels of reticulated platelets• May present in the fetus or neonate• WASP is a signaling molecule which regulates

the actin cytoskeleton– WAS platelets do not aggregate

Immune Thrombocytopenic Purpura

• Accelerated platelet destruction due to the production of platelet autoantibodies

• 1 per 100,000 people• Patient present with mild to severe hemorrhage

– Otherwise normal CBC

• One readily measurable marker – platelet count• Viral infection is thought to serve as an initiator• Best treatment is to raise the platelet count

– IVIG (intravenous immunoglobulins)– Binds the host antibody rendering it unable to bind its

antigen

Bernard-Soulier Syndrome

• Autosomal recessive• Thrombocytopenia perhaps due to decreased

platelet survival• Megakaryocyte demarcation membranes are

abnormal and fragmented• Platelets are giant (~20uM)• Moderate to severe bleeding is associated• Platelet transfusions are possible• GP 1b-IX-V complex is affected

– Binds vWF and is necessary for attachment to the endothelium

Glanzmann Thrombasthenia

• Autosomal recessive• Moderate to severe bleeding disorder• Platelet count is normal• Defect in the integrin αIIbβ3

• Platelets will not aggregate (will not form a thrombus)

• Platelets will not spread to cover a wound• Good target for gene therapy• Main form of treatment is a platelet transfusion

– Problematic due to # of platelets required and immune rejection

Platelet Transfusions

• Biggest obstacle is getting enough platelets to perform the transfusion

• Platelets can be isolated and stored in anti-coagulants

• If stored for more than 3 days the recipient has a decreased chance for recovery

• Platelet ½ life drop to 3 days• Very difficult to store platelets

– Unagitated platelets die quickly– Some platelets activate– Storage temperature 20º-24ºC

HIV

• HIV patients develop chronic immune thrombocytopenic purpura

• Platelet counts can drop to as little as 16,000 per uL– Due to increased destruction

• Splenectomy is good option for these patients• AZT increases platelet counts as well• AIDs patients have more polyploid megakaryocytes

– So no defect in megakaryopoiesis

• However, platelet production is severely inhibited– Proplatelet production is perturbed– Not due to HIV infection of the Mks

Cardiopulmonary Bypass

• 2000 per day performed• “Bypass” partially occluded arteries with a vein• Need a thrombocytopenic environment to reduce

thrombus formation– Mild thrombocytopenia occurs upon surgery,

mechanism unknow– Dilute the blood with isotonic solution

• Excessive bleeding post-op– Up to 1L in 24 hours– Prolonged bleeding up to 72 hours

• Both create severe thrombocytopenia following surgery

• Heparin-induced thrombocytopenia

Thrombocytopenia in Pregnancy

• Found in 7% of pregnant women

• Fetus can develop thrombocytopenia which is directly related to the mother

• No way to test the fetal platelet count

• Petechiae develop near mucus membranes

• Increased morbidity to the mother during C-section

• Increased risk to fetus during natural birth

• Generally not treated