A. Normal Physiology
[Figure] "Blood Cell Development"

1. Also called thrombocytes
2. Platelets are non-nucleated cells with adhesive properties and granules
3. Derived from mesenchymal bone marrow precursors called megakaryocytes
   1. Megakaryocytes are derived from pluripotent bone marrow stem cells
   2. Megakaryocytes sit next to sinusoids in bone marrow
   3. Platelets are derived from break-off of megakaryocyte cytoplasm
   4. Thus, platelets are pieces of cytoplasm
4. Development [6]
   1. Megakaryocyte development is mainly controlled by thrombopoietin (TPO)
   2. TPO is a ligand for the c-mpl surface receptor on megakaryocytes
   3. Interleukins 6 and 11 are also important for development of megakaryocytes
   4. Stem cell factor (SCF, ancestim) stimulates all lines including platelets [3]
   5. TPO levels are high or inappropriately normal in reactive thrombocytosis
   6. TPO levels are also elevated or inappropriately high in essential (clonal) thrombocythemia
   7. c-Mpl abnormalities in essential thrombocythemia leads to reduced binding of TPO
   8. TPO not yet FDA approved to accelerate platelet recovery after chemotherapy [6]
5. Platelet Surface Molecules
   1. The glycoprotein (GP) receptors mediate adhesion to subendothelial tissue
   2. GP Ib/V/IX is constutively active receptor for von Willebrand Factor (vWF)
   3. GP IIb/IIIa binds to fibrinogen; 50-80,000 copies on surface
   4. GP Ia/IIa is an active receptor for collagen (along with GP VI)
   5. GP IIb/IIIa deficiency is called Glanzmann's thrombasthenia with sporadic bleeding
6. Other Platelet Receptors [4]
   1. Important for activation of platelets
   2. Low Affinity Purinergic Receptor (Type 2) - binds ADP (adenosine diphosphate)
   3. High Affinity Purinergic Receptor (P2Y1) - binds ADP
   4. Novel P2Y12 (ADP) receptor which inhibits adenylyl cyclase (reduces cAMP levels)
   5. Thromboxane A2 Receptor (TBX A2)
   6. Serotonin Receptor
   7. Epinephrine Receptor
   8. Thrombin Receptor
   9. Collagen Receptor
7. Platelet Activation
   1. Platelets normally circulate for ~10 days in an inactive, disc form
   2. On activation, they change shape to a compact spherical form with long extensions
   3. Platelet cytoplasm is rich in actin and myosin which bring about shape changes
   4. Clot "retracts" using the same actin-myosin motors
   5. Granule contents may be released on activation of platelets
   6. Granules contents stimulate normal recruitment and activation of additional platelets
8. Two Types of Platelet Granules
   1. Dense granules contain adenosine diphosphate (ADP) and calcium, other small molecules
   2. These other molecules include serotonin, thromboxane A2, prostacyclin
   3. These factors reinforce platelet activation and platelet-surface coagulation reactions
   4. Alpha granules secrete many proteins including platelet factor IV, vWF, fibrinogen, ' Clotting Factor V

B. Role in Clotting [5]

1. When blood vessels are traumatized, the endothelial lining is disrupted
   1. Very high shear conditions, especially in microcirculation cause vascular damage
   2. Vascular damage leads to endothelial cell breakdown and exposure of subendothelium
   3. Collagen is a major constituent of subendothelial region
2. Platelets adhere well to Subendothelial Components
   1. These include Collagen types I, III (interstitial) and IV (basement membrane)
   2. Platelets localize to the plug within seconds, narrowing the stream of leaking blood
   3. A series of integrins on the platelet membrane mediate adhesion and aggregation
   4. Glycoprotein IIb/IIIa (GP IIb/IIIa) is last step in binding to a variety of proteins
   5. GP IIb/IIIa binds to fibrinogen (and other proteins) and localizes clot to damaged area
   6. GP IIb/IIIa also binds to vitronectin and von Willebrand factor (vWF)
   7. Antibodies to GP IIb/IIIa are extremely potent inhibitors of platelet actions
   8. RGD (arginine-glycine-aspartate) peptides block platelet binding to fibronectin
3. Enhancement of Platelet Aggregation
   1. Thromboxane A2 (also potent vasoconstrictor)
   2. Adenosine Diphosphate (ADP)
   3. Thrombin (and thrombin bound to thrombomodulin)
   4. Serotonin
   5. Epinephrine
   6. Platelet activating factor (PAF)
   7. Vasopressin
   8. Exposed collagen (wounds, ruptured atheromata)
   9. Soluble CD40 ligand (CD154) activates platelets and is elevated in acute thrombosis [7]
4. Platelet-Subendothelial Interaction [8]
   1. von Willebrand Factor (vWF) is required for platelet aggregation
   2. vWF binds to platelet GP Ib/IX receptor and this complex binds exposed collagen
   3. Bound platelets than release granule contents including ADP and other contents (above)
   4. Platelet granule content deposition and shape changes strongly activated by thrombin
   5. ADP binding to platelets activates GP IIb/IIIa receptor
   6. GP IIb/IIIa (an integrin) binds to fibrinogen and other proteins
5. A lipid surface is required for rapid coagulation, and platelets provide this function
   1. In platelet poor plasma, coagulation times are on the order of 2-4 minutes
   2. In the presence of sufficient platelets, coagulation times are ~1 minute
6. Within 1 minute of vessel damage, in the presence of platelets, fibrin strands form
   1. Fibrinogen is bound by platelet GP IIb/IIIa
   2. The platelet plug remains intact for a few hours
   3. It is eventually completely replaced by a complete fibrin clot

C. Diseases Associated With Platelets

1. Thrombocytopenia
   1. Too few platelets
   2. Platelets which are present usually have good activity
2. Thrombocythemia
   1. Too many platelets
   2. Platelets which are present usually have poor clotting activity
   3. One of the myeloproliferative diseases
3. von Willebrand Disease - platelets do not clot properly
4. Megakarocytic Leukemia (M7)
5. Dysfunctional Platelets
   1. Drug induced - aspirin, clopidogrel, ticlopidine
   2. Chronic renal failure (uremia) - increased bleeding times, normal platelet numbers
   3. Cardiac and other bypass surgery

D. Platelet Transfusions [9]

1. Storage at room temperature with stirring is required to prevent aggregation
2. Manipulation of Platelets
   1. WBC depletion - irradiation (UVB treatment), filtration (high losses)
   2. Goal is to reduce alloimmunization which is caused by contaminating donor lymphocytes
   3. HLA Matching
   4. Overall alloimmunization risk is 3-5% with leukocyte depleted platelets [10]
3. Unit definition: ~4x10exp(11) platelets per 6 random units or 1 pheresis unit
4. Very common febrile / rigor reaction to platelets
5. Indications
   1. <5K/µL - should be given
   2. 5-30K/µL - given for prophylaxis or for active bleeding
   3. Chemotherapy prophylaxis - given for platelet counts <10K/µL (as safe as <20K/µL) [11]
   4. Major surgery prophylaxis - given for platelet counts <50K/µL
   5. Enhanced platelet destruction - given at 50K/µL with microvascular bleeding
   6. Contraindicated in TTP generally
   7. Platelet dysfunction - aspirin effects, uremia, vWF deficiency, others
   8. For platelet dysfunction, consider giving desmopressin (DDAVP; see below)
6. TPO may reduce the need for platelets but is not yet FDA approved [6]

E. Platelet Blocking Agents

1. Types of Platelet Blocking Agents
   1. Cyclooxygenase Inhibitors - aspirin, others
   2. Inhibitors of Platelet P2Y12 Receptors - ticlopidine, clopidogrel [12]
   3. Inhibitors of Platelet Adhesion - glycoprotein IIb/IIIa blockers
   4. Direct thrombin inhibitors - antithrombin, argatroban
   5. Specific Thromboxane Inhibitors - in development
   6. Unclear Mechanism - dipyridamole (Persantine®)
   7. These agents tend to be more useful for arterial than for venous thromboembolic disease
2. Other clotting inhibitors
   1. Heparin - enhances antithrombin efficacy
   2. Warfarin - inhibits vitamin K dependent clotting factors
   3. Prostacyclin - blocks platelet aggregation, counteracts TBX A2
   4. Nitric oxide - mediated via increases in cGMP levels
   5. TPA / streptokinase - activates fibrinolysis cascade

F. Assessment of Platelets and their Function

1. Peripheral smear - number, size of platelets
2. Bone Marrow Biopsy (Aspiration) - number and appearance of megakarycytes
3. Bleeding Time (Whole Blood Clotting Time)
   1. Mainly measures platelet function (clot formation); normal 3-7 minutes
   2. Useful for platelet counts >100K/µL
   3. Normal bleeding time (platelets 10K-100K/µL) = 30.5-(platelet count per µL / 3850)
   4. Bleeding Time may be affected by fibrinogen levels, vWF levels, other anti-coagulants
   5. Prolonged Bleeding Time: Uremia, Dysglobulinemia, Liver Disease
4. HemoSTATUS® [13]
   1. Measures effects of platelet activating factors on kaolin-activated clotting time
   2. Clot ratios correlate with peri- and postoperative blood loss
   3. Also detects improvement in platelet function after desmospressin or transfusions
5. Von Willebrand Factor Levels

References

1. Schafer AI. 2004. NEJM. 350(12):1211
2. George JN. 2000. Lancet. 355(9214):1531
3. Blaise D, Faucher C, Vey N, et al. 2000. Lancet. 356(9138):1325
4. Solet DJ, Zacharski LR, Plehn JF. 2001. Am J Med. 111(1):45
5. Vorchheimer DA, Badimon JJ, Fuster V. 1999. JAMA. 281(15):1407
6. Kaushansky K. 2006. NEJM. 354(19):2034
7. Heeschen C, Dimmeler S, Hamm CW, et al. 2003. NEJM. 348(12):1104
8. Sharis PJ, Cannon CP, Loscalzo J. 1998. Ann Intern Med. 129(5):394
9. College of American Pathologists. 1994. JAMA. 271(10):777
10. Trial to Reduce Alloimmunization to Platelets Study Group. 1997. NEJM. 337(26):1861
11. Rebulla P, Finazzi G, Marangoni F, et al. 1997. NEJM. 337(26):1870
12. Sharis PJ, Cannon CP, Loscalzo J. 1998. Ann Intern Med. 129(5):394
13. Despotis GJ, Levine V, Saleem R, et al. 1999. Lancet. 354(9173):106