Platelet function and defects

Question 1

Platelet granules

Answer 1

• Dense bodies (smaller, fewer): ADP, ATP, serotonin, Ca, histamine
• Alpha granules (larger, more): adhesive proteins (fibrinogen, vWF, P-selectin, ect), coagulation factors (V, XI, fibrinogen, PAI1), growth/angiogenesis factors (PDGF, TGF-b, PF4)
• Lysosomal granules
• Granules secreted into canalicular system: contiguous w/ plasma membrane, increases the platelets surface area when activated

Question 2

Platelet receptors

Answer 2

• GP=glycoprotein
• GPIb/IX binds vWF
• GPIIb/IIIa binds fibrinogen and vWF
• GPIa/IIa binds collagen
• GP VI binds collagen
• ADP receptors
• PARs bind thrombin
• Thromboxane A2 receptors
• Integrins bind fibrinogen

Question 3

Arachidonic acid metabolism

Answer 3

• Platelets utilize phospholipiases to cleave off arachindonic acid from their membranes, then turn it into TxA2 via COX pathway
• TxA2 is platelet activator and vasoconstrictor
• Endothelial cells use the same mechanism to make arachidonic acid but then send it through the prostacyclin synthase pathway to make prostacyclin
• PCL is a vasodilator

Question 4

Platelet procoagulant factors

Answer 4

• “Factor III”: translocation of phosphatidyl serine (PS) from inner to outer membrane
• Fibrinogen
• vWF
• Factor V
• Factor XIII
• P-selectin for microparticle fusion

Question 5

Platelet formation

Answer 5

• Anucleate fragments of megakaryocytes, diameter of 3 microns, volume of 7fL
• Due to increase in megakaryocytic size and number
• TPO is most important regulator of megakaryocytic development and proliferation
• TPO is mostly synthesized in liver and secreted at fairly constant rate
• At some point a megakaryocytic stops dividing and undergoes endomitosis (repeated cycles of DNA replication w/o cell division) until it is up to 64N
• Then the cell extends proplatelets (pseudopodia) into sinuses in BM and platelets are broken off into the blood (each proplatelet makes 1-2k platelets)

Question 6

TPO

Answer 6

• Thrombopoietin binds to MPL receptor on platelets and megakaryocytes
• Levels vary according to platelet and megakaryocytic mass
• TPO production in hepatocytes can be increased by IL6
• # of platelets depends on the size of the meg, which depends on the ploidy of the meg

Question 7

Thrombocytopenia

Answer 7

• Usually either secondary to aplastic anemia (decreased production) or increased destruction (autoAbs to platelets)
• Can also be from other causes (ineffective production, sequestering in spleen, dilution)
• In aplastic anemia there is a reduced # of megs, thus a higher level of TPO since there are fewer receptors binding TPO
• In ITP (idiopathic thrombocytopenic purpura, an autoimmune-mediated destruction of platelets), the Abs are mostly targeting the platelets. Thus the megs are still able to bind the TPO
• There will be an increase in TPO production due to low platelet levels, and an increase in megs in response to the TPO. But overall the excess TPO will be bound by the excess megs and the TPO level will look normal (possibly slightly low)

Question 8

Increasing platelet production

Answer 8

• There is an increase in platelet production in ITP even though the platelet levels are low (due to Ab-mediated destruction)
• The higher TPO levels lead to a larger # of megs, an increase in the ploidy of megs, and thus an increase in meg size & volume
• It also leads to a decreased maturation time of the megs
• The platelets released are larger, younger, and far more effective than the older platelets

Question 9

Platelet sequestering

Answer 9

• Platelet lifespan is 10 days
• 30% of platelets are in the red pulp of the splenic cords (only recover 70% of platelets in normal people)
• In splenectomized pts you can recover 100% of platelets

Question 10

Evaluation of thrombocytopenia

Answer 10

• Can be due to decreased production: small platelets, lower # of megs, usually BM problem
• Can be ineffective production: usually associated w/ megaloblastic anemia
• Can be increased destruction: big platelets, increased # of megs, can be immune or non-immune mediated
• Can be redistribution: enlarged spleen due to portal HTN, storage disease, or BM disorder
• Can be dilution: due to many blood transfusions
• Lower limit of platelets is 150K (upper limit: 400K)

Question 11

Pseudothrombocytopenia

Answer 11

• Natural Ab in blood binds to platelets at room temp and the platelets aggregate
• Artifact of the blood drawing process, to Dx must do blood smear and look at the thin edges

Question 12

Platelet size

Answer 12

• Normal MPV is 7-9fL
• With increased consumption the MPV goes up
• With decreased production the MPV goes down

Question 13

Cytokines on megakaryocyte development

Answer 13

• Endomitosis stimulated by TPO, IL3, IL6, IL11
• The cytokines are released during inflammation, thus chronic illness can lead to elevated platelet levels
• During maturation megs begin to express platelet-specific receptors and granules, also platelet mitochondria, membranes, and lysosomes

Question 14

Sources of fibrinogen and vWF

Answer 14

• Fibrinogen: majority made in liver (some from platelets)

- vWF: majority made in endothelium (some from platelets)

Question 15

Platelet adhesion

Answer 15

• Endothelial injury leads to release of vWF, which adheres to sub endothelial collagen
• The vWF binds to platelets’ GPIb/IX receptor, causing the platelet to “roll”
• This slowing of the platelet leads to stable binding of the subendothelial collagen to the GPVI receptor
• High shear in arteries cause conformational change in vWF to enhance its binding to GPIb/IX (these interactions do not require energy)
• Once GPVI binds to collagen it induces the expression of GPIa/IIa, which also binds to collagen and stabilizes platelet adhesion

Question 16

vWF and adhesion

Answer 16

• Synthesized mostly in endothelial cells
• Stored in weibel-palade bodies (in endothelium and platelets) as large, highly adhesive multimers (long, “sticky” form)
• Constitutively secreted, but is cleaved in circulation by ADAMTS13 to smaller less active form
• Has several binding domains: GPIb/IX, collagen, VIII (prevents rapid clearance of VIII), GPIIb/IIIa, ADAMTS13, heparins
• In vWD factor VIII will be low, leading to a prolonged aPTT

Question 17

Platelet activation inducers

Answer 17

• Collagen binding to GPVI (adhesion)
• vWF binding to GPIb/IX (adhesion)
• Thrombin (activated by developing clot)
• ADP (RBCs, damaged tissue, activated platelets)
• TxA2
• Epinephrine

Question 18

Platelet activation suppressors

Answer 18

• Endothelial NO
• Endothelial prostacyclin
• CD39 (breaks down ADP)

Question 19

Result of platelet activation

Answer 19

• Causes ADP release
• Platelet shape change (extends canalicular system in-folds outwards to create pseudopodia)
• Expression of GPIIb/IIIa (leads to aggregation)
• Expression of P-selectin
• TxA2 production and release
• Inhibition of cAMP (platelet antagonist)

Question 20

Platelet aggregation

Answer 20

• Activated platelets recruit and activate other platelets, gradually accumulating and aggregating to form the plug
• The expression of GPIIb/IIIa binds fibrinogen (in veins) and vWF (in arteries) and cross links platelets
• Aggregation serves as a surface for thrombin generation (surface receptors for VIIIa, expression of PS, fusion of microvesicles)
• Expression of PS from inner to outer membrane enabled by scramblase nz (ATP and Ca dependent), only happens when platelets are activated

Question 21

Platelet adhesion defects

Answer 21

• Defective vWF (function, amount)
• Defective GPIb/IX (absent, or enhanced binding and clearance of vWF-platelet type vWD)
• Defective collagen or GPVI

Question 22

Lab evaluation for platelet adhesion defects

Answer 22

• Bleeding time, PFA100
• Ristocetin cofactor assay (RCA): induces conformational change in vWF to enhance its binding to GPIb/IX
• If RCA is abnormal there is a problem with vWF (either mutated or too low)
• Collagen binding assay: measures interaction btwn collagen and vWF

Question 23

vW disease (vWD)

Answer 23

• Autosomal dominant disorders of mucocutaneous bleeding, menorrhagia (primary hemostatic defect)
• Dx: vWF:Ag levels increased by infection and pregnancy or estrogen, type O blood has lower vWF:Ag levels
• Can have different types (1, 2, or 3)

Question 24

Type 1 vWD

Answer 24

• Partial quantitative deficiency of vWF due to decreased production
• Most common

Question 25

Type 3 vWD

Answer 25

- Complete quantitative deficiency of vWF due to no production - Clinically looks very similar to hemophilia A by lab tests, can distinguish by location of bleeding (vWD 3 bleeding from mucocutaneous areas)

Question 26

Type 2 vWD

Answer 26

- All are qualitative variants of vWF - Type 2A: absence of high molecular weight vWF multimers (only makes short chains) - Type 2B: same as 2A but with increased affinity for platelet GPIb/IX receptor (leads to thrombocytopenia) - Type 2M: abnormal function of vWF not caused by absence of HMW multimers - Type 2N: qualitative variant w/ markedly decreased affinity for factor VIII (looks hemophilia A)

Question 27

Lab evaluation of vWD

Answer 27

- vWF:Ag measures the amount of circulating low to high MW multimers (very low levels associated w/ bleeding) - Ristocetin cofactor assay (RCA): functional assessment of vWF binding to GPIb/IX to form platelet plug (doesn't use patients plts). Looks at pts' vWF, not GPIb/IX (normal plts) - If does not appear normal it means that there is a defect in vWF or vWF is too low - vWF multimer assay: HMW multimers are most potent, decreasing or absent HMW multimers can cause bleeding - Factor VIII level: will be decreased in vWD (VIII relies on vWF to not be degraded) - Ristocetin-induced platelet aggregation (RIPA): assesses sensitivity of platelet GPIb/IX-vWF binding (uses patient's plasma and platelets, looks at both vWF and GPIb/IX). Used to Dx type 1 vWD (low, occ. N), type 2 vWD (low in all except 2B where it is high), absent in type 3 vWD, Bernard soulier (GPIb/IX defect) - PFA100 (or bleeding time): will be prolonged in vWD but not in hemophilia A (a way to distinguish the two)

Question 28

vWD Rx

Answer 28

- DDAVP (desmopressin): stimulates endothelial release of vWF - Is a natural antidiuretic (causes you to retain water) and can cause hyponatremia - This can be worsened by vomiting, IV fluids - Cryoprecipitate: VIII, vWD, fibrinogen, XIII - Purified vWF:VIII - Antifibrinolytic agents may help control mucosal bleeding

Question 29

Bernard soulier syndrome (adhesion defect)

Answer 29

- Autosomal recessive deficiency in GPIb/IX - Causes mild-moderate thrombocytopenia, large platelets - RIPA will be low - Cannot correct the RIPA w/ addition of normal plasma (unlike in vWD)

Question 30

Inherited platelet activation defects

Answer 30

- Storage pool defects: alpha or dense granule deficiency - Deficiency of alpha granules: gray platelet syndrome (lifelong mucocutaneous bleeding) - Proteolysis of alpha granules due to excess platelet plasminogen activator: quabec platelet disorder (delayed mucocutaneous bleeding) - Platelet receptor disorders: ADP, collagen, TxA2, epinephrine - Defects in COX or arachidonic acid (can't make TxA2) - Defects in intracellular signaling

Question 31

Glanzmann's thrombasthenia (aggregation defect)

Answer 31

- Autosomal recessive defect in platelet GPIIb/IIIa - Moderate-severe mucocutaneous bleeding - Normal platelet count, often associated w/ Fe deficiency anemia - Have prolonged bleeding time/PFA100 (primary hemostatic defect) - Absence of aggregation to any agonist except ristocetin - Confirm Dx w/ flow cytometry w/ Abs to GPIIb/IIIa

Question 32

Platelet aggregometry

Answer 32

- Measure of platelets ability to precipitate out of solution ("aggregate") - In normal blood adding ADP, epinephrine, collagen, arachidonic acid, or ristocetin will all cause the platelets to aggregate - In bernard soulier syndrome, everything is normal except ristocetin does not induce aggregation (no GPIb/IX receptor for vWF to bind to) - In storage pool defects there is reduced response to everything except ristocetin (lower levels of receptors) - In glanzmann's there is no GPIIb/IIIa receptor so there cannot be activation-induced aggregation, however ristocetin still induces aggregation through GPIb/IX and vWF binding

Question 33

Glanzmann's thrombasthenia pathogenetics

Answer 33

- Several genetic mutations involving GPIIb/IIIa (40% are compound heterozygotes) - 3 different phenotypes based on the abundance of GPIIb/IIIa (>20% of normal receptor abundance means there is something wrong w/ the receptor) - Excessive bleeding variable, even w/in same genotype - Moderate to severe spontaneous mucocutaneous bleeding can occur - Menorrhagia common, can have severe bleeding after trauma or postpartum

Question 34

Glanzmann's thrombasthenia Rx

Answer 34

- Hormonal Rx in women - Platelet transfusion after trauma or before surgery - Fibrinolytic inhibitors - Recombinant VIIa

Question 35

Acquired platelet function defects

Answer 35

- Medications: aspirin irreversibly inhibits COX, preventing formation of TxA2 (prevents platelet activation/aggregation) - Other medications either also prevent platelet activation, or they are a GPIIb/IIIa antagonist and they prevent aggregation - Renal failure (uremia inhibits platelet function) - Paraproteinemia: IgM interfere w/ platelet interactions w/ fibrinogen/vWF - Cardiopulmonary bypass: platelets activated in pump, dysfunctional thereafter - Acquired vWD of aortic valve disease: consumption of HMW multimers by platelet activation (pulls the HMW multimers out of solution)