Hemostasis Week 3

Thrombopoiesis steps

1. Hematopoietic stem cell

2. Myeloid progenitor

3. mega/erythro progenitol

4. megakaryoblast

5. promegakaryocyte

6. megakaryocyte

7. platelets

Platelets under microscope vs electron microscopy

microscope: purple dots smaller than RBC

electron microscope: resting platelet = flat/circular, activated = spiderlike

GPIIb/IIIa on a resting platelet are activated by what?

Agonist

Fibrinogen can connecting PLTs by which receptor on PLTs' surface?

GP IIbIIIa

PLT morphology:

- peripheral zone refers to ___

PLT surface

PLT morphology:

- Structural zone is _____, it helps maintain ___ shape of the resting PLTs and participate in shape change of activated PLTs

cytoskeleton, discoid

PLT morphology

- membrane system is _____

the tubular system on membrane; canalicular system

PLT morphology

- Organelle zone includes M_____, G______ and G______ (which including three types ___,___, and ____)

mitochondria, glycogen, granules; lysosome, alpha, dense

Receptors on PLT surface, which is the primary one?

- Thrombin is the strongest angonist

- Thrombin > ADP, collagen & epinephrine

PLT FXN disorders

-Acquired

Drugs , storage defect, systemic disorders & hematological disorders

PLT FXN disorders

Example of systemic disorders

Uremic, antiplatelet antibodies, cardiopulmonary bypass, liver disease

PLT FXN disorders

Example of hematological disorders

Myeloproliferative disorders, myelodysplasia, leukemia, dysproteinemias, acquired von Willebrand disease

Inherited platelet function disorders based on the fxns are divided into three types:

adhesion, aggregation, activation

Which defect/disorder is associated with adhesion?

GPIb - vWF defect = Von willebrand dz & Bernard-Soulier Syndrome

Which defect/disorder is associated with aggregation?

GPIIb - IIIa defect = Glanzmann thrombasthenia

Which defect/disorder is associated with activation?

• AA metabolism defect = [cyclo-oxygenase defect ASA-like] ASA= Aspirin

• Granule release defect = [storage pool deficiency & release defect]

• cytoskeleton regulation = [Wiskott-Aldrich syndrome]

• phosphatidylserine expose = [Scott syndrome]

Factors to consider for platelet disorder eval

clinical picture/history, CBC/DIFF for PLT #/size/morph, Platelet function assay, esoteric testing

global screening tests for PLT fxn are

bleeding time and Platelet function assay PFA-100

Antiplatelet therapy monitoring assay

- PLTMAP

- VerifyNow

Esoteric test for PLT fxn

- electron microscopy

- PLT flow cytometry

- genotyping studies

specimen requirement for PLT fxns (hint TEG specimen)

- centrifuge?

- refrigerated, frozen, room temp

- mixing? using tube system to transport to Lab

- time limited?

- whole blood, no centrifuge

- Room temp only

- NO mixing or tubing system

- within 30 mins is the best, < 4hrs

Bleeding time determines _____

- time to form a PLT plug

bleeding time evaluates

PLT fxn & vascular contribution to hemostasis

Bleeding time test involves making a puncture wound in a superficial area of the skin and monitoring the time needed for bleeding to stop. What are some limitations of this test?

NO standardization for direction of incision/bloting off blood, NO precision, NO sensitivity

PFA-100

- Principle

- What active the PLTs?

- detect PLT plug forming time

- the membrane contain Collagen + Epinephrine or ADP

- when plug form, covers the aperture, the blood flow stops

- vWB binds to collagen, and bonded vWB binds to PLTs & PLTs are activated by epinephrine or ADP

PFA-100 curve

flow rate rapidly increases then gradually decreases as closure time extends

Interpreted PFA-100 result

- EPI & ADP both normal

- normal

Interpreted PFA-100 result

EPI abnormal; ADP normal

pt with Aspirin

Interpreted PFA-100 result

EPI, ADP both abnormal

vWD, Glanzmann's thrombasthenia, Bernard Soulier or PLT defects

PFA-100 tests, the abnormal closure time (CT) is related to which type of vWD

- 2A,2B, 2M & 3

which vWD type has normal PFA-100 result? why?

2N, 2N defect is decreased affinity to Factor VIII; it has normal PLT binding affinity

(T/F)Closure time is related to PLT count only

False,

since RBCs help to form the plug, LOW HCT <30%, the CT is prolonged

The PFA-100 result looks like the pic with a seesaw tracing and prolonged CT, what might cause it?

HCT is too low <30%

PLT is too low <100K

why should PFA-100 results be interpreted with caution?

test not diagnostic/sensitive for mild platelet disorders

Specific assays of PLT fxn are _____ studies, includes:

Aggregometry studies

- light transmission LTA

- lumi

- whole blood impedance

PRP vs PPP

PPP - PLT poor plasma, 100% of PLT is in aggregation form > settles to bottom of tube

PRP - PLT rich plasma, 0% of PLT is in aggregation form

- what is measured in LTA test?

- what is report as result in LTA test?

- LTA test measure the clearance of plasma is compared with PPP by optical density

- reported as PLT aggregation percentage %

- eg. pt's result Agonist + PRP compare to PPP(100%) is 90% then we report the pt's LTA test result is 90% PLT aggregation

the LTA tracing, B,D went up due to?

- ______ (increase / decrease) turbidity

- what happened to the PLTs in B & D

- increased turbidity

- B: shape change, D: PLT granules secretion

- What is A-B-C-E

- What is A-B-C-D; what causes ABCD situation happen ?

- initial wave of aggregation followed by 2nd wave of aggregation, irreversible

- For weak agonists [ADP & EPI], secondary wave of aggregation not happen and primary wave disagrregate

Mechanism of ASA in PLT activation

Aspirin (Acetylsalicylic acid or ASA) inhibits PLT COX1 (cyclo-oxygenase), which is important for TXA2 production (Thromboxane A2).

- it's irreversible inhibition

- TXA2 is needed for PLT activation

- the second wave of aggregation can be inhibited by Aspirin

why low dose of Ristocetin 0.5-0.6 mg/mL is used in the test panel?

diagnostic for type 2B vWD

- _____ (weak/strong) will have biphasic tracing for ADP and epinephrine

Weak agonist

- which wave could dissolve with Aspirin present?

the 2nd wave of aggregation

LTA: lack of agglutination with Ristocetin. Possible diganosis?

Von Willebrand Disease or Bernard Soulier syndrome (plt defect)

LTA: agglutination only with Ristocetin. Possible diagnosis?

Glanzmann’s thrombasthenia (GP2P3a receptor defect) or Afibrinogenemia (no fibrinogen for plt adhesion)

LTA: ADP disaggregation tracing

failure of granule release via platelet storage pool disorder/defect;

platelet agglutination = insensitive to platelet storage pool disorder

LUMI-aggregometry principle

After PLT aggregation, ____ is released from dense-granule.

_____ will react with this molecule and give chemiluminescence.

LUMI-aggregometer used to simultaneously measure ______ & _______

- ATP

- firefly luciferin

- PLT aggregation and ATP secretion of dense-granule ATP

WBIA test principle

when PLT agg, neg charged surface attached to electrical sensors and the impedance between the sensor enhances platelet agglutination = area under curve

WBIA test

- result report as _____

- PLT fxn is _______ to the result

- need duplicate test, so getting a ____ is important

- good for _____ therapy monitoring

- AUC, area under curve

- proportional

- baseline = PLT fxn before therapy

- antiplatelet

Whole blood impedance method limitations

• not personalized assay

• need to compare with wide normal range

which method correlated well with clinical antiplatelet drug therapy?

whole blood impedance method

When WBIA result is below referance range…

suggests poor platelet function vai antiplatelet drugs, defects, or thrombocytopenia

Three main tests in platelet transmision electron microscopy (PTEM)

- whole mount : quantitate dense granule #

- thin : visualize ultra-structures such as alpha granules and inclusions

- buffy coat/Particle TEM: for WBC aberrant inclusions

which test is gold standard for assessing platelet structures especially dense & alpha granules

platelet transmission electron microscopy

Gray platelet is due to?

alpha granule deficiency

which granules contains protein molecules + more visible with Wright-Geimsa stain?

alpha

which granules contains nonprotein molecules + high content of calcium?

dense

FC gamma II receptor

- which molecule can activate PLT via this receptor?

- associate with _____ dz

- Heparin & PF4 complex

- HIT, heparin induced thrombocytopenia

What is essential for platelet function?

platelet surface receptors

____ is the test of choice for diagnosing platelet surface receptor deficiencies

platelet flow cytometry

what receptor it is & what dz associate with its deficiency

- GPIIb/IIIa

- fibrinogen; Glanzmann's thrombasthenia

what receptor it is & what dz associate with its deficiency

- GP Ib/IX

- von Willebrand; Bernard - Soulie syndrome

what receptor it is & what dz associate with its deficiency

- GP Ia/IIa/VI

- collagen; abnormal PLT aggregation response to collagen

what receptor it is & what dz associate with its deficiency

platelet CD62P

P-selectin; platelet activation def

____ & ___are used for monitoring PLT fxn & antiPLT drug therapy

TEG & ROTEM

Thromboelastography & Rotational Elastometry

What test/tests used to

- provide global information on the dynamics of clot development, stabilization and dissolution that reflect in vivo hemostasis

TEG/ROTEM

TEG/ROTEM is used to assess the contribution of

fibrinogen-platelet interaction

TEG/ROTEM is a Modified method for _____________ _____________

antiplatelet monitoring

What test/tests used to

- converting mechanical strength b/t fibrinogen-PLT into electrical signal

TEG/ROTEM

TEG/ROTEM is a whole blood based assay where whole blood is added to a heated cuvette at 37C. using the TEG/ROTEM device, movement is initiated and as the blood clots, fibrin strands form btw TEG and ROTEM. In other words,

this assay can convert mechanical strength interactions of fibrin to electrical signals

TEG Parameters

- Reaction Time

- Angle & Kinetics

- Maximum Amplitude

- Lysis 30

- RT: time from test start to initial fibrin formation = clotting time

TEG Parameters

- Reaction Time

- Angle & Kinetics

- Maximum Amplitude

- Lysis 30

- Angle& K: Fibrinogen activity; how fast to form and how strong the fibrin is

TEG Parameters

- Reaction Time

- Angle & Kinetics

- Maximum Amplitude

- Lysis 30

- MA the interaction b/t PLT & fibrin @ a maximum strength, 80% of MA contribute from activated PLTs, 20% from fibrin = MA is mostly reflect the PLTs fxn

TEG Parameters

- Reaction Time

- Angle & Kinetics

- Maximum Amplitude

- Lysis 30

- lysis 30: percent lysis 30mins after MA; not a reliable parameter, instrument cannot differentiate real clot lysis vs artifitial effect ; not sensitive to fibrinolysis

In TEG result, high RT means?

RT is proportional to clotting time of PTT reflect low Factor activity = hypocoagulable

in TEG result, high MA and normal Angle means?

MA: hyperplatelet function = hypercoagulability

Angle: normal fibrinogen

in TEG result, low MA and normal angle means?

MA: low platelet function = hypocoagulable

Angle: normal fibrinogen

in TEG result, low angle means?

low fibrinogen level

in TEG result, low RT and high MA and angle means?

RT: fast clotting time

MA: increased platelet/fibrinogen

Angle: increased fibrinogen level

= platelet and enzymatic hypercoagulability

in TEG, high LY30, normal MA means?

increased clot formation and fibrinolysis > primary fibrinolysis

secondary fibrinolysis based on

increased clot formation and relatively increase fibrinolysis = increased MA + increased fibrinomatic parameters (Angle, kinetics)

T/F: TEG is NOT sensitive to Von Willebrand Disease and antiplatelet therapy. Normal results could be observed.

True

TEG result

- Angel 8.8 NR 53-72

- MA 11.5 NR 50-70

what to expect?

low FIB & PLT (PLT

PLT Mapping assay

- used to monitor?

- agonist used?

- unit of result

- antiplatelet therapy; modified test based on TEG

- Arachidonic Acid (AA) & adenosine diphosphate (ADP)

- % inhibition

VerifyNow

- measure PLT response to ___,___,&____ antiplatelet agents

- method, clotting, antibody, optical

P2Y12 inhibitiors, aspirin, GP IIb/IIIa

- optical measure

What proteins/factors does warfarin decrease the synthesis of?

Factor II, VII, IX, X, Protein C and S

warfarin inhibits ______, which leads to Vitamin K depletion, and further decreasing Vitamin K dependent clotting factor, preventing clot forming

Vitamin K epoxide reductase

What are the functional vitamin K-dependent clotting factors

II, VII, IX, X

Wafarin-induced skin necrosis occurs due to ________

acquired protein C deficiency

Warfarin treatment affects Protein______ & Factor _____ most because they have the shortest half-life time among Vitamin K dependent factor/protein

- Protein C & Factor VII

Wafarin-induced skin necrosis occurs after _____ (how long) after drug therapy or with ______ initial dose

3-5 days after drug therapy OR with High initial dose

CON of using Warfarin

- ______ (Narrow/wide) Therapeutic range

- _____ (slow/fast) onset , offset action

-_____ (few/multiple) interactions

- _____ & ______ are the major interactions as drugs

- ______ may increase warfarin activity

-______ may decrease warfarin activity

- does it need monitor?

- Narrow

- Slow

- 727 drugs; multiple

- Aspirin, Ibuprofen

- cooked onions

- broccoli, kale, spinach (rich in VitK)

- required monitoring

What are the ideal attributes for anticoagulants

Oral administration, rapid onset, wide therapeutic range, predictable therapeutic effect, no food/drug interactions, no monitoring required, defined pharmacokinetics in renal/hepatic disease, reversible, cost effective

WHat is DOACs and the example of it? (D-, R-, A-)

and they inhibits IIa or Xa?

Direct Oral AntiCoagulants

- Dabigatran (IIa) - Pradaxa

- Rivaroxaban (Xa) - Xarelto

- Apixaban(Xa) - ELIQUIS

- Edoxaban (Xa) - Savaysa

mechanism of DOACs vs Warfarin

- directly inhibit either Factor IIa (Thrombin) or Factor Xa

- warfarin lowers the functional lvl of all the VitK dependent clotting factors (2,7,9,10,CS)

For Factor Xa & IIa inhibitors, there are two catogories:

Direct & Indirect

- traditional / new anticoagulants

- reversible / inreversible

- inhibits free factor/ bound factor

- catalytic/ stoichiometric

- ATIII dependent/ independent

Which ones apply to direct factor Xa and IIa inhibitors?

• new anticoagulants

• specific and reversible inhibition of a single factor

• inhibits bounded and free

• stoichiometric

For Factor Xa & IIa inhibitors, there are two catogories:

Direct & Indirect

- traditional / new anticoagulants

- reversible / inreversible

- inhibits free factor/ bound factor

- catalytic/ stoichiometric

- ATIII dependent/ independent

Which ones apply to indirect factor Xa and IIa inhibitors?

• traditional anticoagulants except Warfarin

• irreversible ATIII-mediated inhibition of factor IIa or Xa

• inhibits only free factor

• Catalytics = accelerate the binding of anti-thrombin with Factor IIa or Xa

Examples of Indirect ATIII-dependent factor Xa inhibitors

• Unfractionated Heparin (usually we call Heparin)

• LMWHs including: Enoxa-, Dalte-, Tinza- + -parin

• Pentasaccharides: Fondaparinux

Examples of direct factor Xa inhibitors

• Apixaban(Eliquis)

• Rivaroxaban (Xarelto)

• DU176b Edoxaban (Savaysa)

Examples of Vitamin K antagonists for factor Xa

Warfarin depletes factor Xa, no inhibit