Harvey Chapter 7 Evaluation of Hemostasis PART I: Platelet Disorders (with Schalm's) AKG

Question 1

Where is vWF stored?

Answer 1

Endothelial Weibel-Palade bodies (only trace amounts in platelets)

Question 2

vWF circulates in multimers of various sizes bound to ____.

Answer 2

FVIII (preserves half life of FVIII) - hence ‘factor VIII related antigen’

Question 3

vWF binds to subendothelial collagen and undergoes a conformational change to be able to bind to _____ on platelets.

Answer 3

GP1b

Question 4

____ multimers are most effective for wWF adhesion to platelets.

Answer 4

HMW

Question 5

This molecule breaks down multimers of vWF into smaller ones.

Answer 5

ADAMST13

Question 6

In type __ vWD, there is a partial quantitative deficiency (<50%) of wWF, but the remaining wWF remains in all multimer sizes with normal structure and function.

Answer 6

Type I

Question 7

Which vWD has a functional loss in vWF (loss of large/HMW vWF multimers)?

Which assay is used to detect functional loss?

Answer 7

Type 2A

Functional assay: collagen binding assay (vWF needs to be functional in order to bind to collagen)

Question 8

Which type of vWD results in complete vWF deficiency (<0.1%)?

Answer 8

Type 3

Question 9

Which vWD subtypes are recessive vs dominant inheritance?

Answer 9

Type 1 – dominant

Type 2A and Type 3 – recessive

Question 10

Type 1 vWD leads to _____ bleeding, while type 2A and type 3 lead to _____ bleeding.

Answer 10

Type 1 - mild to severe

Type 2A and 3 - severe

Question 11

Type 1 and 2A vWD will have _____ VWF:Ag; type 3 vWD will have ____ VWF:Ag.

How do you differentiate type 1 from 2A?

Answer 11

Low

Absent

Collagen binding assay (VWF:CB) - need HMW multimers of vWF to bind to collagen. HMW multimers are present in type 1, but not in type 2A.

You can also use protein electrophoresis and western blots to look at vWF multimers - but these tests are mainly done for research, and not common clinically.

Question 12

____ and ___ are the two best quantitative assays for measuring VWF antigen (VWF:Ag)

Answer 12

ELISA and latex immunoassay

Question 13

What does it mean to have 120% of vWF antigen? Is this clinically significant?

Answer 13

Platelets have 20% mroe than the average pool. Not clinically significant.

Question 14

What are the three categories of primary hemostasis disorders?

Answer 14

1. Thrombocytopenia
2. Platelet function (issue with platelet aggregation)
3. vW disease

Question 15

What are two POC assays that serve as screening tests for vWD if you suspect bleeding disorder and platelet count is normal and coag profile is normal?

Answer 15

BMBT

Platelet function analyzer (PFA-100)

Question 16

PFA-100 measures platelet plug formation in whole blood under _____. The endpoint (closure time) is the occlusion of an aperture coated with _____ (mimics exposure of subendothelium when endothelium gets injured) and platelet clumping agonist (____ or _____).

Answer 16

High shear flow

collagen

ADP or epinephrine

Question 17

PFA-100 will be prolonged by which subtypes of vWD?

Is this test specific?

Answer 17

all vWD types

this test is nonspecific, and will be affected by thrombocytopenia and anemia

Question 18

What is the difference between an intrinsic and extrinsic platelet disorder?

Why is it important to classify between the two?

Answer 18

An intrinsic platelet disorder is a defect in platelets causing platelet dysfunction or thrombocytopenia.

An extrinsic platelet disorder is a defect in serum or subendothelial collagen affecting platelet adhesion (e.g., vWD, hypo/dysfibrinogenemia).

You will see petechia and ecchymotic hemorrhage and prolonged mucosal bleeding times and normal coagulation profile with both.

Transufsion products for intrinsic vs extrinscic disorders differ.

Question 19

Inherited macrothrombocytopenia occurs in this dog breed _____, and is the result of a _____ deficiency. Is it associated with clinical bleeding?

Answer 19

Cavalier King Charles Spaniels

Beta-1-tubulin deficiency - not associated with clinical bleeding

Question 20

Glanzmann Thrombasthenia is a mutation resulting in the absence (< 5%) of ______ receptor. This results in a functional issue with platelet ____. There is no clot retraction and platelets fail to aggregate in response to any/all agonists (even thrombin).

Answer 20

GPIIb-IIIa (aka αIIb-ßIII) –> this is the fibrinogen receptor, and results in an issue with platelet bridging.

Question 21

Cyclic hematopoiesis is an inherited disorder of ______ that affects three cell types (____, _____, and ____). Platelet counts are _____ and platelet function is ______. There is an absence of ____ granules and defective reactivity to platelet agonists. The mutation was found in gene _____.

Answer 21

Grey collies

neutrophils, reticulocytes, platelets

Plt counts are normal, platelet function is decreased

Dense granules

AP3ß1

Question 22

This syndrome involves abnormal leukocyte, melanocyte, and platelet granulation and results in increased infections and bleeding diathesis. It alters platelet function by having absent ___ granules. These granules contain ADP which is responsible for _____ of platelets in response to ____. The mutation is found in the ____ gene. Species affected?

Answer 22

Chediak-Higashi syndrome

Dense granules

aggregation

collagen

LYST gene (lysosomal trafficking regulator)

Question 23

Calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) thrombopathias is a platelet _____ disorder that results in a conformation change in ______ (therefore it does not affect #, just function).

It mostly affects ____, and often is referred to as canine thrombopathia in literature. Other breeds/species affected include ____, ____, and ____.

There is ______ platelet aggregation responses to ADP/collagen.

There is ____ platelet aggregation response to thrombin, making clot retraction assays ____ becuase they use thrombin.

Answer 23

Signal transduction

GPIIß-IIIα

Bassets (1/3rd are carriers); Eskimo, Lanseers, Simmental Cattle

absent platelet aggregation in response to ADP/collagen

mildly decreased platelet aggregation in response to thrombin; making clot retraction assays normal.

Question 24

List the contents of platelet dense granules (5)

Answer 24

ADP

ATP

Serotonin (5-HT)

Calcium

Magnesium

Question 25

This canine congenital platelet dissorder associated with bleeting is a _____ disorder and causes decreased/absent activation of beta-integrins on leukocytes and platelets. It is found in this canine breed.

Answer 25

kindlin-3; signal transduction disorder German Shepherd

Question 26

This intrinsic platelet disorder is more specifically a procoagulant expression disorder that occurs in the _____ and results in lack of phosphatidylserine expression on the outer membrane, and membrane microvesiculartion failure upon activation with calcium ionophore. Platelet aggregation, BMBT, and clot retraction times are \_\_\_\_. It causes problems because platelets are unbale to generate and support a surface for coagulation protein activation (so kind of more a secondary hemostasis problem). Which flow cytometry marker can be used to detect it?

Answer 26

Scott syndrome GSD Normal **Annexin V** - on flow, it is markedly **reduced**.

Question 27

List differntials for acquired platelet dysfunction/hypofunction:

Answer 27

* Uremia * Antiplatelet antibodies * Infection/inflammation/neoplasia (hyperglobulinemia - globulins coat platelet surface and impaire adhesion and aggregation) * Increased fibrinolytic products (FDPs) - increased fibrinolysis or decreased clearance secondary to liver disease * Snake evenomation (modulation of ADP/TXA2 release; ROS; interaction with receptors) * NSAIDS: COX inhibitors (decreased TXA2 which is a potent platelet agonist), aspirin, and phenylbutazone causing transient platelet dysfunction in horses

Question 28

Platelets have an open canalicular system that is continuous with surface membrane in which veterinary species?

Answer 28

Small animals, but NOT in horses and cattle

Question 29

What is the mean platelet life span (dogs, cats, horses, cattle)?

Answer 29

4-6 days

Question 30

Splenectomy _____ lifespan of platelets.

Answer 30

increases (spleen removes aged platelets)

Question 31

Thrombocyte counts are ___ in non-mammlian than mammalian species.

Answer 31

Lower

Question 32

How do platelets generate ATP?

Answer 32

Krebs cycle and oxidative phosphorylation (have mitochondria) AND anaerobic glycolysis

Question 33

Majority of adenine nucleotides (~ 2/3rds) is stored within ____ of platelets. ADP/ATP ratio is \_\_\_, which is opposite to the cytoplasm.

Answer 33

dense granules; 1.5; in cytoplasm you have more ATP\>ADP

Question 34

Activated platelets secrete _____ from α-granules, which binds heparin-like moelcules on endothelial surfaces and displaces ______ to promote coagulation.

Answer 34

PF4 (CXCL4); displaces antithrombin

Question 35

Platelet activation results in the translocation of PS from the inner surface to the outer membrane surface. PS on the outer membrane will bind to ___ and serve as a nucleation site for the assembly of these two main coagualtion factor complexes ____ and \_\_\_\_\_. This promotes accelerated coagulation.

Answer 35

Calcium tenase (IX + VIII) --\> activates FX prothrombinase (X+V) --\> activates FII

Question 36

In addition to clotting, platelets are important for maintaining normal vascular integrity. One important protective vasoactive molecule stored and secreted from platelets is \_\_\_\_.

Answer 36

Sphingosine-1 phosphate

Question 37

Under high shear conditions, _____ and ____ will bind to platelet GP1b/IX/V. Under low shear/static conditions, platelets adhere directly to collagen in subendothelium using ____ and ____ receptors.

Answer 37

vWF and thormbospondin-1 GPVI and GP1a-IIa receptors

Question 38

NO and PGI2 serve as potent vaso\_\_\_\_ and ____ platelet activation.

Answer 38

vasodilators inhibit

Question 39

Anaplasma platys infection in dogs results in cyclic ____ and concurrent ____ that occur at 1-2 week intervals.

Answer 39

parasitemia and thrombocytopenia

Question 40

How to dx anaplasma platyis infection?

Answer 40

A. platys is cross reactive with A. phagocytophilum in Snap4Dx PCR and 16SrRNA gene squencing IFA for anitbodies against A. platys - will cross react with other rickettsial diseases

Question 41

Pseudothrombocytopenia is most common when using which anticoagulant? How does this occur?

Answer 41

Most commonly with EDTA Hidden epitopes are unmasked in GpIIbIIIa d/t calcium chelation --\> results in auto-antibody mediated platelet clumping.

Question 42

Thrombocytosis may result in pseudo\_\_\_\_\_ in serum becuase ___ is released during clotting. This is only significant with thrombocytosis because reference intervals of this analyte are accounted for with normal thormobocyte concentrations.

Answer 42

hyperkalemia; potassium

Question 43

TPO is involved in fragmentation from megakaryocytes. T/F

Answer 43

False

Question 44

What are causes for pseudothrombocytosis?

Answer 44

* Small RBCs * Fragmented RBCs * Hemolyzed RBCs * Leukocyte fragments * Particulate cell debris

Question 45

What causes physiologic thrombocytosis?

Answer 45

Epinephrine release (~30% of platelets are stored in the spleen)

Question 46

List the drugs responsible for drug-induced thrombocytosis.

Answer 46

Vincristine Epinephrine Endogenous glucocorticoids (NOT exogenous GCs)

Question 47

Reactive thrombocytosis caused by ___ and ___ is the most common cause of thrombocytosis. What cytokine is the main mediator?

Answer 47

INflammation and neoplasia; IL-6

Question 48

Iron deficiency may result in thrombocytosis. T/F

Answer 48

True

Question 49

Thrombocytosis is a common feature of all chronic myeloproflierative diseases, so it is difficult to differentiate essential thrombocythemia from polycythemia vera or CML. T/F

Answer 49

True

Question 50

LIst the main mechanisms that can cause thrombocytopenia (give specific examples under each mechanism)

Answer 50

1. Decreased production (hypoplastic BM, myelophthisis, FeLV, FIV) 2. Increased platelet utilization (DIC, thromboembolism, hemangiosarcoma, vasculitis, inflammation) 3. Increased platelet destruction (primary vs secondary IMT) 4. Sequestration of platelets (splenomegaly from hemolytic anemia/immune-mediated dz; congestion; splenic torsion; hypothermia causes sequestration in the liver) 5. Massive external hemorrhage (esp. with rodenticide tox, accentiuated by dilutional effect of platelet poor fluid) 6. Infections 7. Drug and chemical induced (chemo, cephalosproins, sulfonamides, griseofulvan, NSAIDs, methimazol and propylthiouracil, azathioprine, phenobarbital, antiviral drug ribavirin, toxins) 8. Neoplasia (lymphomas and hemangiosarcoma MC) 9. Inherited macrothrombocytopenia in CKC spaniel dogs

Question 51

How does hypthermia in dogs cause thrombocytopenia?

Answer 51

sequestration of platelets in the liver

Question 52

WHich inflammatory cytokines inhibit platelet production? Which promote platelet aggregation? Which promotes platelet production? Which enhances platelet phagocytosis?

Answer 52

IL-10 and IFN-a PAF IL-6 M-CSF and GM-CSF

Question 53

List the drugs and chemicals that result in thrombocytopenia.

Answer 53

* Cancer chemotherapeutic agents * Antimicrobials: cephalosporins, sulfonamides, dapsone, griseofulvin * NSAIDs: phenylbutazone, carprofen * Antithyroid drugs: methimazole, propylthiouracil * Immunosuppressive azathioprine * Exogenous or endogenous estrogens in dogs and ferrets * Dextroamphetamine and amphetamine * Phenobarbital * Antiviral drug ribavirin * Antipsoriasis cream capcipotriol * Toxins: Trichothecenes, bracken fern, phenol, fluoroacetate, *Macrozamia* seeds

Question 54

Platelet aggregate formation in cats can be decreased with ____ vacuum tubes containing platelet inhibitors.

Answer 54

CTAD (citrate, theophylline, adenosine, dipyridamole)

Question 55

Which breeds are platelet counts generally lower?

Answer 55

Polish Ogar Dogs Greyhounds Cavalier King Charles spaniels

Question 56

Normal to decreased MPV does not rule out thrombopoiesis. This is becuase dogs with IMT often have normal to low MPVs due to the presence of platelet \_\_\_\_.

Answer 56

Fragments

Question 57

MPVs are higher in \_\_\_thyroid cats and lower in \_\_\_thyroid dogs.

Answer 57

higher hyperthyroid cats lower in hypothyroid dogs

Question 58

Platelet function analyzer (PFA-100 is an in-vitro modified bleeding time test to assess platelet function. It uses ___ plasma and depends on platelet adhesion, secretion, and aggregation. Blood is drawn through a ____ shear rate through an aperture caoted with agonists (\_\_\_ and ___ or ____ and \_\_\_). The closure time is recorded as the time required for the aperture to be ____ with platelet aggregates and for blood flow to \_\_\_. Decreased platelet counts and anemia will result in ____ closure times.

Answer 58

PAF is an in-vitro modified bleeding time test that uses **citrated** plasma and depends on platelet adhesion, secretion, and aggregation. Blood is drawn at a **high shear rate** through an aperture coated with agonists (**collagen and epinephrine – CEPI; or collagen and ADP – CADP**). The closure time is recorded as the time required for the aperture to be **occluded** with platelet aggregates and for the blood flow to **stop**. While this analyzer assesses platelet function, decreased platelet counts will result in **prolonged “closure”** times, as well as a low hematocrit – limiting the applicability of the PFA-100 in anemic patients.

Question 59

Platelet aggregometers that use platelet-rich plasma are evaluated via ____ whereas those that use whole-blood are evaluated using \_\_\_\_\_\_.

Answer 59

spectrometry electrical impedence (as activated platelets aggregate to two electrodes - impedence increases)

Question 60

The three main aggregating agents used to evaluate platelet aggregateion are:

Answer 60

ADP, collagen, epinephrine

Question 61

What is the traditional gold standard platelet function test? Explain how the test works.

Answer 61

Light-transmission aggregometry (LTA) - after agonist is added to the sample, responsive platelets change shape from discoid to tiny spheres --\> causes a transient decrease in light transmission. This is followed by increase in transmitted light as platelets continue to aggregate. As the size of the platelet clumps grow, more light is transmitted until a sustained plateau is reached representing maximal, irreversable platelet aggregation.

Question 62

\_\_\_\_ is a modified technique of light-transmission aggregometry (LTA) which can detect _____ resulting from the release of ATP in the aggregation reaction mixture. ATP release is quantified through its reaction with a chemiluminsecent reagent called \_\_\_\_. This technique can also measure platelet _____ using whole blood in dogs.

Answer 62

Lumi-aggregometry Chrono-lume Secretion