1ry Hemostasis

hemostasis vs thrombosis

hemostasis is physiological

thrombosis is pathological

primary hemostasis (1ry HS) consists of ___ & ___.

vascular & plt plug

secondary & tertiary hemostasis (2ry & 3ry HS) consist of ___

fibrin clot formation

inhibition & lysis

endothelial cells function

innermost layer of blood vessels

barrier b/t blood & interstitial spaces

regulates HS by inhibit/promoting clots

stores vWF

blood vessel (BV) injury → what are the steps to prevent bleeding?

1. vasoconstriction (slow blood flow)

2. diversion of blood flow

3. activation of plt (adhesion → aggregation)

4. activation of coag system

5. feedback amplification (more of) plt & coag activation

resting endothelium (coag not activated)

plt activation is inhibited by

• PGI2, NO, ADPase

coag is inhibited by

• AG

• protein C & S

activated endothelium (promotes clot formation)

Von Willebrand Factor (vWF) function

• what does it bind to?

• where is it stored?

“anchor in HS”: binds to damaged collagen upon injury

• unfolds from spherical → linear shape → recruits plts

• binds to collagen, plts, factor VIII (vWF=carrier)

• stored in megakaryocytes, plts, endothelium

plt receptor: GP1b binds to vWF that’s anchored to the collagen → plt activation → plt aggregation via TAX2, ADP → recruit fibrinogen (mesh) = plt plug

Von Willebrand Disease (vWD)

• what is it?

• what type of bleeding?

m/c inherited bleeding DO

• defect in 1ry HS: dec adherence to vascular injury → inadequate plug

type: mucocutaneous/superficial

• easy bruising, epistaxis (nosebleeds), subcut ecchymosis (under skin bleeding), GI bleeds, bleeding oral procedures

what are the subtypes of vWD?

usually autosomal dominant

mild dz

• type 1 = dec qty of vWF

moderate dz (dec qty & quality)

• type 2A

• type 2B/plt type = gain of fxn plt binding

• type 2M = dec collagen GP1b binding

• type 2N = dec binding to fVIII → mimics hemophilia A

severe dz (near absence of vWF)

• type 3: lethal bleeds, → fVIII will also decrease

acquired vWD

• malignancies, prosthetic heart valves

• resemble Type 1 or 2A

vWD testing: von Willebrand antigen

detects if protein is there, but not how it’s working

vWD testing: vWF activity (Ristocetin, GP1b, etc)

tells you how well vWF binds plts

vWD testing: factor VIII C assay

vWF binds to fVIII !

relationship b/t vWF and fVIII?

vWF is the carrier in the bloodstream for fVIII; and vWF protects fVIII from degradation

if vWD type 2N: dec binding to fVIII → mimics Hemophilia A (defic of fVIII)

other vWD testing:

vWF multimer

PFA 100 - replaces bleeding time

plt aggregation - Ristocetin induced plt aggregation (RIPA)

PT/PTT

how does the vWF Ristocetin cofactor activity test (RIPA) work?

Ristocetin exposes GP-1 binding site for vWF → plts agglutinate via GP1b forming bridges

• tests how well vWF can bind to plt

→ aggregometer measures change in light transmission compared to a standard curve

• vWD indicated if dec optical density

discontinued bc labor intensive → vWF latex agglutination

the RIPA was replaced by what test?

vWF latex agglutination

• test is automated using ACL top

• rgt = vWF Ab-coated latex-GP1b → pt’s vWF will bind → optical density recorded

ex of vWD types lab results

vWD treatment

cryo (rare)

Amicar or TXA (fibrinolytics)

Desmopressin (contraindicated in type 2B vWD)

vWD concentrates

??

vWD vs Bernard Soulier syndrome?

BSS have large plts and thrombocytopenia, vWD should not

type 2 vWD may mimic hemophilia A, so always run ___.

PT and PTT screening assay w vWD panel

vWF multimer analysis function

indicates vWF multimers that are formed via disulfide bonds

can detect if dec in functioning vWF → vWD type

** can also detect ultra-large vWF multimer = spontaneous plt aggregation → thrombotic thrombocytopenic purpura (TTP)

can you exclude presence of mild vWD based on vWF tests alone?

no, in inflammatory states (pregnancy), vWF Ag, vWF activity, and fVIII (acute phase reactants) are abnormally high

• so in mild vWD → vWD may present w normal vWF levels

acute phase reactants are _

plasma proteins that inc/dec in response to inflammation

ex) vWF, fibrinogen, fVIII

plts facts

• resting unactivated plt are disc shaped anucleated cell fragments that come from megakaryocytes

• life span = 7-10 days

• 2-4um in size

• N: 150k-400k plt/mL

plt periphery

glycocalyx: creates neg surface charge & adsorbs plasma proteins (vV, vWF, fibrinogen)

plasma membrane: phospholipid bilayer, cholesterol, proteins

plt membrane receptors & binding sites

GPIb/Ix: or vWF

GPIIb/IIIa: fibrinogen

GPIa/IIa: collagen

plt organelles

mitochondria

dense granules

alpha granules

lysosomes

peroxisomes

plts: dense granules store mediators of plt function and HS which are ___

ADP: aggregation

serotonin: vasoconstruction

Ca2+

plts: alpha granules store variety of bioactive substances ie _.

vWF

fibrinogen

plt factor 3 & 4: thrombin generation, heparin neutralizing

factor V

fibrinolytic factors (plasminogen)

steps of activated plt stages

1. adhesion

2. shape change

3. activation

4. secretion

5. aggregation

→ 1ry hemostatic plug

thrombocytopenia is caused by

• decreased production:

• inc consumption:

• induced by:

m/c plt DO

plt count <150k

• caused by dec production: aplastic anemia, radiation

• inc consumption: splenic hepatic sequestration, ITP/TTP, HUS, bleed, DIC, NAIT (maternal fetal)

• drug induced (heparin induced)

qualitative plt disorders

Bernard-Soulier syndrome: defective GPIb/IX, giant plt

Glanzmann thrombasthenia: defective GPIIb/IIIa (after plt activation, for plt aggregation)

delta-storage pool disease: dense granules defic

gray plt syndrome: alpha granule defic; agranular plt

defective thromboxane A2 synthesis: defic of cyclooxygenase or TXA2 synthase

→ all inc PFA (closure time)

Bernard-Soulier syndrome

defective GPIb/IX → abnormal RIPA

giant plt

**giant St. Bernard dog

Glanzmann thrombasthenia

defective GPIIb/IIIa → abnormal w ADP, collagen, epinephrine

delta-storage pool dz

dense granule defic → no ADP, serotonin → same w Glanzmann

GrAY plt syndrome

alpha-granule defic → agranular plt

normal plt aggregation

defective thromboxane A2 (TXA2) synthesis:

• what is the reaction of TXA2 synthesis?

• what OTC medicine inhibited TXA2 production?

defic of cyclooxygenase (COX1) or TXA2 synthase → abnormal 2ry plt aggregation

TXA2 = plt activator and BV vasoconstrictor (injury → plt produce TXA2)

in plt: arachidonic acid --(COX1)→ TXA2

• inhibited by Aspirin!!

adhesion disorders

• first 3 already mentioned, what is dz of collagen defect?

von Willebrand dz

Bernard-Soulier syndrome: defic of GPIb

Glanzmann’s thrombasthenia: absence or defic of GPIIb/IIIa , fibrinogen binding site

Ehlers-Danlos Syndrome (Indian Rubber man): collagen defect, prevents binding or vWF/plt to collagen fiber → vWF can’t anchor :((

release reaction defects

• Aspirin therapy: inhibits production of TXA2 which prevent plts aggregation

• m/c plt defect

  • storage pool disease: plts lack dense granules → no ADP

hypofibrinogenemia

congenital DO w low levels of fibrinogen = lack glue for aggregation

or

congenital afibrinogenemia: absence of fibrinogen

May-Hegglin anomaly

giant plts (cookies), Dohle bodies (croissant), thrombocytopenia

**May is a baker, uses EGG

Hermansky-Pudlak syndrome

dec ADP in dense granules

** my hermana LAKs a(D)p

Wiskott-Aldrich syndrome

dec dense granules (no ADP, serotonin) & thrombocytopenia

** Whisky → xs alcohol → ruins SD (can’t get dense…)

Chediak-Higashi syndrome

storage pool defect

LYST gene mutation → abnormal giant lysosomes

defective phagocytes → recurring infns

defective melanin production → albinism

TAR baby syndrome

storage pool defect

Alport’s disorder

DO of basement membranes

thrombotic DO or plts

plts are “hyperaggregable” → interact w relatively normal BV wall → sclerotic vessel dz, thrombosis ie stroke, deep vein thrombosis (DVT), pulmonary emobolism, retinal vein occulsion

testing for plt quality & quantity

plt count

bleeding time (discontinued)

plt aggregation

plt function analysis (PFA)

thromboelastography: TEG or ROTEM

plt function analysis (PFA) is

in vitro bleeding time

doesn’t assess for vascular/collagen bleeding

screening test!

plt inhibition test

• assess __ receptor inhibition? by what type of therapy?

• what does the above receptor bind to? → plt aggregation

optical detection system to assess P2Y12 receptor inhibition, reported in P2Y12 reaction units (PRUs)

• P2Y12 receptor: key in plt aggregation in response to ADP

• measures extent of ADP-induced plt clumping of fibrinogen-coated beads (plt aggregation)

• can monitor anti-plt therapy (ie Plavix)

thromboelastography (TEG)

evaluate formation, strength, lysis of clot

uses:

• monitor anticoag therapy (warfarin/heparin)

• assess DIC

• trauma/surgery bleeding guiding

rotational thromboelastometry (ROTEM)

similar to TEG, but difference in way machine works (more commonly used in Europe)

similar uses as TEG

common agonists (enhancers) used for plt aggregation

• ADP concentrated

• ADP diluted

• epinephrine

• collagen low vs high

• Ristocetin high

• Ristocetin dilute

• arachidonic acid

• thrombin

important plt DO table and plt aggregation patterns

plt aggregation curves

• y-axis: OD dec, meaning inc plt aggregation

• x-axis: time (min)