Lecture 1 - hemostatis part 1

the process by which the body stops bleeding after an injury;

BLOOD

Stop

Clotting; it is always ongoing but at low levels, unless a big injury occurs.

bleeding

- Produces a clot to stop the bleeding
- Confines the clot to the site of injury
- Dissolves the clot as the wound heals

Initiate clotting if required

Prevent undesirable clotting.

Clot breakdown.

By desquamation (shedding of the inner lining of blood vessels) and small injuries to blood vessels ; involves vascular intima and platelets

By large injuries to blood vessels and surrounding tissues; involves platelets and coagulation system

rapid, short-lived response: Primary Hemostasis
delayed, long-term response): Secondary Hemostasis

Procoagulant substanaces exposed or released by damages or activated endothelial cells

the activator, tissue factor, is exposed on cell membranes.

Tissue factor and undergo vasoconstriction

platelet adhesion, aggregation and secretion

zymogens, cofactors, control proteins, and fibrinogen

fibrin clot breakdown.

help circulate blood throughout body ;
3 layers:
inner layer - tunica intima
middle layer - tunica media
outer layer - tunica adventitia

vascular endothelium ; yes it releases proteins based on need.

muscle depending on BV type and size
smooth muscle cells contract = vasoconstriction

Sub-endothelium
Support a surface protein called Tissue factor
- Procoagulant - important for the initiation of coagulation.

RBCS
WBC
platelets

add bulk and structural integrity to final fibrin clot

help stimulate wound healing; monos and lymphs have tissue factor on surface that can trigger coagulation

procoagulant only -- initiate and control hemostasis; they adhere, aggregate and secrete their granule contents to help form a clot.

Prekallikrein
FXII - 12
FXI - 11
FIX - 9
FX - 10
FVII - 7
PROTHROMBIN
FXIII - 13

Antithrombin
Heparin cofactor II
TFPI
Protein C
alpha2 - macroglobulin
alpha1 - antitrypsin
ZPI

HMWK
Tissue factor
FVIII - 8
FV - 5
Protein Z
Protein S
Thrombomodulin

CFU - GEMM ;
IL-3, TPO, AND MEG-CSF

BFU- Meg - clones hundreds of daughter cells
CFU - Meg - clones a dozen daughter cells
LD-CFU-Meg - undergoes the first stage of endomitosis

DNA is duplicated without cell division

TRUE

30-100 mm; largest normally occurring cells in the marrow; only polyploid cells in body

~ 2000-4000 platelets

the process of producing platelets (also known as thrombocytes), the cell fragments essential for blood clotting, within the bone marrow

a network of internal membranes found within megakaryocytes (MKs), the cells that produce platelets;
biologically identical to MK plasma membrane and form basis for fragmentation into single PLTs;
DMS dilates and tubules called 'proplatelet processes' develop

squeeze through or between endothelial cells and break off or shed into the central vein of BM

the MK nucleus is reabsorbed by macrophages.

MPL - expressed in ALL maturation stages
CD34 - only on MK progenitors
CD41 - all positive except BFU - Meg, αIIb portion of αIIbβ3
CD42 - MKI through PLT positive, GPIb portion of vWF receptor (GPIb/IX)
PF4 - MKI through PLT positive
vWF - MKI through PLT positive
Fibrinogen -

TPO, thromobopoietin; produced by liver (also by kidney and smooth muscle cells

to circulating platelets via membrane receptor

TPO increases in circulation

Stem cells to differentiate into MK progenitors -> MK progenitors to differentiate into MK -> Proliferation and maturation of MK -> platelet release from MK.

Normal and activated state

ADP
Serotinin
Collagen (GP VI, GPIa/IIa)
vWF (GP Ib/IX/V)
Fibrinogen (GP IIb/IIIa)
Thrombin
Epinephrine

membrane-bound compartments within platelets that store and release various molecules crucial for hemostasis (blood clotting)

alpha granules and dense granules

50-80/plts;
PROTEINS:
Fibrinogen
Factor V
beta-thromboglobulin
HMWK
PAI-1
Plasminogen

2-7 per platelets;
ADP
ATP
Serotonin
Ca2+
Mg2+

Adhesion - adhere to site of injury/disruption
Aggregation - aggregate to with other
Secretion - secrete their granule contents

Platelets bind collagen directly through GPVI and GPIa/IIa or indirectly through vWF (which binds GPIb/IX/V on platelets), ADP TXA2, "inside-out", "outside-in" signaling .

Platelets bind to each other through GPIIb/IIIa, shape and membrane platelet flip-flopping.

Platelets release granular contents (alpha and dense)
Mostly coagulation proteins
Launching platform for secondary hemostasis

Either platelets or vascular (rare) abnormalities

can be either qualitative or quantitative.
Qualitative - number of platelets are abnormal; function is normal
Quantitative - platelets are low or high

decreased platelets

increased platelets

For ex: post-surgery, post-splenectomy, IDA, inflammation or disease, strenuous exercise
Not associated with thrombosis or hemorrhage
Disappears when underlying condition under control

Essential thrombocythemia, Polycythemia Vera, CML, or primary myelofibrosis.
Can cause either thrombosis or bleeding
Marked and persistent elevations in platelet count are seen.

< 150 x 10^9/L

< 100 x 10^9/L

2-5 years old, following viral infections or vaccinations (MMR, DTP, polio)

3-4% cases: < 10,000uL

- Severe thrombocytopenia, signs of hemorrhage, normal RBCS and WBC in CBC
- Severe thrombocytopenia, sudden hemorrhage, no family history

3 weeks

NO, is in mostly adults ( woman > men)

Mutation in genes encoding the subunits of GPIIb/IIIa (Fibrinogen & vWF receoptor)

Platelet numbers and morphology are normal
Aggregometry: no aggregation in response to all activating agents (ADP, collagen, thrombin, epinephrin, AA), normal response to ristocetin (which is similar to vWF).

Rare disorder of platelet adhesion ; autosomal recessive inheritance

Usually in early childhood; Ecchymosis, epistaxis, and gingival bleeding

Rare, autosomal dominant disorder
Exact frequency unknown

Thrombocytopenia (low platelet count)
Characteristic abnormally enlarged or misshapen platelets (giant PLTs seen)

- Thrombocytopenia with giant PLTs
- Basophilic inclusions (called Dohle-like bodies) within the granulocytes and occasionally monocytes.

- PLTS function usually normal
- Also classified as Inherited Giant PLT syndrome.

Yellow - blood culture; sterile
Light blue - 3.2% sodium citrate
Red
Yellow
Green
Lavender - EDTA
Gray

sodium citrate - blue top tube

bleeding time - OG test of platelet function now replaced by:
PFA - 100 Analyzers - automated platelet function testing
Platelet Aggregometry Studies - measure how well PLTs aggregate together to form clots

Citrated whole blood - is aspirated at high shear rates through disposable test cartridges
Test cartridges contain 2 membranes: one coated collagen/epinephrin (CEPI) and the other one coated with collagen/ADP (CADP) and an aperture <- AGONISTS

Time required for a PLT plug to occlude aperture (small opening)

Assessment for inherited, acquired, or drug-induced platelet dysfunction
- can be used as an initial screening test for patients with impaired primary hemostasis (Von Willebrand Disease) <- Disease where vW is missing or doesn't work. ... quite successful
- to monitor DDAVP (desmopressin) therapy (DDAVP is a medication that helps release vWF from cells; used in partients before surgery to reduce bleeding)
- to check how aspirin affects platelets (Aspirin blocks platelet activation, so this test can show if the medication is working; also helps identify if aspirin is causing bleeding problems)

how well PLTs aggregate together to form clots

MUST NOT ACTIVATED PLATELETS
USE LARGER BORE NEEDLE !!!!

Spin low to produce PLATELET RICH PLASMA (PRP)
- allow platelets to regain function - let sample site for ~ 30 minutes
- Test within 4 hrs of collection to avoid spontaneous in vitro platelet activation and loss of normal activity
-chilling destroys PLT activity - samples held at 15-25oC until tested.

PRP in a cuvette with a plasticized magnetic stir-bar:
-keeps platelets in suspension at 800-1200 RPM (gentle speed)
- warm to 37 degrees celsius for 5 minutes.
Photometer directs light through cuvette to photodetector
- OD of PRP is high
- Baseline established = 0% light transmission
-add agonist (PLT activator or aggregating agent)

platelets shape change - increase in %T
platelets start aggregating - increase in %T
platelets form large aggregate - up to 100%T

-Resting platelet; stable baseline
- Shape change
- Primary Aggregation
-Secretion
-Secondary aggregation

Platelet secretion/aggregation defects → e.g., Storage pool disease (dense granule deficiency)

Platelet-related acquired disorders → e.g., HIT (Heparin-Induced Thrombocytopenia)

Vascular disorders → e.g., abnormal vessel wall function that affects platelet interaction

Bleeding time or PFA-100 → Prolonged/abnormal

Platelet aggregation studies → Decreased secondary wave of aggregation with some agonists (e.g., ADP, epinephrine, collagen)

Normal response with ristocetin

May have prolonged BT or abnormal PLT function testing

Physicians work to rule out other causes for bleeding.

Leads to excess anticoagulant (e.g., sodium citrate) relative to plasma, which prolong clotting times like PT and aPTT falsely.

Clotting has already occurred, so fibrinogen and clotting factors are consumed, making the sample unacceptable for testing. Results will be invalid or falsely shortened

Hemolysis releases intracellular contents (like ADP and potassium) which can interfere with platelet function tests, activate coagulation, or affect optical readings. Results may be falsley shortened or prolonged, depending on the test.

Causes optical interference in photo-optic instruments used in coagulation testing. It can lead to inaccurate results due to turbidity or color interference.

Prolonged stasis causes hemoconcentration, which elevates proteins like fibrinogen and factors V and VIII, possibly falsely shortening clotting times (e.g., shortened aPTT)

Cold temperatures can activate Factor VII (7) and cause platelet activation, leading to inaccurate results, especially in platelet function or factor assays.

Room temperature above 25oC can cause degradation of labile factors (especially factor V and VIII), leading to falsely prolonges PT and aPTT.