CLS 442 Lecture 1 Intro to Hemostasis, Primary, PLTs

Hemostasis definition

Complex physiologic process that keeps circulating blood in a fluid state

Hemostasis maintains balance between ____ & ____

Coagulation; Hemorrhage

Components of hemostasis system (3)

• Procoagulant properties

• Anticoagulant properties

• Fibrinolytic/Fibrinolysis properties

Hemostasis Complex Cascade & Purpose (3)

• Primary Hemostasis- Formation of Platelet Plug

• Secondary Hemostasis- Formation of Fibrin clot

• Fibrinolysis- Break down of Fibrin Clot

Primary Hemostasis (4)

• activated by desquamation & small injuries to BVs

• Vascular intima and PLTs involved

• Rapid

• Procoagulant substances exposed or released by damage or activated ECs

Secondary Hemostasis (4)

• activated by large injuries to BVs and surrounding tissues

• Coag sys and PLTs involved

• Delayed

• Tissue factor(trigger) is exposed on cell membranes

What are the 5 components Hemostasis involves to stop bleeding?

• BVs

• PLTs

• Plasma components

• Fibrinolytic proteins

• Inhibitors

BVs contain _______ and undergo _______ ______

Tissue Factor; vasoconstriction

What are the plasma components?

• Zymogens

• Cofactors

• Control Proteins

• Fibrinogen

3 BVs Layers

• Tunica intima- Inner layer

• Tunica media- Middle layer

• Tunica adventitia- Outer layer

Tunica intima (Vascular Endothelium) releases what proteins based on need?

• Anticoagulant

• Procoagulant

• Fibrinolytic

What occurs in the tunica media?

Smooth muscle cells contract (Vasoconstriction)

• TM contains muscle depending on BV type & size

Tunia adventitia supports the surface protein ______ ______

Tissue Factor

Component roles of vessels (10)

• ADAMTS13

• ECs

• EPCR

• PAI-1

• PGI2

• TAFI

• TF

• TFPI

• TPA

• VWF

RBCs add ____ & ____ ____ to final fibrin clot

bulk; structural integrity

WBCs help stimulate _____ _____

wound healing

______ & ______ have Tissue Factor on surface that can trigger coagulation

Monos; Lymphs

PLTs are ______ only meaning they don’t prevent clotting or break down the clot, instead they _____, _____, and ______ secrete their granule contents to help form a clot

procoagulant; adhere; aggregate; secrete

What are the 7 control proteins?

• AT

• Heparin cofactor II

• TFPI

• PC

• a2-macroglobulin

• a1-antitrypsin

• ZPI

PLTs are produced by _______ through _______

megakaryocytes; endomitosis

MK progenitors (3)

• BFU-Meg

• CFU-Meg

• LD-CFU-Meg

Endomitosis is when…..

DNA is duplicated without cell division

Through endomitosis, MKs become _____. Most have a ploidy of ____ (range ___ - ___)

polyploid; 16N; 4N; 64N

_____ are the largest normally occuring cells in the marrow ranging from ____ - ____

MKs; 30mm; 100mm

What is the only polyploid cell in the body?

MKs

MK 8N or 18=6N can produce ______ - ______ PLTs

2000; 4000

Thrombopoiesis

prod. & release of PLTs from MKs

Demarcation System(DMS)

• part of Thrombopoesis(PLT prod.)

• plasma membrane invades the cytoplasm in a series of channels

• biologically identical to MK plasma membrane

• form basis for fragmentation into single PLTs

DMS dialates & tubules called _______ _______ develop

proplatelet processes

What squeezes through or between endothelial cells and break off or shed into the central vein of BM?

Proplatelet processes

In Thrombopoiesis, after the cytoplasm is released as platelets the MK nucleus is reabsorbed by ________

macrophages

What are the 7 markers for identification of MK & PLT?

• MPL

• CD34

• CD41

• CD42

• PF4

• VWF

• Fibrinogen

Receptor for TPO

MPL

Markers spef. to PLT lining

• CD34

• CD41

• CD42

• PF4

• VWF

• Fibrinogen

What is Thrombopoietin(TPO)?

• Hormone prod. by liver, kidney, and smooth muscle cells

• Attaches to circulating PLTs via membrane receptor

• ↓PLTs = ↑ circulating TPO

• stimulates PLT prod.

TPO induces what? (4)

• Stem cells to differentiate into MK progenitors

• MK progenitors to differentiate into MK

• Proliferation and maturation of MK

• PLT release from MK

• in synergy with various other cytokines (IL-3, IL-6, IL-11)

What are the 4 structures of PLTs?

• Membrane structure

• SCCS

• DTS

• PLT granules

What is the size of a platelet?

• 2-4 microns

• can be 20 microns= Giant PLT

what are the 7 a granules(50-80/plt)?

• Fibrinogen

• Factor V

• vWF

• B-thromboglobulin

• HMWK

• PAI-1

• Plasminogen

What are the 5 dense granules(2-7/plt)?

• ADP

• ATP

• serotonin

• Ca2+

• Mg2+

what are initiate and control hemostasis and are always ready to react?

PLTs

What is Adhesion?

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

What is Aggregation(Key Step)?

PLTs binds to each other through GPIIb/IIIa, shape change, membrane PL flip-flopping

What is Secretion?

• PLTs release granular contents (a and dense)

• Mostly coag proteins

• Launching platform for secondary hemostasis

What is the receptor for fibrinogen?

GP IIb/IIIa(helps binding)

What is the direct and indirect receptor for collagen?

Direct- GPVI and GPIa/IIa

Indirect- through vWF(which binds GPIb/IX/V on PLTs)

what carries and protects coagulation FVIII for 12 hours?

vWF

What are primary hemostasis disorders?

• either PLT or Vascular(MAHA, DIC) abnormalities

• can be qualitative(pH don’t function properly) or quantitative

• can be congenital or acquired

• manifest as mucocutaneous bleeding(GI, nose bleed)

What are the common presentations of Primary Hemostasis Disorders?

• Brusing

• Petechiae-small

• Purpura

• Ecchymosis- diffused

• Epistaxis-nose bleed

• Gingival bleeding

ALL strong indicators or Primary Hemostasis Disorders

Reactive thrombocytosis

• secondary to other conditions

• not associated with thrombosis or hemorrhage

• disappears when condition is under control

• ex. post-surgery, post-splenectomy, IDA, inflammation/disease, strenuous exercise

Myeloproliferative disorders

• can cause either thrombosis or bleeding

• persistent elevations in PLT count seen

Thrombocytopenia

• PLT count <150×10⁹ /L or <150,000 /µL

• clinically significant bleeding= <100,000 /µL

• surgery, PLT transfusion= <50,000 /µL UNLESS 13 or 18 yrs old

What are the pathophysiologic processes that result in Thrombocytopenia?

• Impaired or decreased PLT prod.

  • low BM MKs

  • ineffective thrombopoiesis

• Increased/Accelerate PLT destruction

  • by immunologic responses or mechanical damage, consumption, or sequestration

• Abnormal PLT distribution

  • Splenic sequestration due to splenomegaly

  • splenomegaly,hyposplenomegaly ↑PLT

Immune thrombocytopenic purpura (ITP)

• destruction of PLTs by an AB-mediated mechanism (Ab against GP II/IIIa, GPIbIX/V, GPIaI/Ia)

• In children (2-5 yrs old), following viral infections or vaccinations (MMR, DTP, polio)

• KEY feat.: thrombocytopenia (3-4% cases: risk of spontaneous bleeding:nose, GI)

• most acute(children) cases recover in abt 3 wks

• Chronic ITP mostly in adults(women)

What are the certain diagnosis of acute ITP in a child? (2)

• Severe thrombocytopenia, signs of hemorrhage, normal RBC and WBC in CBC

• Severe thrombocytopenia, sudden hemorrhage, no family history

Why does ITP occur?

• AB binds to PLT

• PLT destroyed by spleen in the peripheral blood by macrophages

• BM senses & accelerates PLT prod. to compensate

• ↑PLT prod. in BM due to destruction

what is the treatment to ITP?

Immunosupressants, Drugs(Steroids, Coritsol, IVIG)

A patient has ITP and is RH+, what is the likely treatment?

Rhogam

What are the 4 quantitative primary hemostasis disorders?

• Reactive thrombocytosis

• Myeloproliferative disorders

• Thrombocytopenia

• ITP

What are the 3 qualitative platelet disorders?

• Glanzmann’s Thrombasthenia

• Bernard Soulier syndrome

• May-Hegglin Anomaly

What is Glanzmann’s Thrombasthenia?

• extremely rare coagulopathy

• hemorrhage may be severe

• all types of bleeding: epistaxis, ecchymosis, hemarthrosis, subcutaneous hematoma, menorrhagia, and GI and UT hemorrhage, have been reported

• manifests in neonatal period or infancy

• mutation in genes encoding the subunits of GPIIb/IIIa (Fibrinogen and vWF receptor)

What is the lab diagnosis of Glanzmann’s Thrombasthenia?

• PLT count and morphology both normal

• Aggregometry: no aggregation in response to all activating agents (ADP, collagen, thrombin, epinephrin, AA), normal to ristocetin

What is Bernard Soulier syndrome?

• Rare disorder of PLT adhesion

• Common in early childhood

• Ecchymosis, epistaxis, & gingival bleeding

• Mutation in GPIba of the GP Ib/IX/V receptor complex (vWFR): defective PLT adhesion

What is the lab diagnosis of BSS?

• Thrombocytopenia (↓PLT survival/prod)

• Giant PLTs

• Aggregometry: Normal response to ADP, collagen, thrombin, epinephrin, AA, but lack of aggregation in response to ristocetin

What is May-Hegglin Anomaly

• not that severe compared to other Quali.PDs

• exact frequency unknown

• Characteristic abnormally enlarged or misshapen PLTs

• On PBS:

  • Thrombocytopenia with giant PLTs

  • Basophilic inclusions (Döhle-like bodies) within granulocytes and occasionally monocytes

• PLT function normal

May-Hegglin Anomaly is also classfied as…..?

Inherited Giant PLT syndrome

PLT anti coag of choice, RI, size, MPV?

• EDTA

• RI: 150,000-450,000/µL

• SIze: 2-4µm

• MPV: 2-20 fL (20 usually=giant)

3 tests for platelet function

• Bleeding time

• PFA-100 Analyzers

• PLT Aggregometry studies

What is PFA-100?

• Automated sys. for analyzing platelet aggregation

• Citrated whole blood is aspirated at high shear rates thru disposable test cartiridges containing 2 membranes, 1 coated with CEPI and another with CADP

• Agonists induce adhesion, activation, & aggregation

• Time required for PLT plug to occlude aperture is indication of PLT function

  • screening, not diagnostic

  • RI used

• in vitro; # of hematocrit & PLLT can affect results

What does PFA-100 analyze?

• assesses for inherited acquired, or drug-induced PLT dysfunction

• can be used as initial screening for impaired primary hemostasis (VWD)

• monitors DDAVP therapy in pre surgical patients

• assesses PLT dysfunction from Aspirin

What is DDAVP

antidiuretic drug that releases VWF from cells as side effect to assist with mucocutaeneous bleeding

What is PLT aggregometry?

• measure’s PLTs ability to aggregate to form clots

• KEY: sample procurement

  • dont’ activate PLTS

  • use larger bore needle

  • light blue tube

• Spin low for PRP

  • let PLTS sit for 30 mins to regain funciton

  • test within 4 hours of collection avoid in vitro PLT activation and losss of normal activity

What is the testing procedure for Optical aggregometer?

• PRP in cuvette with plasticized, magnetic stir bar

• suspend PLTS gently at 800-1200 rpm

• warm to 37C for 5 mins

• photometer directs light through cuvette to photodetector

• OD of PRP= high

• baseline established= 0% light transmission

In optical aggregometer, if you add agonist(PLT activator or aggregating agent), what occurs (3)?

• PLT shape change- increase in %T

• PLTs aggregate- increase in %T

• PLTs form large aggregate- up to 100%. T

Storage Pool Defects- Dense granules

• Prolonged BT

• Abnormal PFA-100

• ↓PLT aggregation to some agonists

  • WHY?- has ADP= aggregation

• a or dense granules affect secretion

HIT

• early monitoring using 4Ts scoring sys.

• Enzyme immunoassay to ID Ab

• Confirmatory PLT activation Assay (Serotonin Release Assay(Radioactive PLTs)

• Rapid PLT agglutination immunoassay also available

• PF4= develop thrombocytopenia= risk for thrombosis

Vascular Disorders

• may have prolonged BT or abnormal PLT function

• other coag tests usually normal

• normal- primary hemostasis disorders=vascular