Platelet Physiology😍

Antithrombotic and Anticoagulation

• Sub endothelium 
• Pro-thrombotic

• Endothelium
• Highly anti-thrombotic.
• [As long as endothelium is intact - Clotting does not occur]
• Secretes
• Prostacyclin
• Nitric oxide
• NO is the endothelium derived relaxing factor.
• ADPase

• Anticoagulant effect of endothelium is by:
1. Thrombomodulin
2. Heparin like molecule
3. Tissue factor pathway inhibitor

Factors Regulating Anticoagulation Pathways

1. Plasminogen activator inhibitor

2. Alpha 2 antiplasmin

3. Thrombin activatable fibrinolysis inhibitor

Role of Vitamin K in Coagulation

• Acts as a coenzyme for
• gamma carboxylation /
• post translational carboxylation.

• This activates:
• Clotting Factors 2, 7, 9 & 10.
• Protein C & S.
• Osteocalcin.

• Inactive Epoxide form → Active hydroquinone form of Vitamin K
• by Epoxide reductase.

Megakaryocyte

• Precursor or "mother" of platelets.

• Multi-nucleated cell.

Normal Platelet Count and Function

• Normal platelet count: 1.5 to 4.5 lakhs per mm cube.

• Normal function: To make a blood clot.

• Normal platelet adhesion is prevented by endothelium.

Steps of Blood Clot Formation

• Tikka (Ticagrelor) in a Can (Cangrelor) → Reversible

• PAR-1 (Protease Activator Receptor) INHIBITOR:
• AtoPaxar
• VoraPaxar

• Process:
• Vasoconstriction → Platelet Adhesion → Platelet Secretion → Platelet Aggregation → Clotting System ++

1. Vasoconstriction

• Temporary stop in blood flow.

• Mediators:
• Serotonin (from platelets)
• Endothelin

2. Platelet Adhesion

• Occurs at injury site.

3. Platelet Activation

• Conformational changes

4. Platelet Secretion

• Platelets release granule contents.

• Two types of granules present: 
• Alpha granules
• Delta granules

• Alpha granules:
• Release most contents
• Examples: 
• P-selectin, vWF, Platelet Factor 4, PDGF, Factor 5, Factor 8, Fibrinogen, Fibronectin
• Release "P" and "F" (factor) related substances.
• HIT is against Platelet factor 4

• Delta granules (or Dense granules):
• Mnemonic: "DENSE CASHE"
• Serum Calcium
• Adenosine (ADP, ATP)
• Serotonin
• Histamine
• Epinephrine

5. Platelet Aggregation

• Platelets stick to each other.

• Requires:
• GP2B3A receptors on platelets.
• Fibrinogen linking platelets.
 

• Platelet plug is the primary hemostatic plug.

• Mnemonic: For steps after 2 and 3, use receptor GP2B3A

6. Activation of Clotting System (Secondary Hemostatic Plug)

• Platelets flip Phosphatidylserine (PS) to outside.

• Flipped PS has a negative charge.

• Negative charge activates clotting system → fibrinogen (from alpha granules) → Fibrin → forms stable blood clot → Secondary hemostatic plug.

The Clotting System Pathways

• Consists of intrinsic, extrinsic, and common pathways.

COAGULATION CASCADE

• Positive feedback

• Endothelial injury 
• Lot of Tissue Factor (3) is produced
• In vivo, Extrinsic cascade > Intrinsic
• Factor 10a produced → activates intrinsic cascade
• by back amplification.

Pathways

1. Extrinsic Pathway
• Activated by tissue injury (external).
• 7, 3
• Initiated by Tissue factor
• 3 → 3a
• 7 → 7a
• 3a + 7a + Ca → 10a (activate factor 10)
• "Calcium is at the connections".
• Mnemonic:
• 73 year old not in tour → external
• "7 + 3 is also 10".

2. Intrinsic Pathway
• CONTACT ACTIVATION IN LABS
• 8, 9, 11, 12
• 12a → 11a → 9a → 8a + Calcium → 10a
• Mnemonic: 8th 9th 11th 12th in tour
• Initiated by negatively charged surfaces
• from phosphatidylserine
• Kininogen/Kallikrein 
• Amplifies intrinsic pathway

3. Common Pathway
• Starts after factor 10 activation
• 10 → 2 → 1
• 10a + 5 + Calcium + Phospholipid = 2a
• 2a + 13a + 5a = 1a

4. Fibrin Stabilization
• Fibrin (1a) + 13a + Calcium → Stable clot
• Factor 1a
• Fibrin clot
• Factor 13:
• Laki-Lorand factor / fibrin stabilizing factor

Diagnostic Tests for Bleeding Disorders

• APTT and PT → in Light blue Vacutainer

• Mnemonic:
• APTT:
• Apt techaer
• Safeayitt (Cephalin) kondupovan (Kaolin) → tourinu (78,9,11,12)
• 2-4 days tour → 26 to 40 sec
• PT
• Part time → for 73 yr old
• 11:00 → 16:00 hrs → 11-16sec

Bleeding - Approach

1. Platelet Issues

• Bleeding time ↑

Decrease in number (Thrombocytopenia):

• Isolated: Often due to TTP, ITP.

Pancytopenia:

• Often due to Acute leukemia, Myelodysplastic syndrome (MDS), Aplastic anemia.

Functional issue of platelets:

Inherited:

• Bernard-Soulier Syndrome
• Defective GP 1B9 receptor.
• Abnormal Ristocetin
• Aggregation
• Giant platelets
• Mnemonic: "B for B" (Bernard-Soulier, Big platelets).

• Glanzmann's Thrombasthenia
• Defective GP2B3A receptor.
• Abnormal ADP/collagen
• Aggregation
• Mnemonic: To differentiate from Bernard-Soulier (GP1B9), "B comes first, G comes later".

• Alpha Granule Defect
• Known as Gray Platelet Syndrome.
• Mnemonic: "Alpha granule problem, aged white hair... Gray platelet syndrome".

• Delta Granule Defect
• Known as Hermansky-Pudlak Syndrome.
• A/w oculocutaneous albinism.

Acquired: 

• CKD

• Drugs like ecospirin.

DIAGNOSTIC APPROACH

Bleeding can be due to	Features
Platelet issue	Small bleed like Mucosal bleed
Clotting factor	Hematoma, joint bleed

1. Number

• Thrombocytopenia
• A/c → Child < 6m
• C/c → Old > 6m

2. Function

• PFA 100 analysis
• Platelet function analysis
• Best Screening Test
• Calculates aperture closing time
• (time for platelets to form a clot)
• Platelet Fault analysis
• (If prolonged → Defective platelet function)
• -ve → Normal (<80sec)
• +ve → Abnormal (>80sec)

If Abnormal PFA → RAT

• Ristocetin Agglutination Test

• Check adhesion: Adhesion factor
• Negative
• Adhesion defect → vWF, BS
1. Add plasma
• Normalise → vwF (due to gain of factor 8)
2. Check APTT
• ↑↑ → vwF
• Normal → BS
• Positive
• Normal adhesion
• So check aggregation

If RAT Positive → Secondary wave of aggregation

• Absent/defective waves → Aggregation defect → GT

• Waves partially normal → Activation defect → GP, HP

---

Level Affected	Defect	Disorder
Adhesion	Gp Ib–IX defect ↳ Bernard–Soulier syndrome vWF defect ↳ von Willebrand disease	• Abnormal PFA • RAT Negative
Activation	Alpha granule defect ↳ Grey platelet syndrome Delta granule defect ↳ Hermansky–Pudlak syndrome	• RAT Positive • Secondary wave of aggregation ↳ Waves partially normal
Aggregation	Gp IIb–IIIa defect ↳ Glanzmann thrombasthenia	• RAT Positive • Secondary wave of aggregation ↳ Absent/defective waves

Feature	BSS	Glanzmann
Defect	Gp Ib–IX	Gp IIb–IIIa
Key Features	Large platelets	ㅤ
Problem	Adhesion defect (Ristocetin)	Aggregation defect (ADP / Collagen)
Bleeds	Platelet bleeds	Severe bleeding  ↳ Recurrent episodes ↳ Heavy menstrual bleeding

NOTE

• Blue → Normal sample

• Red → Patient sample

Plasma Used	Test
PRP – Platelet-Rich Plasma	• Platelet aggregation studies
PPP – Platelet-Poor Plasma	• PT / APTT / coagulation studies • Reference for 100% light transmission in Platelet aggregation

• 51 (HLA B51) yr old Nolan (Anti enolase) on a Bus (behcets)

• rF 7a or aPCC
• rFVIIa bypasses 8 & 9

Bypassing Agents

• Recombinant Factor VIIa (rFVIIa)
• Activates Factor X → Xa
• Bypasses factors VIII and IX

• Activated Prothrombin Complex Concentrate (aPCC / FEIBA)
• rFVIIa preferred when aPCC contraindicated (e.g., DIC risk).

Scenarios (Lab Test Interpretations)

Oozing from puncture sites d/d

• DIC

• Acute hemolytic transfusion reaction

Factor 12 deficiency

• Doesn’t cause bleeding in vivo

Hemostatic Disorders Summary

Thromboelastography (TEG)

Significance

• Tests efficiency of the entire blood coagulation pathway.

• Evaluates:
• Clot formation (all phases).
• Fibrinolysis.

Thromboelastography Parameters

• React (R) freshly (FFP)

• Alpha (α) Run (Kinetic) → Cry (Cryo)

• MAP (Ma → P)

• Fibrinolysis → TXA

Von Willebrand Disease (VWD)

• Platelet + clotting factor defect → vWD..

• "Combo problem"

Key Exam Lines

• Most common inherited bleeding disorder = VWD.

• Most common type = Type 1

• Severe type & AR= Type 3

• Unique point: Bleeding time ↑ + aPTT ↑ (PT normal).

• Ristocetin test abnormal in VWD.

• Type 2N → AR
• mimics hemophilia A
• RIPA Normal
• vWF → Normal → BT Normal
• F8 release affected → aPTT ↑↑

Von Willebrand Factor (vWF)

• Gene: Produced by the chromosome 12p gene.

• Storage: 
• Stored in the sub endothelium inside Weibel Palade bodies.

• Structure: 
• vWF initially produced as monomer.
• Disulphide bonds form multimers
• Released into circulation.
• Proteolyzed by ADAMTS 13 (vWF metalloproteinase)

• Platelet adhesion
• Endothelial injury → Subendothelium exposed → releases large numbers of high molecular weight vWF → Blood hits vWF → conformational change in vWF.

vWF binds to	Collagen Receptor	Platelet Receptor
via	gp6	gp1b9
• vWF globular form → Converted to Filamentous form.
• Mnemonic:
• "111 makes it step number one"
• GP 1B9 binds to 1 von Willebrand factor for step number 1)

Lab Findings

• ↑ Bleeding time (platelet adhesion defect).

• APTT prolonged (↓ FVIII)

• PT normal.

• Platelet count normal (except Type 2B ↓).

• Abnormal ristocetin-induced platelet aggregation (RIPA).
• RIPA → Normal in 2N

Diagnosis

• Gold standard:
• ↓↓ vWF antigen level
• ↓↓ Ristocetin cofactor activity
• ↓↓ Factor VIII activity
• vWF multimer analysis (for subtype classification).

Management of VWD

• TOC: vWF concentrates.

• DOC: Desmopressin 
• (also called Diamino Dimethyl arginine vasopressin - DDAVP).
• Releases vWF and FVIII from endothelium.
• Useful in Type 1 (contraindicated in Type 2B).

• Also with anti-fibrinolytics (tranexamic acid) and cryoprecipitate

Idiopathic Thrombocytopenic Purpura (ITP)

• Immune-mediated platelet destruction
• Caused by autoantibodies against:
• Gp IIb-IIIa

• Impaired platelet release from megakaryocytes

Bone Marrow Evaluation

• Not routinely required

• If done:
• Increased megakaryocytes
• ↑ Megakaryocytic precursors

Clinical Presentation

• Common manifestations: Petechiae, Ecchymosis, Epistaxis.

• Usually normal on general physical examination.

• Absence of:
• Fever, Pallor, Jaundice.
• Splenomegaly 
• Bleeding into joints.
• Lymphadenopathy 

ITP Classification and Features

• Platelet count ↓
• due to antibodies against GP2B3A.

• Bleeding time ↑

Feature	Acute ITP	Chronic ITP
Demographics	Seen in children	Seen in adults ↳ Female > Male
History	Follows viral infection	No viral history
ㅤ	Platelet Count < 50,000	Platelet Count < 10,000
Duration	Self-limiting	Persists for > 6 months
Treatment	No long-term treatment needed	• Requires steroids  • Splenectomy

Management of Adult ITP

• 1st episode of ITP
• If no significant bleeding
• Wait and watch

• 1st line treatment:
• Oral Prednisolone 2 mg/kg/day for 5-7 days.
• If there is no response → IV Ig or Anti-D.

• Relapsed Cases (2/3rd of patients):
• Steroids + Azathioprine.

• Refractory Cases
• Rituximab is considered if there is no response.
• Splenectomy in refractory cases.

• Fostamatinib

• Maintenance
• Thrombopoietin receptor agonists - TPO agonists
• Eltrombopag (Oral).
• Romiplostim (Subcutaneous injections).

• Avoid Platelet transfusion