Blood Clotting (Coagulation)

The process of blood clotting and then the subsequent dissolving of the clot, following repair of the injured tissue, is termed Haemostasis. A problem with the coagulation system is called a Coagulopathy.

i.e. Depletion of a factor (absence)

Deficiency of a factor (decreased amount) Dysfunction of a factor (not working)

John Santangelo

John Santangelo
Bone Marrow

Platelets
Small, granulated bodies that aggregate at the site of injury

Non-nucleated Half-life of 4 days

Count- 150 400 x 109/L of blood

Made from megakaryoblasts in Bone marrow

Thrombopoiesis
Pluripotent stem cell Committed stem cell Megakaryoblast Megakaryocyte Platelets

Megakaryocytes
Giant cells- 35-160m in diameter Multinucleated

Forms 2000-4000 platelets Platelets formed by pinching off of cytoplasm Megakaryocyte

Thrombopoietin Produced in kidney and liver Cause megakaryocyte maturation

Bound to platelets- feedback

Platelet morphology
Non-nucleated, Colorless disc, 2-4 m in diameter
Cell membrane: 6 nm in thickness Phospholipids important in initiation of Coagulation Receptors for ADP, vWF, collagen, fibrinogen, thrombin

Functions of platelets 1.Hemostasis 2. Coagulation of blood 3. Clot retraction 4. Phagocytosis 5. Storage and transport- serotonin & histamine

Primary Haemostasis
Dependent on Platelets and Von Willebrand Factor (vWF)
Platelets gather and attach to vWF

vWF is needed for platelet adhesion

Platelets degranulate after attachment and release ADP and Thromboxane which attracts more platelets Forms a platelet plug Requires endothelial damage to adhere

Secondary Hemostasis

Platelet aggregation initiates secondary haemostasis through the coagulation cascade Coagulation cascade is initiated by the intrinsic or extrinsic pathway The final cascade results in fibrin deposition cross-linking platelets and clot formation

Thrombocytopaenia:
A reduction in platelets below the Normal Reference Range (NRR)

Bone marrow depression

Hypersplenism
Unknown cause

Viral infections

Idiopathic Thrombocytosis: Adrenalin

Splenectomy

Epinephrine administration

Platelet NRR = 150 400 x 109/L

Composition of Blood Cellular part: 1. Red Cells (erythrocytes) 2. White cells (leukocytes) 3. Platelets (thrombocytes) Liquid part: Proteins, enzymes, chemicals, coagulation factors, antibodies, and many more. The cells float in this liquid part. If blood is anticoagulated, it is called Plasma.

If it is allowed to clot, it is called serum.

Serum is unsuitable for coagulation studies as the coagulation factors have been used up in the clot.

Vivo:
Inside the body Vitro: Outside the body

Thrombocytosis: An increase in platelets above the Normal Reference Range (NRR)

Thrombocytopaenia: A decrease in platelets below the Normal Reference Range (NRR)

Thromboasthenia: Abnormal platelet function

Normal Reference Range for platelets:

150 400 x 109/L Platelets are also called, Thrombocytes

Epistaxis: Bleeding nose Menorrhagia: Excessive bleeding during menstruation

Petechiae: Bleeding under the skin. (small red spots)

Purpura: Bleeding under the skin. (the small red spots join)

Petechiae

Purpura

Liver

Femur

The initial phase of the coagulation process is vascular constriction.

This limits the flow of blood to the area of injury.

Next, platelets become activated by thrombin and aggregate at the site of injury, forming a temporary, loose platelet plug.

The protein fibrinogen is primarily responsible for stimulating platelet clumping.

Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release the nucleotide, ADP and the eicosanoid, TXA2 (both of which activate additional platelets), serotonin, phospholipids, lipoproteins, and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug.

To insure stability of the initially loose platelet plug, a fibrin mesh (also called the clot) forms and entraps the plug.
If the plug contains only platelets it is termed a white thrombus. If red blood cells are present it is called a red thrombus.

Finally, the clot must be dissolved in order for normal blood flow to resume following tissue repair.
The dissolution of the clot occurs through the action of the conversion of plasminogen to plasmin.

Two pathways lead to the formation of a fibrin clot: The intrinsic pathway and The extrinsic pathway.

These two pathways converge on a common pathway that leads to clot formation.
Both pathways are complex and involve numerous different proteins termed clotting factors.

Hemostasis
Injury to vessel Constriction of injured vessel (vasospasm) Platelet adhesion Platelet activation and aggregation Temporary platelet plug formation Activation of clotting factors Fibrin network True clot

Factor I - fibrinogen Factor II - prothrombin

Coagulation (Clotting) factors

Factor III - tissue thromboplastin (tissue factor) Factor IV - ionized calcium ( Ca++ ) Factor V - labile factor or proaccelerin Factor VI - unassigned Factor VII - stable factor or proconvertin Factor VIII - antihemophilic factor

Removing any factor will result in non stop bleeding

Factor IX - plasma thromboplastin component, Christmas factor

Factor X - Stuart-Prower factor Factor XI - plasma thromboplastin antecedent

Factor XII - Hageman factor

Factor XIII - fibrin-stabilizing factor

When blood is collected into a tube it clots. It clots faster in a glass tube because of the rough surface of the glass. When collected into a plastic tube it takes much longer because the plastic is has a smooth surface.

The plastic tubes are coated on the inside to promote clotting

When the skin is cut it provides a rough surface which promotes clotting as well as the cut tissue releasing tissue factor. To perform a coagulation test, unclotted blood is needed.
Coagulation test should be done within 4 hours of collection

The easiest way to stop blood clotting in the tube is to remove one of the clotting factors.

The easiest clotting factor to remove it CALCIUM. (Factor IV)

Note that we use Roman Numerals for the clotting factors. The blood collected from a patient to perform Coagulation studies is placed into a tube containing 3.8% Sodium Citrate. Sodium Citrate is an anticoagulant The ratio of Sodium Citrate to Whole Blood is 1:10 0.5mL Sodium Citrate + 4.5mL Whole Blood The Citrate combines with the Calcium making it unavailable for clotting. Therefore the blood remains liquid.

In the laboratory, calcium is added to this blood and the time it takes to form a clot in the plasma is measured.
Note that plasma is used to perform the test not whole blood or Serum.

All the coagulation factors are produced in the Liver except the following:

VIII & vWF (Von Willebrand Factor )

Factors II VII IX X are Vitamin K dependent

The normal bacterial flora in the bowel (E Coli) produces Vitamin K

It is also available from plant and animal sources

In the newborn, there is no normal bowel flora so a newborn is often vitamin K deficient. Newborn babies are given vitamin K soon after birth to prevent bleeding. Broad spectrum antibiotics also deplete the normal E Coli bowel flora.

Normally the blood remains liquid inside the blood vessels.

There are two systems that initiate blood clotting: The Intrinsic System and the Extrinsic System.

The Intrinsic pathway can be initiated by events that take place within the lumen of blood vessels.
The Intrinsic pathway requires only elements (clotting factors, Ca++, platelet surface etc.) found within, or intrinsic to the vascular system. The Extrinsic pathway is the other route to coagulation. It requires Tissue Factor (tissue thromboplastin), a substance which is "extrinsic to", or not normally circulating in the vessel. Tissue Factor is released when the vessel wall is ruptured.

Tests performed to diagnose a coagulopathy

Prothrombin Time (PT) To measure the Extrinsic system Will be abnormal if there are deficiencies in the following factors: I, II, V, VII, X

Activated Partial Thrombin Time (APTT)

To measure the Intrinsic system Will be abnormal if there are deficiencies in the following factors: I, II, V, VIII, IX, X, XI, XII Thrombin Time (TT) Will be abnormal if there are deficiencies in the following factors: I

Test for Common pathway:

Both A.P.T.T. and P.T. There is NO single screening test for the Common Pathway

Tests performed to diagnose a coagulopathy - cont

D-Dimer: When the clot breaks down (fibrin degrading) it releases fibrin degradation products. D-Dimer measures these breakdown products. Platelet Aggregation test: The platelet aggregation test aids in the evaluation of bleeding disorders by measuring the rate and degree to which platelets form a clump (aggregate) after the addition of a chemical that stimulates clumping (aggregation).

Several different substances called agonists are used in the test.

These agonists include adenosine diphosphate, (ADP) epinephrine, thrombin, collagen, and ristocetin. An agonist is a chemical that binds to a receptor of a cell and triggers a response by that cell.

Old Tests

Bleeding time Tests vascular integrity and platelet function Incision on volar aspect of the forearm 1mm deep and 1 cm long BP cuff inflated to 40 mmHg Normal < 8 minutes Borderline 8-10 minutes Abnormal 10 + minutes Affected by ASA (acetylsalicylic acid, Aspirin ) (permanent) and NSAIDs

Thromboasthenia
Platelet function disorder Tests:

Old Tests:
Bleeding & Clotting time

New Test:
PFA 100 Plate Function Analysis 100

Normal Reference Range: The Normal Reference Range for a particular test or measurement is usually defined as the prediction interval between which 95% of values of the healthy population fall into, whatever the distribution of these values. In case of normal distribution, it can alternatively be defined as the interval limited by 2 standard deviations from either side of the mean.

It is sometimes referred as "reference interval", normal range or normal values (and sometimes "usual" range/values). All these terms should be discouraged as not everyone outside the interval is abnormal, and people who have a particular condition may still fall within this interval. Therapeutic range: The range of concentrations at which a drug or other therapeutic agent is effective with minimal toxicity to most patients.

A minimum of 100 patients are selected to establish a Normal Reference Range

Total = 95.4%
Within 2 Standard Deviations