BLOOD

Part 2
MHR

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 Platelets (Thrombocytes)
• Small, disc shaped, granulated, active structure
• Non-nucleated (not reproduce)
• 2-4 micron (cell fragment)
• Life span=short (5-9) days

Steps Of production
• Pluripotent (uncommitted stem cells) → myeloid stem cell → megakaryoblast→ megakaryocytes
→platelets

Structure of Platelets

A. Platelet Membrane:
• Phospholipid bilayer membrane
with negatively charged Glycoprotein coat
(to prevent adherence to normal endothelium), adhere to injured endothelium
• Contain > 50 glycoprotein receptors for:
▪ Collagen (GPVI)
▪ Von Willebrand factor (GP Ib),
▪ Fibrinogen (GP 11b/111a),
▪ Thrombin, ADP
▪ Thromboxane A2 (TXA2).
• Plasma membrane has an open canalicular system:
▪ pathway for uptake of calcium and release of intracellular substances.

B. Cytoplasm: contain
• Cytoskeleton of microtubules,
• Golgi, Mitochondria, Lysosome , Glycogen, residuals of endoplasmic reticulum
• Contractile proteins (actin, myosin) →enable active platelet to change shape
• Granules: see later

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 HEMOSTASIS

Definition prevention of blood loss after injury, Consists of:

A. Local VC of injured vessel

B. Formation of Temporary hemostatic plug
(primary hemostasis)
C. Conversion of temporary platelet plug into definitive clot via fibrin thread
(blood coagulation) (Secondary hemostasis).
D. Dissolution of Clot via plasmin & repair of vascular endothelium and smooth muscles
by PDGF and vascular endothelial growth factor (VEGF) released from the platelets

I. Local Vasoconstriction: immediate (may be so marked & close the lumen),

VC due to:

1. Nervous reflexes: initiated by stimulation of pain receptors in subendothelial layer of traumatized vessel.
2. Local myogenic contraction due to direct trauma to the smooth muscles
3. Humoral (Chemical) substances: ADP, serotonin, thromboxane A2 secreted from platelets.
4. Endothelin: is a vasoconstrictor, secreted from injured endothelial cells.

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II. Formation of Temporary Hemostatic Plug: (platelet reactions) ((primary hemostasis)
Can stop blood loss completely in small injury

1. Platelet adhesion: via receptors to exposed sub-endothelial collagen & von Willebrand factor.
o Von Willebrand factor: glycoprotein formed by endothelial cells & found in subendothelial tissue
Also present in plasma, bound to factor VIII
2. Platelet activation
• caused by adhesion →that initiate intracellular signaling→ cause platelet activation
→platelet swell, change shape, put out pseudopodia
→release granules
• Activation is enhanced by ADP, thrombin
3. Platelet Release reaction: (Ca++ dependent reaction)→ release of
➢ Dense granules: non-protein e.g.
❖ Calcium (cause more release)
❖ ADP (activation, aggregation, fusion of platelet)
❖ Serotonin (reinforce and maintain VC)
➢ Alpha granules: ) proteins(
❖ Clotting factors: as fibrinogen, factor V, von-Willebrand factor and factor Xlll.
❖ Platelet-derived growth factor (PDGF) → stimulate growth and multiplication of
endothelium, smooth muscles and fibroblasts.
❖ Platelet activating factor (PAF): help platelet aggregation
Membrane phospholipids
PAF (phospholipase enzyme)
Arachidonic acid
Cyclooxygenase
Prostaglandin

Thromboxane synthase Prostacyclin synthase

(Of platelets) (Of healthy endothelium)

Thromboxane A2 Prostacyclin
1. VC 1. VD
2. ↑Platelet release 2.  Platelet release
3. ↑Platelet aggregation 3.  Platelet aggregation

✓ Prostacyclin opposite to thromboxane A2→ keeps platelet plug localized to site of injury &
Prevent clot spread to healthy area
✓ Aspirin inhibits COX→↓ thromboxane A2 & prostacyclin
✓ Endothelial cells can produce new COX within hours, Platelets cannot.
✓ Daily intake of aspirin →↓clot formation & prevents myocardial infarctions.

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4. Platelet aggregation (platelet plug formation)
❖ by ADP, PAF, thromboxane A2→ cause platelets to bind to each other through fibrinogen receptors
(Gpll/llla).
❖ Receptor- bound fibrinogen acts as a bridge between two Gpll/llla molecules on adjacent platelets.
❖ platelet aggregation → cause release of the granules
→more ADP, Thromboxane A2,
→more aggregation
→self propagating process
→results in formation of a platelet plug.

5. Platelet pro-coagulant activity:

▪ platelet factor 3 (PF3) is exposed on platelet membrane
▪ → provide ideal surface for concentration & activation of clotting factors
(platelets act as catalysts for coagulation)

6. Platelet irreversible fusion: by ADP, thrombin & platelet enzymes.

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 III. Blood Coagulation (Clotting) (secondary hemostasis)
o Conversion of temporary platelet plug into definitive clot via production fibrin thread (stabilizes plug)
o By clotting factors" plasma proteins" (mainly -globulins), synthesized by the liver
o Proteolytic inactive enzymes,
o When activated→ activate other →cascade of reaction → end in clot formation
Coagulation factors are classified into 3 groups:
Fibrinogen group Prothrombin group Contact Group
Factor I (fibrinogen), V, VIII, XIII Factor II (Prothrombin),VII, IX, X factors XI and XII
Activated by thrombin Need vitamin K for synthesis in Activated by contact to
liver electronegative charge

Mechanism of Coagulation
A. Intrinsic Pathway occurs both in vivo and in vitro

Contact with subendothelial collagen (in vivo)

Contact with -ve charged wet surfaces e.g. glass (in vitro)
1. XII XIIa
Accelerated by (HMW) kininogens & plasma kallikrein (protein)

2. XIIa activate XI
3. XIa activate IX
4. IXa form complex with VIIIa (activated by thrombin (separate it from VWF) PL+ Ca activate X

5. Xa in presence of (phospholipid+ Ca + Va) activate Prothrombin into thrombin

B. Extrinsic Pathway occurs only in vivo

• Tissue trauma ( vascular or extravascular )→ release thromboplastin (tissue phospholipid ,TPL,

factor III) → activate VII
• TPL+ VIIa in presence of phospholipid (TPL) + Ca → activate IX & X
• Xa in presence of (phospholipid+ Ca + Va) activate Prothrombin into thrombin

The Common Pathway

• Thrombin convert Soluble Fibrinogen polymer into insoluble fibrin monomer
• Fibrin monomer polymerize spontaneously → loose mesh of fibrin
• Loose mesh is converted into tight mesh (strand) by XIII
(XIII= Fibrin stabilizing factor (activated by thrombin) in presence of Ca +2)
• Net: formation of clot= meshwork of fibrin threads, run in all directions (Platelets, blood cells
and plasma entrapped in the clot).

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Interaction between two systems
1. In the body, following injury, Clotting initiated by both systems simultaneously.
2. Extrinsic system is rapid (15 sec) & and extensive, intrinsic system is slow (1-6 min).
3. Extrinsic system →
• Leads to formation of thrombin rapidly→ activates factors VIII & V leading to activation of intrinsic
• VIIa (extrinsic)→ activates IX (intrinsic factor) , X (common).
4. When critical amount of thrombin is formed, vicious circle develops causing more blood clotting
5. Ca++ is required for all the reactions except the first 2 steps in intrinsic system

Anticlotting Mechanisms = (Physiological limitations of blood coagulation)

Aim prevent blood clotting in healthy blood vessels

(only clots in pathologic conditions as injury to the vessel wall or atherosclerosis)
1. Healthy endothelium: is antithrombotic and anticoagulant tissue →preventing blood clotting
• Acts as a barrier between blood & subendothelial collagen, →preventing activation of
platelets and coagulation factors.
• Endothelial surface is smooth and negatively charged repelling the negatively charged
platelets and coagulation proteins.
• Produce NO and prostacyclin (PG12) = antiplatelet agents.
• Express thrombomodulin on their surface →forms complex with thrombin, →activates
protein C , together with its cofactor protein S →inactivates Va and VIIla.
• Produce tissue plasminogen activator (TPA) = part of fibrinolytic system → activates
plasminogen to plasmin→ that degrades fibrin into fibrin degradation products (FDPs).
N.B. Streptokinase, urokinase (bacterial enzymes), Human TPA produced by rDNA
techniques, is given soon after onset of myocardial infarction (within 6 hours) → lyses
clots in coronary arteries.
2. Heparin: present in small amounts in the blood and combines with antithrombin III to inactivate
factors IX, X, Xl & Xll
3. Liver: inactivates any activated Coagulation factors
4. Continuous normal flow rate of blood is important because stasis allows intravascular clotting ,
(prolonged bed rest after operations can lead to thrombosis .)
5. Balance between TXA2 and prostacyclin limits the clot to the site of injury allowing blood to flow
freely in the vessels.

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6. Fibrinolytic System:
a. All endothelial cells, except those in the cerebral microcirculation, express thrombomodulin
b. Thrombin + thrombomodulin →thrombomodulin-thrombin complex →activates protein C with its
cofactor protein S→
• Inactivate factor V & VIII.
• Inactivate inhibitor of tissue plasminogen activator (TPA)
• ↑formation of plasmin.
c. Plasmin (fibrinolysin): lyses fibrin & fibrinogen→ fibrinogen degradation products (FDP) → inhibit
thrombin.

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 Anticoagulants

Definition substances prevent blood clotting.

A. In vitro anticoagulants prevent clotting outside body, e.g. in test tube:
1. Removal of Ca2+ ions: Oxalates precipitation of Ca2+ as salt
Citrates (used in blood transfusions) deionizing Ca2+ ions
2. Silicon coated (un-wettable) tubes prevent activation of factor XII & platelets.
3. Heparin.

B. In vivo anticoagulants prevent clotting in body.

Heparin Dicumarol
Origin Mast cells and basophils Plant
Mode of Action Facilitates action of Antithrombin III Competitive inhibition of vitamin K:
→ blocks activity of IXa, Xa, XIa, XIIa inhibits formation of II, VII, IX, X,
protein C
Site of Action In vivo and in vitro Only in vivo
Onset Rapid Slow
Duration Short long
Administration Injection Orally
Antidote Protamine sulphate 1% Vitamin K
Blood transfusion Blood transfusion

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 Abnormalities of Hemostasis

A- Conditions that cause excessive bleeding:

1) Thrombocytopenic purpura:
➢ Platelet deficiency < 50,000/mm3 or defective platelet function (thrombasthenia→ causes
subcutaneous hemorrhages (petechial)
➢ Prolongation of bleeding time

2) Vitamin K deficiency:
➢ Vitamin K is fat-soluble vitamin formed by intestinal bacterial flora.
➢ Vitamin K →essential for formation of factors II, VII, IX and X , protein C and S by the liver
➢ Causes of Deficiency:
• Absence of intestinal bacterial flora in newborn infants &
• Prolonged treatment with antibiotics
• Obstruction of bile duct (as bile needed for absorption)
➢ Prolongation of clotting time

3) Hemophilia:
➢ sex-linked Recessive disease (on X chromosome).
➢ carried by female to their males sons
➢ 3 types: Hemophilia A: absence of factor VIII (85%) classic hemophilia
Hemophilia B: absence of IX (10%)
Hemophilia C: absence of XI (5%)
➢ characterized by episodes of hemorrhage in joints or internal organs after minor trauma
➢ Prolongation of clotting time.

B- Conditions cause excessive intravascular clotting: “Thromboembolic Conditions”:

• Slow blood flow as in leg veins due to prolonged bed rest after operations, varicose veins
• Roughness of endothelium in atherosclerosis

C- Conditions→ excessive bleeding & intravascular clotting, (Disseminated

intravascular coagulation) DIC

• Wide spread clotting + bleeding tendency due to consumption of platelets & clotting factors.
• Cause: Due to massive thromboplastin production from necrotic tissues
E.g. retention of dead fetus in uterus for weeks or in septicemia or shock and tissue necrosis

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 Hemostatic Tests

1. Bleeding time
• Time needed for bleeding to stop without clotting (1-3 minutes),
• Depends on platelet count and function,

2- Coagulation time
• Time needed for blood to clot = 3-10 minutes.
• Prolonged in extrinsic and intrinsic system disorders as vitamin K deficiency, hemophilia,
liver diseases.

3- Prothrombin time
• Test for extrinsic pathway (VII)
• Normal value= 15 seconds , prolonged in vitamin K deficiency

4. Activated partial thromboplastin time (APTT)

• Test for intrinsic pathway (VIII, IX, XI, XII)
• Normal value= 30-40 sec., prolonged in hemophilia

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 BLOOD GROUPS
ABO system RBC’s membrane contains either A or B antigen.
Blood Group (phenotype) A B AB (universal recipient) O (universal donor)
Genotype AA or AO BB or BO AB OO
Agglutinogen (antigen) A B A + B No antigen
Agglutinin (antibody) Anti-B Anti-A No antibody Anti-A +Anti-B
% 40% 10% 5% 45%

Importance of blood group determination

1- In Blood transfusion
➢ Cross matching test is done = Recipient serum (antibodies) mixed with donor’s blood (RBC’s)
To avoid incompatible blood transfusion that result in agglutination.
(Reverse rarely occurs due to dilution of donor’s antibodies in large volume of recipient blood)

2- Medico-legal (good negative test): to exclude not to prove paternity since many persons
have the same blood group.

Rh factor: (D factor)
➢ Discovered in Rhesus monkey.
➢ 85% Rh positive. 15% Rh negative.
➢ Both have no anti- D
➢ Only formed in Rh negative persons if receive antigen D (Rh positive blood) (become sensitized)
➢ if that person receives Rh positive blood again→ will result in agglutination and hemolysis of RBC’s

Importance of Rh factors
1. Erythroblastosis Fetalis: (Rhesus hemolytic disease of new born)
➢ Rh +ve male married Rh -ve female → Rh +ve fetus
➢ During delivery: Rh +ve fetal blood → enter maternal circulation
→anti-D (IgG) is formed. (sensitization)
➢ During next pregnancy: anti-D (IgG) cross placenta → causes agglutination & hemolysis of fetal RBC’s
➢ At birth: baby is Anemic, jaundiced (↑bilirubin), Kernicterus (bilirubin cross blood brain barrier)
or born dead
➢ Prevention:
A. Rh -ve female should never receive Rh +ve blood
B. Anti-D is given immediately after delivery to neutralize D antigen & prevent sensitization
1st baby affected in previously sensitized RH -ve mother: via previous transfusion with Rh +ve blood
No fetal complication regarding ABO system as ABO antibodies cannot cross placenta (IgM)
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2. Repeated blood transfusion
• If Rh -ve receive Rh +ve blood→ anti D is formed
• Next transfusion with Rh +ve blood→ result in agglutination.

Blood Transfusion

Indications
1- Hemorrhage (Restore whole blood)
2- Restore one element, e.g. RBCs, WBC’s, platelets, plasma proteins and clotting factors
3- Erythroblastosis fetalis.

Precautions before blood transfusion

1. Compatible (cross matching test), RH negative person is transfused with RH negative blood
2. Free from diseases or contamination.
3. Fresh, not stored > 3 weeks at 4 C, high HB%.

Complications of Incompatible blood transfusion→ agglutination & hemolysis→ lead to

A- Clumping of RBCs →Blockage of Capillaries → backache, joint pain, anginal pain (coronary blocked).
B- Intravascular hemolysis→ release of
1) Histamine→ (VD) →↓ABP (Shock)
2) K→ Hyperkalemia →cardiac arrhythmia.
3) Hb →
• Hemolytic jaundice: bilirubin is produced from Hb→ yellow coloration of skin & mucous membrane
• Filtered by renal glomeruli→ form acid hematin→ blockage of renal tubules →renal failure.

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 Leucocytes (White Blood Cells WBCs)

Mobile units of the body’s protective system.

Steps of production in bone marrow:

interleukins
❖ Multipotential stem cells (uncommitted stem cells) → committed stem cells →
divide and mature into 2 separate stem cell lines:
• Myeloid line→ gives rise to 3 types of granulocytes, monocytes and megakaryocytes.
• Lymphoid line→ give rise to lymphocytes.

Leucocytic count: 4,000-11,000 WBC’s/mm3 blood.

Leukocytosis: leucocytes ≥ 20,000/mm3
▪ causes: in any condition of tissue destruction such as inflammation with pus formation
and myocardial infarction
Leukemia: malignant disease of bone marrow leading to uncontrolled production of abnormal WBCs
→cannot protect the body against infection.
Agranulocytosis: rapidly fatal disease → bone marrow stops producing granulocytes
▪ causes: exposure to radiation or drugs and toxins.

Inflammatory response

Definition
• Response of immune system to tissue injury (may be due to infection, trauma, extremes of heat
and cold and chemical agents)
• Defense mechanism →vital to health.
4 cardinal signs of inflammation
1. Redness: caused by the dilation of small blood vessels around the injury
2. Heat: from increased blood flow to the area
3. Swelling: caused by accumulation of fluid in ISF due to ↑capillary permeability
4. Pain: induced by chemical mediators of inflammation, such as bradykinin, histamine, serotonin,
and prostaglandins
Cells involved: mainly, neutrophils and monocytes.

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 Inflammation mobilizes defensive cells to the site of injury, →kills pathogens, and limits their
spread and initiates tissue repair.
It occurs in any tissue but is most observed in the skin

Steps involved in the inflammatory response:

via action of inflammatory mediators produced at inflammatory site on nearby capillary walls
1. Margination: sticking of neutrophils and monocytes to adhesion molecules expressed by endothelial cells
2. Diapedesis: Both cells squeeze through the pores between endothelial cells
3. Chemotaxis: they move by amoeboid movement towards site of inflammation
• Chemotaxins: substances attract phagocytes to site of inflammation as breakdown products of
inflamed tissues, bacterial toxins
• Negative chemotaxis: bacterial toxins repel neutrophils
4. Opsonization: When immunoglobulin and complement proteins coat the bacteria → make them tasty
to phagocytes
5. Phagocytosis: bacteria bind to receptors on phagocytic cell membrane and is engulfed into the cell
• A phagocytic vacuole is then formed with which lysosomes fuse and release their enzymes to kill
the bacteria.

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