Hematology and Thrombosis

• Circulatory hemostasis is balance between bleeding and clotting

Major Components of Hemostasis

• Vascular System
• Platelet (Thrombocytes)
• Blood Coagulation Factors
• Fibrinolysis and ultimate tissue repair

Minor Factors

• Complement
• Kinin System
• Serine Protease

Processes Involved in Hemostasis After Injury to a Small Vessel

• Blood vessel spasm
• Formation of Platelet plug
• Contact among damaged blood vessel, platelets, and coagulation proteins
• Development of blood clot around the injury
• Fibrinolytic removal of excess hemostatic material to re-establish vascular integrity

Blood Vascular System

• The order from Artery to Vein is: Artery > Arteriole > Capillaries > Venule > Vein

Coats/Tunics of The Blood Vessels

• Tunica Intima (inner tubular cavity): Made up of endothelium (simple squamous epithelial), endothelial cells thickened by subendothelial connective tissue layer

• Tunica media (middle): Thickest coat, smooth muscle + elastic fiber

• Tunica adventitia

• Arterioles are the microscopic continuation of arteries that gives branches to the metarterioles which joins the capillaries

• Venules are microscopically sized veins, and connects capillaries to the vein

• Capillaries + arterioles + venules are the major vessel of microcirculation

• Sinusoids are specialized capillaries found in the BM, spleen and liver

Vasculature Physiology

• Vasoconstriction is a short lived reflex reaction of the smooth muscle in the vessel wall produced by the sympathetic branches of the autonomic nervous system
• Stenosis/ narrowing of the blood vessel is a normal response of the body to the pain

The Role of Endothelium

• The endothelium contains connective tissue such as collagen and elastin
• The endothelium is highly active metabolically and involved in clotting process by producing or storing clotting components
• Plasminogen Activator in the endothelium allows rapid lysis of fibrin clots

Weibel - Palade Body

• The Weibel-Palade Body is an organelle of the endothelial cells discovered by Weibel and Palade in 1964
• The storage granule for Von-Willebrand factor "VWF" (bridge to connect platelet and collagen), a molecule for platelet adhesion
• It is also, a storage for P selectin (a receptor for leukocytes)

Endothelial Functions

• Angiogenesis
• Coagulation
• Inflammation
• Immune response

Endothelial Dysfunction

• Tumor necrosis factor
• Interleukin 1
• Viral infection
• Bacterial toxin
• Cholesterol
• Oxidative modified lipoproteins

Processes if the Endothelium is Disrupted

• Initially, rapid vasoconstriction occurs for up to 30 mins, reduces blood flow and promotes contact activation of platelets and coagulation factors
• 2nd phase - platelets adhere immediately to the exposed sub endothelial connective tissue particularly collagen
• The aggregated platelets enhance sustained vasoconstriction by releasing thromboxane A2 and vasoactive amins (serotonin and amine)
• 3rd phase - coagulation is initiated through both the intrinsic and extrinsic system
• Finally, fibrinolysis occurs to re-establish vascular integrity

3 Essential factors for Vascular Integrity

• Circulating Platelets

• Adrenocorticoids

• Ascorbic acid

• The integrity of arterioles and venules depends on vasoconstriction, formation of platelet plug to the site of injury and formation of fibrin clots

• Arteries are thicker and more resistant to disruption but more dangerous in cases of injury

Platelets

• Megakaryopoiesis: Megakaryoblast > Promegakaryocyte > megakaryocyte > Platelet
• Early precursor is BFU -M and CFU

Mature Platelet

• Younger platelets are larger than the old ones
• Inactive/unstimulated platelets circulates as a thin smooth-surfaced disk

History in Hemostasis

• 2nd century A.D: hemophilia was first recognized

• 12th century A.D: Moises Maimonides reported 2 deaths due to bleeding after circumcision

• 1803: clinical description of families with hemophilia (love of hemorrhage) was first published by Johann Lukas Schonlein

• 1842: platelets were described

• 1905: Theory on blood coagulation was accepted

• 1913: Lee and White blood clotting time was performed

• 1930: Introduction of prothrombin time

• 1940: Platelet count and bleeding time were introduced

• 1964: cascade and waterfall theory was introduced - about fibrin mesh

• Hemostasis is the balance between bleeding and clotting & involve interaction between blood vessel, platelets, coagulation, fibrinolysis and body repair

Process to Maintain Normal Hemostais

• Stop the bleeding by producing clot
• Maintain the clot integrity
• Dissolve the clot and let the tissue repair

Timeline for Platelet Increase

• 5 days = start to increase platelet

• 8-9 days = turnover for megakaryopoiesis

• 12 days = days it takes to maximally optimize platelet production

• 1/3 (33%) of platelets are located on the spleen; meanwhile 2/3 (66%) of them are in the circulation (some books say 30% in the spleen and 70% - in the circulation)

Platelet Normal Values

• SI = 150-450 x 10^9/L (others 200-400x 10^11/L)
• Conventional = 150,000 – 450,000 UL
• Platelet count is Slightly higher in women than in men, and slightly lower in both sexes when over 65 yrs old

Thrombopoietin (TPO)

• A glycoprotein hormone
• Size: 70,000 daltons
• 23% homologous with EPO

Platelet Production Sources

• Liver (most copies/primary)

• Kidney (major for EPO)

• Smooth skeletal muscle

• Stromal cell

• Bone Marrow: Regulates the production of platelets, stimulates and differentiates megakaryocytes

• Blast cells resemble lymphocyte under light microscope, lymphocyte resembles blast cells, agranulocyte, rubricyte, mature/condensed and coarse chromatin

Megakaryocyte Progenitors

• MK-1/ Megakaryoblast is 20-50 UM in diameter, possessing blue cytoplasm at a N:C ratio 10:1

• MK-II / promegakaryocte is 20-60 UM in diameter with an indented nucleus - last stage capable of endomitosis

• MK-III/ megakaryocyte is 40-120 UM in diameter possessing multiples nuclei with no visible nucleoli

• MK-III counts for <0.5% of all the BM cells, at 2-4 of this cell/10x field in a bone marrow aspirate smear and can also be found in the lungs (if necessary can produce blood cells)

• Thrombolite is 1-4 UM in diameter, w/ an Ave size =2.5 UM and is consistent with normal MPV = 8-10 fL

Platelet Characteristics

• Hyalomers, surrounds the chromomere, non-granules
• Average Healthy Human produces 10^8 megakaryocytes producing 10^11 platelets/day
• When differentiating megakaryocyte stages pay more attention to the cytoplasmic appearance rather than chromatin and nucleus (same with other hematologic cells)

Ploidy

• Number of complete sets of chromosomes in a cell

• 2N to 64N (most are 8N and 16N)

• Some megakaryocyte nuclei replicate 5 times reaching 128N (which is unusual but may signal hematologic disease)

• Platelet structure contains anucleate, with a Diameter: 1-4 UM

Platelet Shape and Composition

• Shape: biconvex disc, circular to irregular, lavender and granular under wright-stained smear and purplish blue under wright giemsa

• 60% protein, 30% lipid, 8% carbohydrates, 2% - various minerals, water and nucleotides

• Life span = 10 days, and In the smear = 8-20 platelets/HPO

• Four Areas: Peripheral, Sol-gel, Organelle, Membrane system

Peripheral Zone

• Is composed of: Glycolyx which is a fluffy coat that surrounds the platelets
• This glycocalyx is unique among cellular components
• Glycoprotein receptors of the glycocalyx mediates contract reactions of cell to membrane and the change of cellular shape/ the release of internal components such as serotonin

Sol- gel Zone

• Structural Zone

• Composed of Microfilaments

• Containing protein actin and myosin which, upon simulation of platelets, interact to form actomyosin.

• Contractive protein important in clot retraction

• Protein tubulin -Microtubules- Maintain the platelets disc shape

Platelet Function

• Forms the cytoskeleton of cell
• Provides and maintains the discoid shape
• Maintains the position of organelles
• Microfilaments- secretion of blood coagulation products such as fibrinogen

Organelle Zone Components

• Alpha granules
• Dense/delta granules
• Lysosome
• Glycogen
• Mitochondria

Alpha Granule Contents

• Beta thromboglobulin
• Fibronectin
• Albumin
• Alpha 2- antiplasmin
• Thrombospondin
• Platelet factor 4(PF4)
• PDGF ( platelet derived growth factor)
• Plasminogen
• Factor 1 (Fibrinogen)
• Factor V
• Factor VIII
• Von Wildebrand factor
• High molecular weight Kinninogen (HMWK)
• C1 Esterase inhibitor

Dense/Delta Granule Contents

• Calcium
• Catecholamines
• ADP and ATP
• Pyrophosphate
• Serotonin (5- hydroxy tryptamine)
• Magnesium
• Lysosomal granules contain Neutral protease, Acid hydrolase, Bactericidal enzymes, Glycogen - for energy storage, Membrane phospholipid and Thromboxane A2 precursors

Membraneous System

• Open canalicular system/surface connecting system (Platelet to environment) and Dense tubular system
• Eicosanoid, Vasodilation/Vasoconstriction, Sleep promotion, Pain promotion and Fever promotion

Platelet Funtion

1. Adhesion
2. Activation
3. Secretion
4. Aggregation

Platelet Plug Formation

• Primary - platelets, collagen and von Willebrand factor
• Secondary coagulation – fibrin mesh – clotting factors
  • Platelet to foreign substance and to collagen (a protein in the tissue)

Platelet Activation

• Morphologic and functional changes in platelets, a movement from discoid to spherical with pseudopods
• Requires calcium and actomyosin
• Agonists are substances that indicate platelet activation

Platelet Aggregation / Secretion

• Releases/ secretes granules that forms Platelet plug

Cellular Regulators

• Cellular protease in Lysosome of Granulocytes can limit the spread and formation of clotting and reliquification of clots

Blood Vessels as Natural Anticoagulants

• Substances that are intact with endothelial cells like Prostacyclin (PG 16) aka Prostaglandin 12 which inhibits the activation of platelets, Adenosine, Thrombomodulin, TPA and Von Willebrand Factor, Glycoprotein which are important for platelet adhesion.

Natural Anitcoagulant System

1. Anti-Thrombin III (AT-III)

• Series protease inhibitor that slows major inhibitor of coagulation
• Contains A2 globin glycoperotein, Serpin super family of serine proteinases
• AT - III with Heparin = Rapid inhibitors of Activated factor II, IX, XX,XI, XII which speeds AT - III and thrombin binding complex

2. HC-II

• Endothelial cells have heparin-like molecules that activate AT -III heparin co-factor, and AT - III + HC - II allows rapid inhibition

3. Protein C (APC)

• Alteration in these proteins are associated with thrombosis
• A vitamin K dependent plasma protein synthesized by the the Liver
• Naturally circulates as a zymogen
• Zymogen protein C – activated protein C renders factor V and VIII inactive

4. Protein S

• Co-factor protein C

1st Natural Anticoagulation Mechanism

• Endothelial cell two chemicals nitric oxide and prostacytin (PG1-2) – These two chemicals keep the platelet inactive.

2nd Mechanism: The role of heparin and Antithrombin 3

• Heparin binds with Antithrombin III (AT III)
• When HC II and AT - III combined it renders Factor II, IX, X

3rd Mechanism of Natural Anticoagulation

• Thrombomodulin binds to thrombin then; moon-shaped- passes through thrombin that is attached to modulin
• Prostaglandin 12 inhibits the activation of platelets

4 Stages of the Mechanism of Coagulation

• Vascular Spasm
• Primary Coagulation
• Secondary Coagulator
• Clot Retraction
• Fibrinolysis and Tissue Repair

Vascular Spasm

• Vascular injury exposes sub endothelium with vasoconstriction which follows:

Vascular Spasm Components

1. Endothelins (released damaged endothelial cells which is a chemical signal sent to receptor resulting in each muscle cells contraction and vasoconstriction)
2. Myogenic response (myo means muscle)
3. Pain Receptor Activation which makes pain receptor release cytokines

Primary Coagulation Component

• Platelet Clot formation
• Willbrand factor will attached to collagen which creates a Platelet adhesion factor

Platelet Activation

• Activated platelet releases ADP, T2A2, and Serotonin and causes an Aggregation of platelets by binding to platelet with a bridging protein called Gpllbllla

Secondary Coagulation and the Coagulation Cascade

• Clotting Factors are the main characters of second coagulation which are provided by hepatic cells/
• Endothelial cells also play a role in hemostasis and thrombosis
• The process of blood coagulation is known as the “Coagulation Cascade" : Factor II (Prothrombin)-> Factor Ila (Thrombin)
  • Zymogen (Inactive) to Active Form _ Factor XII-> Factor Xlla

Characteristics of all Coagulation Factors

• A deficiency of the factor generally produces bleeding tendency disorder with the excerptor of factor XII, Prekallikrein (fletcher factor) and high molecular weight kininogen (HMWK) which leads to hemophilia
• The physical and chemical characteristics of the factor are known, including the fact the synthesis of the factor is independent of other protein

3 Groups of Coagulation Factors

1. Fibrinogen Group
2. Prothrombin Group
3. Contact Group

Fibrinogen Group of Coagulation Factors

• Includes: Factors I, V, VII and XIII which get consumed during coagulation
• Factors V and VIII are known to decrease during blood storage in vitro and increase in pregnancy or inflammation as well as through the subsequential use of oral and contraceptive drugs

Prothrombin Group of Coagulation Factors

• Factors II, V, VII, IX and X which are dependent on Vitamin K during synthesis
• Vitamin K : Is available to the body through dietary sources and intestinal bacterial production and Inhibit by warfarin while being stable to remain well in the stored plasma

Contact Group of Coagulation Factors

• Factors XI, XII, Prekallikrein (fletcher factor) and HMWK which are :Involved in the intrinsic pathway and Moderately stable and not consumed during coagulation

Factor I : Fibrinogen

• Fibrin I activated
• Large, stable globin (341, 000 molecular weight)
• Precursor of fibrin which result to clot when fibrinogen is exposed to thrombin 2 which gets consumed in blood coagulation

Factor II Pro Thrombin

• 63,000 molecular weight, stateable protein
• In the presence of ionized calcium, prothrombin is converted to thrombin by enzymatic action of thrombin from both extrinsic and intrinsic sources
• Prothrombin has a half-life of almost 3 days with 70% consumption during clotting

Factor IIa: Thrombin

• Molecular weight of 40,000, activated form of prothrombin
• Proteolytic enzyme that interacts with fibrinogen also a patient platelet aggregating substance and large quantity converted consume in conversion of fibrinogen to fibrin
• One unit of thrombin will coagulate 1mL of standard

Tissue Thromboplastin

• Commonly called Factor III is term given to any non-plasma substances containing lipoprotein complex form tissue which allows any to convert prothrombin to thrombin

Ionized Calcium

• Commonly called Factor IV, is necessary for the activation of thrombin and thrombins conversion

Factor V Proaccerlerin

• Extremely labile-globular protein, that rapidly deteriorates at half life of 16 hrs at most

Proconvertin - Factor VIII

• A beta-globulin is an essential component of the intrinsic thromboplastin-generating mechanism and is not destroyed or consumed in clotting while stable in serum when left in the room

Antihemophilic Factor - Factor VIII

• An acute phase reactant that is rarely consumed and very labile
• Functional components: Factor VIII: C, Factor VIIIC: Ag and Factor VIIIC :RCO

Factor IX – Plasma Thromboplastin Component

• Is a stable protein that is neither consumed during clot nor destroyed for two weeks

Factor X : Stuart Factor - Contains alpha lobin, stable protein that is not consumed during coagulation and is only effective with V and Calcium

Factor XI - Plasma Thromboplastin Antecedent

• Includes Beta globulin and stable factor

Factor XII – Hegeman Factor

• Is stable not consumed

Common Notes on Blood Coagulation

• Intrinsic Pathway
• Extrinsic Pathway
• Common Pathway
• Clot Retraction and Fibrinolysis And Tissue Repair

Explore key components of hemostasis, including vessel wall layers, Weibel-Palade bodies, and the fibrinolytic system. Learn about platelet production, function, and storage, as well as megakaryocyte maturation and characteristics. Discover platelet's role in the inflammatory response.