Hemostasis & Coagulation Ahmad Sh. Silmi Msc Hematology,FIBMS Unit 1 : Primary Hemostasis

Homeostasis Vessels Coagulation Proteins Platelets State of fluid equilibrium within the blood vessels Vessels Platelets Fibrinolysis/Inhibitors Coagulation Proteins

Hemostasis Arrest of bleeding Events preventing excessive blood loss Vascular spasm: Vasoconstriction of damaged blood vessels Platelet plug formation Coagulation or blood clotting

Haemostasis: Vasoconstriction Platelet activation Haemostatic plug Coagulation Stable clot formation Clot dissolution

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Primary hemostasis Involves: Vasoconstriction Platelet adhesion Platelet secretion Platelet aggregation

Vascular System Blood Vessels Arteries Veins Capillaries Carry blood from the heart to capillaries Thickest walls of the vasculature Veins Return blood from capillaries to the heart Thinnest walls of vasculature Capillaries No vessel wall Do not contribute to hemostasis

Vascular System: Blood Vessels Construction Endothelium Single layer of endothelial cells, lining vessels Coated by glycocalyx Protects basement membrane Produces Von Willebrand's factor (vWF), a part of Factor VIII Secretes prostaglandins, plasminogen activators Negatively charged, repels circulating proteins and platelets Subendothelium Smooth muscle and connective tissue with collagen fibers

Vascular System: Blood Vessels Basement membrane Collagen material – stimulates platelets Connective tissue Elastic fibers- provide support around vessels

Vascular System: Blood Vessels Function Endothelium Controls vessel permeability Controls blood flow rate Produces and releases substances that inhibit OR stimulate platelets, coagulation and fibrinolysis Subendothelium Collagen within is whats exposed upon injury

Vascular Endothelium Products: Stimulators Produces vonWillebrand factor (vWF) Helps in platelet adhesion to collagen Carries factor VIII Tissue factor (TF) activates secondary hemostasis via extrinsic pathway Tissue plasminogen activator (tPA) is released activating fibrinolysis

Vascular Endothelium Products: Inhibitors Release of tPA activates release of plasminogen activator inhibitor (PAI-1) to inhibit fibrinolysis Thromomodulin forms a complex with thrombin Platelet aggregation via prostacyclin production

Vascular System: Function Following Injury Initiate hemostasis Vasoconstriction of the arterioles Minimizes blood flow to injured area Prevents blood loss Immediate Short-lived

Vasoconstriction Mechanism Neurogenic factors Regulatory substances Prolong vasoconstriction Serotonin ( made by platelet activation & endothelium) Thromboxane A2 ( made by platelet activation & endothelium) Endothelin-1 (made by damaged endothelial cells)

Vasoconstriction Vasodilation Counteracts Vasoconstriction Endothelial cells Prostaglandin (PGI2)/ Prostacyclin Vasodilates to increase blood flow to bring fresh supplies of clotting substances Inhibits platelet aggregation Contraction of venules Causes gaps between them which pushes fluids causing edema or swelling

Thought question… Think about the last time you cut your finger with a piece of paper. Did your finger bleed immediately? If not, what might have prevented the bleeding?

Answer.. No, the finger probably did not bleed immediately, due to vasoconstriction of the blood vessels

Discussion What actions of the endothelial cells prevent clotting from occurring within the blood vessels?

Answers.. Since the endothelial lining has a negative charge, it normally repels coagulation proteins and platelets in the circulation. It synthesizes products that help to inhibit fibrin formation.

All About Platelets… Second major component of the hemostatic system

Platelets What is a platelet? Small 2-3 µm Anuclear Reddish-purple granules Fragments of megakaryocyte cytoplasm

Platelets Life span 9-10 days Normal Range 150-440 x 109 /L

Platelet: Side note Seen in conditions with increased need and/or destruction Giant platelets Micromegakaryocytes= Dwarf Megs May Hegglin anomaly, Bernard-Soulier syndrome, pregnancy, malignancy Seen in malignant disorders such as CML and MDS

Anatomy of a Platelet Peripheral zone: Responsible for platelet adhesion and aggregation Glycocalyx: Contains glycoprotein receptors: GPIb binds von Willebrand’s factor needed for platelet adhesion to collagen GPIIb/IIIa bind fibrinogen needed for aggregation Bind ADP and thrombin, promoting aggregation Factors I, V, VIII on surface, involved in 2o hemostasis Plasma membrane: Exposed on platelet activation Layer called PF3 (platelet factor) surface for interaction of plasma coagulation factors Initiation of formation of thromboxane A2. This stimulates aggregation and vasoconstriction

Anatomy of a Platelet Structural or Sol-Gel zone: Responsible for platelet retraction/contraction functions and platelet shape Microtubules Cytoskeleton Binding protein Organelle zone: Responsible for storage and platelet release functions Granules Dense bodies, alpha granules, lysosomal granules and microperoxisomes Mitochondria Glycogen

Zone PERIPHERAL Glycocalyx Phospholipid Integral Proteins: Ib/IX, IIb/IIIa Sodium ATPase STRUCTURAL Microtubules: Tubulin Cytoskeleton: Actin/Myosin ORGANELLE Granules: Alpha & Dense Enzymes: Lysosomes & Peroxisomes MEMBRANE Canalicular System – derived from DMS of megakaryocyte Dense Tubular System – calcium storage

Platelet Receptors GPIb/IX – vWF GPIIb/IIIa – Fibrinogen Required for PLT adhesion GPIIb/IIIa – Fibrinogen Required for PLT aggregation Phospholipid (Pl) Bind vitamin K dependent proteins , Ca++ dependent Bind Va and VIIIa (called “PF3” in this context)

Platelet Granules ALPHA DENSE ATP ADP Serotonin Calcium Ions Plasma Derived Fibrinogen – PLT aggrgation Ig Albumin Megakaryocyte Derived vWF – PLT adhesion, VIII carrier Coagulation Factor V Plasminogen – source of plasmin PAI-1 – inhibits activation of fibrinolysis a2-antiplasmin – plasmin inhibitor PF4 – inactivates heparin PLT-derived Growth Factor (PDGF) – tissue repair Thrombospondin – PLT aggregate stabilizer Fibronectin – PLT binding ATP ADP Serotonin Calcium Ions

Diagrammatic Representation of the Platelet

PLATELET STRUCTURE ADP, ATP, Ca2+, serotonin, etc. PF4, ß-TG, PDGF, TGF-ß, fibrinogen, VWF, etc. ADP, ATP, Ca2+, serotonin, etc.

Production of Platelets Made in Bone marrow Need dictates the amount of platelets produced. Stimulus for production is the platelet mass in circulating blood ~ 80 % and megakaryocyte mass in bone marrow Originate from CFU-GEMM to form CFU-Meg Cytokines and growth factors such as IL-3 and GM-CSF influences progenitor stages

Platelet Development Megakaryoblast 10-15 µm Increased nuclear: cytoplasmic ratio Promegakaryocyte 80 µm Dense alpha and lysosomal granules Basophilic megakaryocyte Megakaryocyte

Production of Platelets Precursor Cell= Megakaryocyte Produces about 2000-4000 platelets Platelets are released via sinuses of bone marrow

Production of Platelets Thrombopoietin (TPO) Regulates platelet development Influences all stages of megakaryocyte production Produced in the liver, kidney and spleen

Production of Platelets How does TPO work? Maintains a constant number of platelets in peripheral blood by binding Mp1 (platelet receptor). Bound TPO can not stimulate proliferation of bone marrow progenitor cells The higher the platelet count, the more TPO is bound and stimulation of bone marrow is decreased.

Thought question… If a patient had a low platelet count what will happen?

Answer… TPO increases the number of megakaryocytes in the bone marrow, increases size and DNA count of megakaryocytes and increases maturation rate

Function of Platelets Surveillance of blood vessel continuity Checks endothelial lining for gaps and breaks Fill-in small gaps caused by separation of endothelial cells Formation of primary hemostatic plug Surface for coagulation factors to make secondary hemostatic plug Aid in healing injured tissue

Formation of Primary Hemostatic Plug Once the platelets “normal” environment is changed, they become activated or adhesive Three stages of plug formation: Adhesion Release reaction Aggregation

Stage 1: Platelet Adhesion It is the binding of platelet to non platelet surface: sub endothelial collagen involves changes from a disc shape to a slightly broader, plate like form to increase surface area a number of plasma proteins are required for normal platelet adhesion. Thrombin Fibronectin vWF vWF is the largest component of factor VIII and secreted by platelets and by vascular endothelial cells.

Adhesion cont’d Increase bond that seal platelet to the vessel wall Collagen – vWF –Platelet vWF bridge physical distance between platelate and sub endothelial collagen Increase bond that seal platelet to the vessel wall reversible Adhesion

Adhesion is Reversible & No ADP is released.

Stage 1: Platelet Activation Platelets undergo a shape change from disc to spiny sphere with projections Activation required for 1O hemostatic plug formation Activation continues until Ca ++ threshold met Outcome Activation of GPIIb/IIIa receptors for fibrinogen Secretion of granules within platelets into tissues

Platelet Shape Change George Lancet 2000; 355:1531

Stage 2: Platelet Secretion & Release Requires ATP It is release of contents of the granules of platelet Primarily ADP  stimulates aggregation Cathecolamine (especially epinephrine) and serotonin enhance vasoconstriction Granules trigger a secondary aggregation which is irreversible

Platelets contain 3 types of secretary granules: Lysosome containing acid hydrolyses α-granules containing platelate specific proteins (Plt factor 4, β- thromboglobulin, as well as other proteins such as Platelet derived growth factor and coagulation proteins found in plasma (fibrinogen & von Willebrand’s factor) -granules containing ATP, ADP, Calcium & serotonin

Steps in platelet plug formation: Extension Figure 1B

Granules con’t Factor V: receptor on platelet surface for factor Xa & prothrombin PF4: heparin neutralizing factor ADP: agonist, continues to recruit and stimulate platelets by increasing cytoplasmic calcium

Steps in platelet plug formation: Perpetuation Figure 1C

Stage 3: Platelet aggregation Chemical changes cause platelets to aggregate and stick to one another Newly arriving platelets become activated by agonists Aggregation is triggered by: PLT factor 3, adenosine diphosphate (ADP), Thrombin and Thromboxane A2

Exposure of GPIIb/IIIa sites bind fibrinogen Fibrinogen + activated platelets serves as a bridge between two platelets Calcium must be present Activated platelet membrane generates TXA2 TXA2 stimulates release Irreversible once platelet aggregate they do not disaggregate.

Platelates shed membranes rich in phospholipid (appearance of PLT factor 3 on the PLT membrane) this happens during PLT plug formation serves as a catalytic site for the coagulation proteins Aggregation is also a response to helps initiation of the coagulation mechanism

Aggregation cont’d platelet aggregation

Arachidonic acid Cyclo-oxygenase PLATELET ENDOTHELIAL CELL Thromboxane Cyclic Endoperoxides PLATELET ENDOTHELIAL CELL Thromboxane synthetase Prostacyclin synthetase Prostacyclin (PGI2) Thromboxane (TxA2) Inhibits plt aggregation Vasodilator Enhances plt aggregation Vasoconstrictor

Aggregated Platelets Resting Platelet GPIIb/IIIa (inactive) GPIIb/IIIa Agonist (activated) e.g. ADP TXA2 thrombin Fibrinogen high shear: von Willebrand factor substitutes for fibrinogen

Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets Platelet Aggregation Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets

Lets have a close view about the mechanism

Platelet Activation Pathways (1) ADP Adrenaline COLLAGEN THROMBIN ADP GpIIb/IIIa Aggregation GpIIb/IIIa Aggregation GpIIb/IIIa Aggregation GpIIb/IIIa Adhesion Platelet GpIb Adrenaline Adhesion vWF Exposed Collagen Endothelium

What happens when you have an injured blood vessel? Endothelial cells Coagulation factors… Platelet Exposed sub-endothelium Tissue factor (TF)

How does a platelet plug a hole under high shear stress conditions? -GP Ib binds the exposed vWF on sub-endothelial cells Platelet Platelets roll on vWF and become activated… vWF

Platelet activation Thrombin Agonist eg. thrombin Negative charge (Priming and propagation (Intrinsic pathway) of coagulation) Agonist eg. thrombin Negative charge Activates the coagulation factors and propagates the production of higher levels of thrombin required to support haemostasis PS PS Upon platelet activation, phosphatidyl serine (PS) flips to the outer leaflet of the plasma membrane. PS PS Activation PS PS Thrombin Fibrinogen Fibrin

Platelet activation (continued from previous slide) also results in the release of the constituents of the alpha and dense granules Activation of aIIbb3 allows binding to fibrinogen Alpha granules P selectin (tm) -fibrinogen -PDGF -VWF ADP COX AA TXA2 Aspirin, inactivates cyclooxygenase (COX) Activates platelets Thromboxane A2 Arachidonic acid (AA)

Platelet activation (cont.): - aIIbb3 becomes activated and capable of binding fibrinogen Fibrinogen The co-crosslinking of two or more platelets by aIIbb3 and fibrinogen is termed ‘aggregation’ Platelet

Side note Heparin is used on patients who clot excessively. Endothelial cells make heparin-like molecules and expose them on their surface. PF4 binds these substances. Heparin can complex with bound PF4 and heparin will be neutralized.

Final Stage : Stabilization of Clot AKA: primary hemostatic plug formation Thrombus formation Platelets release Factor V Expose factor III, accelerating coagulation cascade Promote activation of clotting factors

Platelet System: Additional Functions Provides the reaction surface for some coagulation system reactions, as well as platelet factor 3 (PF3) which is platelet phospholipid Supports and maintains endothelial lining Defective hemostasis can occur due to decreased number of platelets (quantitative) abnormally functioning platelets (qualitative)

Factors that activate platelets ADP Thrombin Collagen 5-hydroxytryptamine (serotonin) Thromboxane A2 Mechanical stimuli Many stimuli Several different receptors Multiple signalling pathways

Agonists “a term used to describe substances that can attach to a platelet membrane receptor and activate platelets causing them to aggregate .” McKenzie, SB. Clinical Hematology, Glossary, p. 919

Agonists agonists produced ADP Collagen Thrombin Epinephrine Arachidonic acid Thromboxane A2

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Blood clot