Hemostasis and Thrombosis

Ezekiel T. Arteta As part of the discussion for the Summer Conference Case 2

Hemostasis and Thrombosis
• Hemostasis
– Cessation of bleeding from a cut or severed vessel

• Thrombosis
– When endothelium lining blood vessels is damaged or removed.

• Complex, highly regulated physiological process
– Events • Cellular • Biochemical – Keep blood in liquid state in vasculature – Prevents blood loss following injury through clot formation
Blood Clotting (coagulation)

Hemostasis • Four major physiologic events: – Vasoconstriction of the injured vessel – Platelet plug formation (1o hemostasis) – Formation of Blood clot and Fibrin formation (2o hemostasis) – Fibrinolysis .

Fibrin .BV Injury Neural Tissue Factor Blood Vessel Constriction Platelet Activation Coagulation Activation Primary hemostatic plug Reduced Blood flow Stable Hemostatic Plug Plt-Fusion Thrombin.

Review: Vascular System • Blood Vessels – Arteries • 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 .

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 .Review: Vascular System • Construction – Endothelium • • • • Single layer of endothelial cells.

provide support around vessels .Review: Vascular System – Basement membrane • Collagen material – stimulates platelets – Connective tissue • Elastic fibers.


coagulation and fibrinolysis – Subendothelium • Collagen within is what’s exposed upon injury .Vascular System: Blood Vessels • Function – Endothelium • Controls vessel permeability • Controls blood flow rate • Produces and releases substances that inhibit OR stimulate platelets.

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 .

• Subsequently linked to platelet plug formation.Vasoconstriction • Initial response to vessel injury. – Short-lived • More pronounced in vessels with medial smooth muscles • Dependent on local contraction of smooth muscle. .

Vasoconstriction • Vasoconstriction results from: – Local myogenic spasm – Local autacoid factors from the traumatized tissues and blood platelets – Nervous reflexes .

– Endothelin-1 = synthesized by injured endothelium – Serotonin (5-hydroxytryptamine) = made by platelet aggregation and endothelium – Bradykinin. Fibrinopeptides .Vasoconstriction • Local autacoid factors from the traumatized tissues and blood platelets – Thromboxane A2 = produced locally at the site of injury via the release of arachidonic acid from platelets.

Vasoconstriction • Vasodilators – Prostaglandin (PGI2)/ Prostacyclins • 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. • The extent of VC varies with the degree of vessel injury .

Vasoconstriction .

Platelet Plug Formation Platelets – Anucleate fragments of megakaryocytes – Formed in the bone marrow – Normal value: 150.000 to 400. platelets are normally removed by the spleen. If not consumed in a clotting reaction.000/ L – Average life span of 7 to 10 days – Eliminated in the circulation mainly by the tissue macrophage system – Up to 30% may be sequestered in the spleen. .

myosin. thrombostenin – Residuals of ER and Golgi complex (synthesizes various enzymes and store calcium ions) – Mitochondria = produces ATP and ADP – Enzymes for prostaglandin synthesis – Fibrin-stabilizing factor – Growth factor .Platelet Plug Formation • Contents of Platelets – Actin.

Platelet Plug Formation • Cell Membrane of Platelets – Glycoproteins • Repulses adherence to normal endothelium • Causes adherence to injured areas of vessel wall – There must be exposed collagen • Contents – GPIb binds von Willebrand’s factor needed for platelet adhesion to collagen – GPIIb/IIIa bind fibrinogen needed for aggregation – Bind ADP and thrombin. involved in 2o hemostasis – Phospholipids . VIII on surface. V. promoting aggregation – Factors I.

they become activated or adhesive • Three stages of plug formation – Platelet activation and adhesion – Platelet aggregation – Platelet secretion and release .Platelet Plug Formation • Once the platelets “normal” environment is changed.

• Exposure to surfaces in the tissues causes them to bind to collagen with the presence of von Willebrand factor ( vWF) and Glycoprotein IbIX.Platelet Activation and Adhesion • Platelets attach to non-platelet surfaces. such as collagen fibers in the subendothelium • Platelets move from the blood vessels and into the tissues. making a bridge formation. which triggers a shape change • Reversible • No ADP released .

Platelet Activation and Adhesion • 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 Activation and Adhesion .

Platelet Aggregation • Chemical changes cause platelets to aggregate and stick to one another • Newly arriving platelets become activated by agonists • Exposure of GPIIb/IIIa sites bind fibrinogen • Fibrinogen + activated platelets serves as a bridge between two platelets • Calcium must be present .

Platelet Aggregation • Activated platelet membrane generates TXA2 • TXA2 stimulates release .

α2-antiplasmin. causing vasoconstriction • Granules trigger a secondary aggregation which is irreversible • Granules consist of – Alpha granules: Factor V. serotonin. Factor VIII:vWF.Platelet Secretion and Release • Requires ATP • Platelets release contents of their granules. platelet factor 4 – Dense bodies: ATP. ADP. Fibrinogen. Ca .

continues to recruit and stimulate platelets by increasing cytoplasmic calcium .Platelet Secretion and Release • Granules consist of – Factor V: receptor on platelet surface for factor Xa & prothrombin – PF4: heparin neutralizing factor – ADP: agonist.

Platelet Plug Formation .

in platelet adherence to the subendothelium under conditions of high shear stress that occur in small vessels and stenosed arteries .Impt.

• Activation of Protein Kinase C leads to: – Phosphorylation of pleckstrin – Change of platelet shape – Release of contents of the storage granules – Aggregation .

Platelet Aggregation .

Xa complexes on activated platelets Thrombin Subendothelial Collagen Platelet adhesion secretion Reversible ADP. serotonin. serotonin.Summary and Overview Vascular Endothelial Injury Platelet hemostatic function Vasoconstriction Coagulation activation via tissue factor-factor VIIa IXa. fibrinogen Platelet aggregation secretion Irreversible ADP. fibrinogen Platelet aggregation Fibrinogen PLATELET-FIBRIN THROMBUS . Ca2+. Ca2+.

and from blood proteins adhering to the traumatized vascular wall initiate the clotting process. if trauma is severe. .Blood Coagulation • Clot begins 15 to 20 sec. 1 to 2 minutes if the trauma is minor. from platelets. • Activator substances from the traumatized vascular wall.

IV occasionally identified by roman numeral There’s no VI assigned PLT factor 3.a clotting factors Majority are glycoproteins Majority are synthesized in the liver  Few synthesized in monocytes.k. endothelia.k.a coagulation factors a. III.o o o o o o o o o o Procoagulants a. II. and megakaryocytes Eight circulate as zymogens Four are cofactors Categorized as substrates. Prekallikrein. cofactors. & HMWK are not assigned roman numerals o o • Physical Properties Groupings . or enzymes Nomenclature Roman numerals “a” indicates active form I.

Blood Coagulation Mechanism • Procoagulants – promote coagulation • Anticoagulants – inhibit coagulation • NORMAL= ANTICOAGULANTS PREDOMINATE • INJURY= PROCOAGULANTS are ACTIVATED .

blood cells. and plasma to form the clot. .  formation of prothrombin activator (rate-limiting). – The prothrombin activator catalyzes conversion of prothrombin into thrombin – The thrombin converts fibrinogen into fibrin fibers that enmesh platelets.Blood Coagulation Mechanism • Three essential steps – Rupture of the vessel or damage to the blood  a complex cascade of blood coagulation factors.

• Damage to tissue outside the vessel – Intrinsic • Trauma to the blood itself • Damage to the blood vessel (collagen exposed) .Blood Coagulation • Two Pathways – Extrinsic • Initiation of the fibrin clot in response to tissue injury.

Blood Coagulation .

Intrinsic Pathway Factor XII kallikrein Intact platelets Vascular Endothelial Injury (Collagen is exposed to blood) Damaged platelets Factor XIIa Kallikrein Prekallikrein Release of platelet phospholipids which contains a lipoprotein called platelet factor 3 .

Factor IXa 4. Ca2+ 2. . Factor VIIIa (cofactor) 3.Bradykinin Intrinsic Tenase Complex 1. Factor X Assembly occurs on the platelet’s membrane.

Extrinsic Pathway • Tissue Factor From liver – Phospholipids from the membranes of the tissue – Lipoprotein complex as proteolytic enzyme .

the Gla residues in the amino terminal serve as highaffinity binding sites for Ca2+.Extrinsic Pathway From liver • γ-carboxyglutamate (Gla)-containing zymogens (II. VII. . IX. and X).

• Tissue factor + factor VIIa = TISSUE FACTOR COMPLEX – Also activate factor IX in the intrinsic pathway. .Extrinsic Pathway From liver • Tissue factor acts as a cofactor for factor VIIa.

Extrinsic Pathway From liver • Extrinsic Tenase Complex (activates factor Xa) – Ca2+ – Tissue factor complex – Factor X • Assembly occurs on the platelet’s membrane. .


.• Prothrombinase Complex – Ca2+ – Factor Va – Factor Xa – Prothrombin • Takes place on the membrane of an activated platelet – Has exposed acidic phospholipid phosphatidylserine.

– Cofactor • Binds specifically to the platelet membrane • Forms complex with factor Xa and prothrombin • Inactivated by action of thrombin . spleen and kidney – Found in platelets as well as in plasma.Prothrombin to Thrombin • Factor V – Synthesized in the liver.

γ contains Asp-linked complex oligosaccharides . γ)2 covalently linked by disulfide bonds. Bβ. – Bβ.Fibrinogen to Fibrin • Consists of three nonidentical pairs of polypeptide chains (Aα.

• Contribute to solubility • Prevent aggregation . and tyrosine-O-sulfate in FPB. Glu residues.Fibrinogen to Fibrin – Aα chain = Fibrinopeptide A (FPA). Bβ chain = Fibrinopeptide B (FPB) • Bear excess negative charges at the amino terminal ends of the chains as a result of the presence of Asp.

β.γ)2 – Exposes binding sites for aggregation – RESULT: FIBRIN CLOT . – Generates fibrin monomer (α.Fibrinogen to Fibrin Thrombin • Serine protease • Formed by the prothrombinase complex • Hydrolyzes the four Arg-Gly bonds between the fibrinopeptides and the α and β portions of the Aα and Bβ chains of fibrinogen.

Fibrinogen to Fibrin .

– More stable fibrin clot .Fibrinogen to Fibrin Thrombin • Also converts factor XIII to factor XIIIa. – Has spicific transglutaminase activity – Forms peptide bonds between the amide groups of glutamine and the ε-amino groups of lysine residues.

Fibrinogen to Fibrin .

the fibrin clot is lysed .Fibrinogen to Fibrin • Fibrin is a product formed during hemostasis. tissue repair or inflammation • Fibrin plays a temporary role • Once injury heals.

FXI and FXIII) FVa Prothrombinase .Extrinsic Tenase Ca2+ 2+ Ca FVIIa TF Intrinsic Tenase FIXa Ca2+ FIXa Ca2+ FVIIIa FXa Thrombin Cleaves Fibrinogen Activates Platelets Ca2+ Ca2+ FVa FXa IIa Activates procofactors (FV and FVIII) Activates zymogens (FVII.

Thrombi types • White thrombus – Platelets + Fibrin. particularly in areas with rapid blood flow. with or without injury. poor in erythrocytes – Forms at the site of an injury or abnormal vessel wall. . • Red Thrombus – Erythrocytes + fibrin – Resembles clot in test tube – Forms within vessels with retarded blood flow or stasis.

Thrombi types
• Disseminated fibrin deposit
– In very small blood vessels or capillaries

Plasmin • Responsible for fibrin and fibrinogen degradation • Serine protease • Circulates as plasminogen
– Activated plasminogen are inactivated by α2antiplasmin – Binds to fibrin and is incorporated in clots

• Both plasminogen and plasmin specifically binds lysine residues on fibrin via one of its kringle domains.
– Increasingly incorporates into the fibrin mesh as it cleaves it.

• Activators of plasminogen
– Found in most body tissues – Cleave Arg-Val bond

Fibrinolysis .

etc. macrophages. fibroblasts. – Action: degradation of ECM .Fibrinolysis • Tissue plasminogen activator (t-PA) – From vascular endothelium under injury/ stress. • Urokinase – Precursor: PROUROKINASE – Originally isolated in urine – Synthesized by monocytes. – Catalytically inactive unless bound to fibrin. epithelial cells.

.Fibrinolysis • TAFIa (activated thrombin activatable fibrinolysis inhibitor) – Inhibit fibrinolysis by removing terminal lysines.

Fibrinolysis .

Balance of Hemostasis .

Virchow’s Triad .

• Achieved in two ways: – It circulates as prothrombin • Activated by the coagulation cascade • There are feedback mechanisms at each point in the cascade – Circulating inhibitors .Regulation of Thrombin • To prevent further fibrin formation or platelet activation.

Coagulation Inhibition System • Provides balance and control of clotting mechanisms • Natural inhibitors and anticoagulants circulate in the plasma to: – Prevent clotting when it’s not needed – Limit or localize the clotting that is needed .

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